bdnfpro val met  (Alomone Labs)

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of proBDNF precursor and cleaved BDNFpro domain. αBDNFpro antibody recognizes the furin cleavage site of the prodomain. Western blotting probing recombinant mBDNF, BDNFpro, and proBDNF CR with αBDNFpro and αmBDNF antibodies. b Cortical slices from control mice injected with AAV-GFAP-GFP virus were recorded and fixed 10 min after TBS for immunostaining. z-stack reconstruction shows astrocytes labeled by GFP. Magnification of a single stack from a region of interest (ROI) shows BDNFpro immunoreactivity and BDNFpro/GFP colocalization signal of one GFP-astrocyte delimited by an approximate territory (white dashed). Scale bars: 10 µm. c z-stack reconstruction of BDNFpro/GFP colocalization signals in astrocytes from baseline- and TBS-slices from control mice. The insets show GFP signal. BDNFpro/GFP colocalization was quantified in the whole cell and branches using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 9 cells, 4 slices, 3 mice for baseline; n = 12 cells, 4 slices, 3 mice for TBS). Scale bars: 10 µm. d mBDNF/GFP colocalization was quantified in the whole cell and branches using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 10 cells, 3 slices, 3 mice for baseline; n = 10 cells, 4 slices, 3 mice for TBS). e BDNFpro/GFP and mBDNF/GFP colocalizations in baseline and TBS-slices treated with plasmin were quantified in branches using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 20 cells, 3 slices, 3 mice for baseline BDNFpro/GFP; n = 11 cells, 3 slices, 3 mice for TBS BDNFpro/GFP; n = 21 cells, 3 slices, 3 mice for baseline mBDNF/GFP; n = 13 cells, 3 slices, 3 mice for TBS mBDNF/GFP). f z-stack reconstruction of BDNFpro/GFP colocalization signals in astrocytes from basal- and TBS-slices from tamoxifen-treated p75-flox mice. Insets show GFP signal. BDNFpro/GFP colocalization was quantified using Mander’s overlap ( n = 11 cells, 5 slices, 4 mice for baseline; n = 16 cells, 5 slices, 4 mice for TBS). Scale bars: 10 µm. Data are normalized to baseline and presented as mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Western Blot, Recombinant, Control, Injection, Virus, Immunostaining, Labeling

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Graphical representation of the SIM super-resolution microscope. 3D-SIM image of a GFP-labeled astrocyte in a TBS-slice from control mice. Scale bar: 10 µm. Magnification of a ROI shows BDNFpro/GFP colocalization signal localized in fine membrane extensions of the cell periphery. Scale bar: 200 nm. b 3D-SIM image of the ROI in ( a ); z-axe is visualized in pseudocolor to facilitate microdomains identification. Scale bar: 200 nm. Magnification of microdomains characterized by the typical fingerlike extension (dashed squares 1 and 2) and flat lamellar sheath (dashed squares 3 and 4) are shown. BDNFpro/GFP colocalization is indicated (red arrowheads). Scale bars: 40 nm.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Super-Resolution Microscopy, Labeling, Control, Membrane

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Experimental design linking field-potential with electron microscopy (EM) in layer II/III perirhinal cortex. TBS (10 min)-slices were dissected for EM processing. b Representative EM-image depicts BDNFpro-gold particles at axon bouton (dashed squares 1 to 4) and dendritic spine (dashed squares 5 and 6). Scale bar: 100 nm. Magnification indicates representative areas (dashed squares 1 to 6) in which gold particles (red arrowheads) localization is shown. Scale bars: 10 nm. c Representative EM-image depicts BDNFpro-gold particles (dashed squares 1 to 6) at astrocytic microdomains (light blue) Scale bar: 250 nm. Magnification indicates representative areas (dashed squares 1 to 6) in which gold particles (red arrowheads) localization is shown. Scale bars: 20 nm. d Dot plot depicts the number of BDNFpro-gold particles in whole astrocytes and peri-synaptic astrocytes counted per section ( n = 41 sections, 5 slices, 3 mice). e Dot plot depicts the percentage of BDNFpro-gold particles at peri-synaptic astrocytes ( n = 41 sections, 5 slices, 3 mice). Data are mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Electron Microscopy

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a z-stack reconstruction shows astrocytes labeled by GFP. Cortical slices from control mice injected with AAV-GFAP-GFP virus were fixed 10 min after TBS and processed for immunostaining and confocal analysis. Scale bar: 10 µm. Magnification of a ROI shows one GFP-astrocyte delimited by an approximate territory (white dashed). Scale bar: 10 µm. BDNFpro/GFP and Vamp2/GFP co-localizations signals are shown. Magnification shows representative areas (dashed squares 1 to 4) in which BDNFpro/GFP and Vamp2/GFP signals overlap. Scale bars: 1 µm. b 3D-SIM image of a GFP-labeled astrocyte in a TBS-slice from control mice. Scale bar: 10 µm. Magnification of a ROI shows BDNFpro/Vamp2 colocalization signal. Scale bar: 500 nm. Magnification shows BDNFpro/Vamp2 colocalization signal in fine membrane extensions of the cell periphery (dashed squares 1 to 4). Scale bars: 50 nm. c EM image depicts BDNFpro-gold at astrocytic microdomains (light blue) surrounding an axon bouton. Scale bar: 100 nm. Magnification of the ROI shows gold particles (red arrowheads) in vesicular-like structures. Scale bar: 20 nm. d Digital reconstruction of the image in ( c ). Astrocytic vesicles (black boundary) are shown.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Labeling, Control, Injection, Virus, Immunostaining, Membrane

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of the experimental design. Step I, deletion of p75 NTR in astrocytes from tamoxifen-treated p75-flox mice precludes proBDNF transfer from neurons to astrocyte following TBS. Step II, LV-BDNFpro stop transduction replaces BDNFpro in astrocytes. Schematic representation of the experimental paradigms (right); mice were treated with tamoxifen (−5 to 0), injected with lentiviruses the last day of tamoxifen treatment (0 dptm) and finally recorded (14 dptm). LTP evoked in slices from p75-flox mice and control littermates injected with LV-GFP stop or LV-BDNFpro stop is shown. *** p < 0.001 (unpaired t -test) (p75-flox/LV-GFP stop 102.00 ± 1.85%, p75-flox/LV-BDNFpro stop 177.41 ± 10.74% and control littermates/LV-GFP stop 165.27 ± 2.24% fEPSP 180 min from TBS; n = 10 slices, 6 mice for p75-flox/LV-GFP stop ; n = 9 slices, 6 mice for p75-flox/LV-BDNFpro stop ; n = 6 slices, 4 mice for control littermates/LV-GFP stop ). b Schematic representation of the experimental design. Step II, and I as in ( a ). Step III, LV-TeTN stop transduction in astrocytes prevents from BDNFpro release. Schematic representation of the experimental paradigm as in ( a ). LTP evoked in slices from p75-flox mice and control littermates injected with LV-GFP stop or co-injected with LV-GFP stop /LV-BDNFpro stop and LV-TeTN stop /LV-BDNFpro stop is shown. *** p < 0.001 (unpaired t -test) (p75-flox/LV-BDNFpro stop /LV-GFP stop 167.50 ± 8.78%, p75-flox/LV-BDNFpro stop /LV-TeTN stop 106.26 ± 2.47% and control littermates/LV-GFP stop 151.33 ± 7.39% fEPSP 180 min from TBS; n = 8 slices, 6 mice for p75-flox/LV-BDNFpro stop /LV-GFP stop ; n = 9 slices, 7 mice for p75-flox/LV-TeTN stop /LV-BDNFpro stop ; n = 7 slices, 4 mice control littermates/LV-GFP stop ). c LTP evoked in slices from p75-flox mice and control littermates. Mice were treated with tamoxifen (−5 to 0) and recorded 14 dptm. Slices were perfused (18–28 min) with vehicle or exogenous BDNFpro. *** p < 0.001 (unpaired t -test) (p75-flox/vehicle 103.54 ± 3.43%, p75-flox/BDNFpro 171.09 ± 10.17% and control littermates/vehicle 162.81 ± 9.87% fEPSP 180 min from TBS; n = 8 slices, 4 mice for p75-flox/vehicle; n = 7 slices, 5 mice for p75-flox/BDNFpro; n = 7 slices, 5 mice for control littermates/vehicle). d LTP evoked as in ( c ). Slices were perfused (18–28 min) with vehicle or BDNFpro Val/Met . *** p < 0.001 (unpaired t -test) (p75-flox/vehicle 105.26 ± 1.59%, p75-flox/BDNFpro Val/Met 148.76 ± 8.69% and control littermates/vehicle 147.55 ± 3.55% fEPSP 180 min from TBS; n = 8 slices, 5 mice for p75-flox/vehicle; n = 6 slices, 4 mice for p75-flox/BDNFpro Val/Met ; n = 7 slices, 5 mice for control littermates/vehicle). Data are presented as mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Transduction, Injection, Control

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of the experimental design. Circular DNA probes (−) and (+) are coupled to II° antibody targeting αSorCS2 and αTrkB I° antibody. BDNFpro induces TrkB/SorCS2 complex formation (PLA TrkB/SorCS2 ) that is prevented in the presence of αSorCS2 (blocking) antibody. b Panels show PLA TrkB/SorCS2 signals in primary culture of cortical neurons treated with vehicle or BDNFpro. The insets show reference GFP-neurons. Scale bars: 5 μm. c Panels show a GFP-neuron treated with BDNFpro. Scale bar: 5 μm. Magnification of regions of interest 1 and 2 shows dendritic PLA TrkB/SorCS2 localization (red arrowheads). Scale bar: 1 μm. d Quantification of PLA TrkB/SorCS2 signal in cultured neurons treated with vehicle, BDNFpro (in presence or absence of αSorCS2), mBDNF or proBDNF CR . Data are presented as mean ± SEM; ** p < 0.01 (unpaired t -test) ( n = 111 cells, 3 cultures for vehicle; n = 152 cells, 4 cultures for BDNFpro; n = 98 cells, 3 cultures for BDNFpro/aSorCS2; n = 89 cells, 3 cultures for mBDNF; n = 79 cells, 3 cultures for proBDNF CR ). e z-stack reconstruction showing NeuN, PSD95 and PLA TrkB/SorCS2 signals in baseline and TBS slices. NeuN/PLA TrkB/SorCS2 and PSD95/PLA TrkB/SorCS2 colocalization signals are shown. Scale bars: 40 μm. NeuN/PLA TrkB/SorCS2 and PSD95/PLA TrkB/SorCS2 colocalization was quantified using Mander’s overlap. Data are normalized to baseline and presented as mean ± SEM; * p < 0.05 (unpaired t -test) ( n = 4 slices, 3 mice for each experimental condition).

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Blocking Assay, Cell Culture

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of the experimental design. Step I, deletion of p75 NTR in astrocytes from tamoxifen-treated p75-flox mice precludes proBDNF transfer from neurons to astrocyte following TBS. Step II, LV-BDNFpro stop transduction replaces BDNFpro in astrocytes. Step III, astrocytic BDNFpro provides final increase of TrkB/SorCS2 complexes in dendritic spines and LTP maintenance. b z-stack reconstruction showing NeuN/PLA TrkB/SorCS2 colocalization signal in TBS-slices from p75-flox mice transduced with LV-GFP stop or LV-BDNFpro stop . Scale bars: 40 μm. The insets show the field of analysis. Scale bars: 15 μm. NeuN/PLA TrkB/SorCS2 colocalization was quantified using Mander’s overlap. ** p < 0.01 (unpaired t -test) ( n = 4 slices, 3 mice for each experimental condition). c Dot plot shows quantification of pTrkB/TrkB colocalization in baseline- and TBS-slices from p75-flox mice transduced with LV-GFP stop or LV-BDNFpro stop and control littermates using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 4 slices, 3 mice for p75-flox mice/LV-GFP stop /TBS; n = 4 slices, 3 mice for p75-flox mice/LV-GFP stop /baseline; n = 6 slices, 4 mice for p75-flox mice/LV-BDNFpro stop /TBS; n = 5 slices, 4 mice for p75-flox mice/LV-BDNFpro stop /baseline; n = 5 slices, 3 mice for control littermates/TBS; n = 6 slices, 3 mice for control littermates/baseline). Data are normalized to baseline and presented as mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Transduction, Control

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic diagram depicting the behavioral paradigm used for ORT. Mice were subjected to familiarization (sample phase) with two identical objects (circles). A test phase in which one familiar object (circle) is substituted with a novel one was performed after 10 min (square) and 24 h (triangle). b Schematic diagram depicting the experimental paradigm. p75-flox mice and control littermates treated with tamoxifen (−5 to 0) and injected with LV-GFP stop or LV-BDNFpro stop the last day of tamoxifen treatment (0 dptm) were subjected to ORT (14 dptm). Discrimination index is plotted against time interval between sample phase and test phases. ** p < 0.01 (post hoc Holm–Sidak) ( n = 11 mice for p75-flox/LV-GFP stop ; n = 10 mice for p75-flox/LV-BDNFpro stop ; n = 5 mice for control littermates). c Schematic diagram depicting the experimental paradigm as in ( b ). The dot plot shows mean exploration time of the familiar object and the novel object in the sample phase ( n = 11 mice for p75-flox/LV-GFP stop ; n = 10 mice for p75-flox/LV-BDNFpro stop ; n = 5 mice for control littermates). Data are presented as mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Control, Injection

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of proBDNF precursor and cleaved BDNFpro domain. αBDNFpro antibody recognizes the furin cleavage site of the prodomain. Western blotting probing recombinant mBDNF, BDNFpro, and proBDNF CR with αBDNFpro and αmBDNF antibodies. b Cortical slices from control mice injected with AAV-GFAP-GFP virus were recorded and fixed 10 min after TBS for immunostaining. z-stack reconstruction shows astrocytes labeled by GFP. Magnification of a single stack from a region of interest (ROI) shows BDNFpro immunoreactivity and BDNFpro/GFP colocalization signal of one GFP-astrocyte delimited by an approximate territory (white dashed). Scale bars: 10 µm. c z-stack reconstruction of BDNFpro/GFP colocalization signals in astrocytes from baseline- and TBS-slices from control mice. The insets show GFP signal. BDNFpro/GFP colocalization was quantified in the whole cell and branches using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 9 cells, 4 slices, 3 mice for baseline; n = 12 cells, 4 slices, 3 mice for TBS). Scale bars: 10 µm. d mBDNF/GFP colocalization was quantified in the whole cell and branches using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 10 cells, 3 slices, 3 mice for baseline; n = 10 cells, 4 slices, 3 mice for TBS). e BDNFpro/GFP and mBDNF/GFP colocalizations in baseline and TBS-slices treated with plasmin were quantified in branches using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 20 cells, 3 slices, 3 mice for baseline BDNFpro/GFP; n = 11 cells, 3 slices, 3 mice for TBS BDNFpro/GFP; n = 21 cells, 3 slices, 3 mice for baseline mBDNF/GFP; n = 13 cells, 3 slices, 3 mice for TBS mBDNF/GFP). f z-stack reconstruction of BDNFpro/GFP colocalization signals in astrocytes from basal- and TBS-slices from tamoxifen-treated p75-flox mice. Insets show GFP signal. BDNFpro/GFP colocalization was quantified using Mander’s overlap ( n = 11 cells, 5 slices, 4 mice for baseline; n = 16 cells, 5 slices, 4 mice for TBS). Scale bars: 10 µm. Data are normalized to baseline and presented as mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Western Blot, Recombinant, Injection, Immunostaining, Labeling

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Graphical representation of the SIM super-resolution microscope. 3D-SIM image of a GFP-labeled astrocyte in a TBS-slice from control mice. Scale bar: 10 µm. Magnification of a ROI shows BDNFpro/GFP colocalization signal localized in fine membrane extensions of the cell periphery. Scale bar: 200 nm. b 3D-SIM image of the ROI in ( a ); z-axe is visualized in pseudocolor to facilitate microdomains identification. Scale bar: 200 nm. Magnification of microdomains characterized by the typical fingerlike extension (dashed squares 1 and 2) and flat lamellar sheath (dashed squares 3 and 4) are shown. BDNFpro/GFP colocalization is indicated (red arrowheads). Scale bars: 40 nm.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Microscopy, Labeling

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Experimental design linking field-potential with electron microscopy (EM) in layer II/III perirhinal cortex. TBS (10 min)-slices were dissected for EM processing. b Representative EM-image depicts BDNFpro-gold particles at axon bouton (dashed squares 1 to 4) and dendritic spine (dashed squares 5 and 6). Scale bar: 100 nm. Magnification indicates representative areas (dashed squares 1 to 6) in which gold particles (red arrowheads) localization is shown. Scale bars: 10 nm. c Representative EM-image depicts BDNFpro-gold particles (dashed squares 1 to 6) at astrocytic microdomains (light blue) Scale bar: 250 nm. Magnification indicates representative areas (dashed squares 1 to 6) in which gold particles (red arrowheads) localization is shown. Scale bars: 20 nm. d Dot plot depicts the number of BDNFpro-gold particles in whole astrocytes and peri-synaptic astrocytes counted per section ( n = 41 sections, 5 slices, 3 mice). e Dot plot depicts the percentage of BDNFpro-gold particles at peri-synaptic astrocytes ( n = 41 sections, 5 slices, 3 mice). Data are mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Electron Microscopy

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a z-stack reconstruction shows astrocytes labeled by GFP. Cortical slices from control mice injected with AAV-GFAP-GFP virus were fixed 10 min after TBS and processed for immunostaining and confocal analysis. Scale bar: 10 µm. Magnification of a ROI shows one GFP-astrocyte delimited by an approximate territory (white dashed). Scale bar: 10 µm. BDNFpro/GFP and Vamp2/GFP co-localizations signals are shown. Magnification shows representative areas (dashed squares 1 to 4) in which BDNFpro/GFP and Vamp2/GFP signals overlap. Scale bars: 1 µm. b 3D-SIM image of a GFP-labeled astrocyte in a TBS-slice from control mice. Scale bar: 10 µm. Magnification of a ROI shows BDNFpro/Vamp2 colocalization signal. Scale bar: 500 nm. Magnification shows BDNFpro/Vamp2 colocalization signal in fine membrane extensions of the cell periphery (dashed squares 1 to 4). Scale bars: 50 nm. c EM image depicts BDNFpro-gold at astrocytic microdomains (light blue) surrounding an axon bouton. Scale bar: 100 nm. Magnification of the ROI shows gold particles (red arrowheads) in vesicular-like structures. Scale bar: 20 nm. d Digital reconstruction of the image in ( c ). Astrocytic vesicles (black boundary) are shown.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Labeling, Injection, Immunostaining

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of the experimental design. Step I, deletion of p75 NTR in astrocytes from tamoxifen-treated p75-flox mice precludes proBDNF transfer from neurons to astrocyte following TBS. Step II, LV-BDNFpro stop transduction replaces BDNFpro in astrocytes. Schematic representation of the experimental paradigms (right); mice were treated with tamoxifen (−5 to 0), injected with lentiviruses the last day of tamoxifen treatment (0 dptm) and finally recorded (14 dptm). LTP evoked in slices from p75-flox mice and control littermates injected with LV-GFP stop or LV-BDNFpro stop is shown. *** p < 0.001 (unpaired t -test) (p75-flox/LV-GFP stop 102.00 ± 1.85%, p75-flox/LV-BDNFpro stop 177.41 ± 10.74% and control littermates/LV-GFP stop 165.27 ± 2.24% fEPSP 180 min from TBS; n = 10 slices, 6 mice for p75-flox/LV-GFP stop ; n = 9 slices, 6 mice for p75-flox/LV-BDNFpro stop ; n = 6 slices, 4 mice for control littermates/LV-GFP stop ). b Schematic representation of the experimental design. Step II, and I as in ( a ). Step III, LV-TeTN stop transduction in astrocytes prevents from BDNFpro release. Schematic representation of the experimental paradigm as in ( a ). LTP evoked in slices from p75-flox mice and control littermates injected with LV-GFP stop or co-injected with LV-GFP stop /LV-BDNFpro stop and LV-TeTN stop /LV-BDNFpro stop is shown. *** p < 0.001 (unpaired t -test) (p75-flox/LV-BDNFpro stop /LV-GFP stop 167.50 ± 8.78%, p75-flox/LV-BDNFpro stop /LV-TeTN stop 106.26 ± 2.47% and control littermates/LV-GFP stop 151.33 ± 7.39% fEPSP 180 min from TBS; n = 8 slices, 6 mice for p75-flox/LV-BDNFpro stop /LV-GFP stop ; n = 9 slices, 7 mice for p75-flox/LV-TeTN stop /LV-BDNFpro stop ; n = 7 slices, 4 mice control littermates/LV-GFP stop ). c LTP evoked in slices from p75-flox mice and control littermates. Mice were treated with tamoxifen (−5 to 0) and recorded 14 dptm. Slices were perfused (18–28 min) with vehicle or exogenous BDNFpro. *** p < 0.001 (unpaired t -test) (p75-flox/vehicle 103.54 ± 3.43%, p75-flox/BDNFpro 171.09 ± 10.17% and control littermates/vehicle 162.81 ± 9.87% fEPSP 180 min from TBS; n = 8 slices, 4 mice for p75-flox/vehicle; n = 7 slices, 5 mice for p75-flox/BDNFpro; n = 7 slices, 5 mice for control littermates/vehicle). d LTP evoked as in ( c ). Slices were perfused (18–28 min) with vehicle or BDNFpro Val/Met . *** p < 0.001 (unpaired t -test) (p75-flox/vehicle 105.26 ± 1.59%, p75-flox/BDNFpro Val/Met 148.76 ± 8.69% and control littermates/vehicle 147.55 ± 3.55% fEPSP 180 min from TBS; n = 8 slices, 5 mice for p75-flox/vehicle; n = 6 slices, 4 mice for p75-flox/BDNFpro Val/Met ; n = 7 slices, 5 mice for control littermates/vehicle). Data are presented as mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Transduction, Injection

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of the experimental design. Circular DNA probes (−) and (+) are coupled to II° antibody targeting αSorCS2 and αTrkB I° antibody. BDNFpro induces TrkB/SorCS2 complex formation (PLA TrkB/SorCS2 ) that is prevented in the presence of αSorCS2 (blocking) antibody. b Panels show PLA TrkB/SorCS2 signals in primary culture of cortical neurons treated with vehicle or BDNFpro. The insets show reference GFP-neurons. Scale bars: 5 μm. c Panels show a GFP-neuron treated with BDNFpro. Scale bar: 5 μm. Magnification of regions of interest 1 and 2 shows dendritic PLA TrkB/SorCS2 localization (red arrowheads). Scale bar: 1 μm. d Quantification of PLA TrkB/SorCS2 signal in cultured neurons treated with vehicle, BDNFpro (in presence or absence of αSorCS2), mBDNF or proBDNF CR . Data are presented as mean ± SEM; ** p < 0.01 (unpaired t -test) ( n = 111 cells, 3 cultures for vehicle; n = 152 cells, 4 cultures for BDNFpro; n = 98 cells, 3 cultures for BDNFpro/aSorCS2; n = 89 cells, 3 cultures for mBDNF; n = 79 cells, 3 cultures for proBDNF CR ). e z-stack reconstruction showing NeuN, PSD95 and PLA TrkB/SorCS2 signals in baseline and TBS slices. NeuN/PLA TrkB/SorCS2 and PSD95/PLA TrkB/SorCS2 colocalization signals are shown. Scale bars: 40 μm. NeuN/PLA TrkB/SorCS2 and PSD95/PLA TrkB/SorCS2 colocalization was quantified using Mander’s overlap. Data are normalized to baseline and presented as mean ± SEM; * p < 0.05 (unpaired t -test) ( n = 4 slices, 3 mice for each experimental condition).

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Blocking Assay, Cell Culture

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of the experimental design. Step I, deletion of p75 NTR in astrocytes from tamoxifen-treated p75-flox mice precludes proBDNF transfer from neurons to astrocyte following TBS. Step II, LV-BDNFpro stop transduction replaces BDNFpro in astrocytes. Step III, astrocytic BDNFpro provides final increase of TrkB/SorCS2 complexes in dendritic spines and LTP maintenance. b z-stack reconstruction showing NeuN/PLA TrkB/SorCS2 colocalization signal in TBS-slices from p75-flox mice transduced with LV-GFP stop or LV-BDNFpro stop . Scale bars: 40 μm. The insets show the field of analysis. Scale bars: 15 μm. NeuN/PLA TrkB/SorCS2 colocalization was quantified using Mander’s overlap. ** p < 0.01 (unpaired t -test) ( n = 4 slices, 3 mice for each experimental condition). c Dot plot shows quantification of pTrkB/TrkB colocalization in baseline- and TBS-slices from p75-flox mice transduced with LV-GFP stop or LV-BDNFpro stop and control littermates using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 4 slices, 3 mice for p75-flox mice/LV-GFP stop /TBS; n = 4 slices, 3 mice for p75-flox mice/LV-GFP stop /baseline; n = 6 slices, 4 mice for p75-flox mice/LV-BDNFpro stop /TBS; n = 5 slices, 4 mice for p75-flox mice/LV-BDNFpro stop /baseline; n = 5 slices, 3 mice for control littermates/TBS; n = 6 slices, 3 mice for control littermates/baseline). Data are normalized to baseline and presented as mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Transduction

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic diagram depicting the behavioral paradigm used for ORT. Mice were subjected to familiarization (sample phase) with two identical objects (circles). A test phase in which one familiar object (circle) is substituted with a novel one was performed after 10 min (square) and 24 h (triangle). b Schematic diagram depicting the experimental paradigm. p75-flox mice and control littermates treated with tamoxifen (−5 to 0) and injected with LV-GFP stop or LV-BDNFpro stop the last day of tamoxifen treatment (0 dptm) were subjected to ORT (14 dptm). Discrimination index is plotted against time interval between sample phase and test phases. ** p < 0.01 (post hoc Holm–Sidak) ( n = 11 mice for p75-flox/LV-GFP stop ; n = 10 mice for p75-flox/LV-BDNFpro stop ; n = 5 mice for control littermates). c Schematic diagram depicting the experimental paradigm as in ( b ). The dot plot shows mean exploration time of the familiar object and the novel object in the sample phase ( n = 11 mice for p75-flox/LV-GFP stop ; n = 10 mice for p75-flox/LV-BDNFpro stop ; n = 5 mice for control littermates). Data are presented as mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFpro Val/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNF CR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Injection