Journal: bioRxiv

Article Title: Neurogenesis-mediated circuit remodeling reduces engram reinstatement and promotes forgetting

doi: 10.1101/2023.10.10.561722

Figure Lengend Snippet: ( a ) Experimental design. RV expressing ChR2-GFP or GFP alone was micro- injected into the DG. Blue laser stimulation was delivered daily for 15 d, and morphology of infected adult-generated granule cell assessed 15 d later. ( b ) Representative images from hippocampus showing (i) dendritic morphology (scale bar = 50 µm), (ii) dendritic segments (scale bar = 10 µm) and (iii) LMTs (scale bar = 10 µm) of infected adult-generated granule cells. Photostimulation did not affect ( c ) survival of adult-generated granule cells ( t 7 = 0.36, P = 0.73), but increased ( d ) total dendrite length ( t 10 = 2.35, P < 0.05), ( e ) dendritic complexity (ANOVA, main group effect: F 1,8 = 20.47, P <0.005; main distance effect: F 24,216 = 31.57, P < 0.0001; group × distance interaction: F 24,216 = 6.19, P < 0.0001), ( f ) spine density ( t 8 = 4.61, P < 0.005), and ( g ) LMT density ( t 7 = 3.17, P < 0.05). ( h ) (left) Experimental design. WT and n-ChR2 mice were treated with TAM, trained in contextual fear conditioning, and tested 15 d later. Optogenetic stimulation was delivered daily for 15 days, starting immediately following training. (right) Optogenetic activation did not induce forgetting at the shorter delay in n-ChR2 mice ( t 14 = 0.35, P = 0.73). ( i ) (left) Experimental design. WT and n-ChR2 mice were treated with TAM, trained in contextual fear conditioning, and tested 30 d later. Optogenetic stimulation was delivered daily for 15 d, starting one day following training. (right) Optogenetic activation induced forgetting in n-ChR2 mice ( t 15 = 2.75, P < 0.05).

Article Snippet: We used a CAG promoter-driven GFP (Addgene #16664), GFP-IRES-Cre (Addgene #48201), or ChR2-GFP (Addgene #114367).

Techniques: Expressing, Injection, Infection, Generated, Activation Assay

Journal: Scientific Reports

Article Title: Expression of a Barhl1a reporter in subsets of retinal ganglion cells and commissural neurons of the developing zebrafish brain

doi: 10.1038/s41598-020-65435-w

Figure Lengend Snippet: Barhl1a:GFP in the preoptic diencephalon marks populations of mitotic progenitors and differentiating neurons. Images represent single confocal Z-stacks through the diencephalon of Tg ( barhl1a:GFP;atoh7:gap43-RFP ) embryos at 35 hpf ( A, A’ ) and 30 hpf ( B, C ). Frontal view, dorsal is up. Embryos in ( A - B’ ) have been immunohistochemically stained with anti-GFP, anti-HuC ( A , A’ ) and anti-pH3 ( B , B’ ) antibodies (in red) as well as counterstained with DAPI (in blue). (A’) magnifications of the images in (A) in correspondence with the barhl1a:GFP expressing cell bodies. (A) yellow arrowheads point at the optic nerves from each side marked by Atoh7:gap43-RFP (in gray). White arrows point at Barhl1a:GFP cells co-labelled with HuC, a marker of differentiating neurons located in the basal half of the neuroepithelium. In (B) and its magnification ( B’ ), Barhl1a:GFP cells are also co-labelled by pH3, which marks mitotic neurons located at the apical surface of the neuroepithelium (white arrowhead in B’). ( C ) 30 hpf Tg ( barhl1a:GFP ) embryo injected with the gfap:lssKate DNA construct shows sparse LssKate labelling in the diencephalon (in red); which co-localize with GFP (green). Scale bars: (A–C) 50 µm, (A’,B’) 20 µm.

Article Snippet: In a first step the gfap promoter driving GFP was subcloned into a vector containing IsceI sites using NotI and XhoI restriction enzymes (NEB #R3189 and #R0146).

Techniques: Staining, Expressing, Marker, Injection, Construct

Journal: Molecular psychiatry

Article Title: Functional implications of a psychiatric risk variant within CACNA1C in induced human neurons

doi: 10.1038/mp.2014.143

Figure Lengend Snippet: a . Construction of lentivirus vectors for generating iNs from human fibroblasts. b–e. Human iNs have neuronal morphology and express several neuronal markers, Tuj1 ( b ), MAP2 ( c ), NeuN ( d ) and hSynapsin promoter driving DsRed ( e ) three weeks following induction. f. Human iNs co-cultured with human astrocytes express a presynaptic marker, VGLUT1. g. RT-PCR analyses reveal human iNs express neuronal markers including FOXP2 , TBR1 , GRIN1(NR1) , GABRA6 , GABRB2 , NTRK2 (TrkB), and SLC17A7 (VGLUT1) , but not an inhibitory neuronal marker, SLC32A1(VGAT) . h. Representative traces showing action potentials with injected currents. Scale bars, 50 μm.

Article Snippet: The human synapsin I promoter driving DsRed in a lentiviral plasmid (Addgene Plasmid 22909: pLV-hSyn-RFP) was obtained from Addgene.

Techniques: Cell Culture, Marker, Reverse Transcription Polymerase Chain Reaction, Injection

Journal: Cells

Article Title: Astrocytic CD44 Deficiency Reduces the Severity of Kainate-Induced Epilepsy

doi: 10.3390/cells12111483

Figure Lengend Snippet: GFAP-Cre recombinase viral approach to target CD44 in adult dentate gyrus astrocytes. ( a ) Scheme of bilateral intrahippocampal administration of AAV vectors (CTRL: AAV5. gfaABC1D ::GFP; GFP localizes in the cytoplasm of astrocytes, and AsKO: AAV5. gfaABC1D ::GFP-Cre; GFP-Cre localizes in cell nuclei of astrocytes) into the brains of CD44 fl/fl mice. ( b ) CD44 level in protein extracts from DG of CTRL and AsKO animals (Western blot). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control protein. ( c ) Quantitative analysis of Western blot results ( n = 6 independent animals, Student’s t -test, *** p < 0.001, data presented as mean ± SEM) ( d ) CD44-immunoreactivity in astrocytic s100β profiles from the ML of the DG in CTRL and AsKO mice (see panel f for representative immunostaining) (Student’s t -test, *** p < 0.001, data presented as mean ± SEM). Representative immunofluorescence micrographs of GFP (green), astrocyte marker s100β (white), and CD44 (red) expression in the hippocampus of CTRL and AsKO mice for matching triple stain images shown at ( e ) a lower magnification or ( f ) the close-ups reflecting green squares on panel ( e ). Arrows point to astrocytes (s100β staining) that were or were not transfected (green arrow for GFP-expressing and white arrow for non-expressing, respectively). ML—molecular layer, GL—granular layer. Scale bar: ( e ) 200 μm and ( f ) 20 μm.

Article Snippet: To produce animals with the astrocytic knockout of CD44 (AsKO), one group was stereotaxically injected with 500 nL of AAV5 vector with Cre recombinase–AAV5. gfaABC1D ::GFP-Cre (construct: Bryan Roth, AAV Serotype 5, UNC Vector Core, Chapel Hill, NC, USA, AAV vector with ITR2 where the human gfaABC1D promoter [ ] drives the expression of eGFP attached to the N-terminus of Cre recombinase), whereas the control (CTRL) group was injected with 500 nL of AAV5. gfaABC1D ::GFP vector (Construct: Bryan Roth, Addgene plasmid #50473; AAV Serotype 5, UNC Vector Core, AAV vector with ITR2 where the human gfaABC1D promoter drives the expression of eGFP).

Techniques: Western Blot, Control, Immunostaining, Immunofluorescence, Marker, Expressing, Staining, Transfection

Journal: Cells

Article Title: Astrocytic CD44 Deficiency Reduces the Severity of Kainate-Induced Epilepsy

doi: 10.3390/cells12111483

Figure Lengend Snippet: CD44 deletion in astrocytes reduces reactive astrogliosis following kainate-induced epilepsy. Representative images of astrocytes (GFAP, white), CD44 expression (red), GFP (green), and nuclei (Hoechst, purple) in the hippocampal ML of the DG of CTRL and CD44 AsKO mice before ( a ) and after ( b ) 4 weeks post-KA administration. Scale bar: 20 μm. Quantitative analysis of GFAP-immunoreactive area (µm 2 ) in the ML of the DG in CTRL and CD44 AsKO mice 4 weeks after administration of ( c ) SA (Kolmogorov-Smirnov test, p = 0.41, data presented as median with IQR) or ( e ) KA ( n = 6 independent animals in each group, Student’s t -test with Welch’s correction, ** p = 0.004, data presented as mean ± SEM). Quantitative analysis of CD44-immunoreactivity in astrocytic GFAP profiles in the ML of the DG in CTRL and CD44 AsKO mice 4 weeks after administration of ( d ) SA (Student’s t -test with Welch’s correction, *** p < 0.001, data presented as mean ± SEM) or ( f ) KA (Student’s t -test with Welch’s correction, ** p = 0.001, data presented as mean ± SEM).

Article Snippet: To produce animals with the astrocytic knockout of CD44 (AsKO), one group was stereotaxically injected with 500 nL of AAV5 vector with Cre recombinase–AAV5. gfaABC1D ::GFP-Cre (construct: Bryan Roth, AAV Serotype 5, UNC Vector Core, Chapel Hill, NC, USA, AAV vector with ITR2 where the human gfaABC1D promoter [ ] drives the expression of eGFP attached to the N-terminus of Cre recombinase), whereas the control (CTRL) group was injected with 500 nL of AAV5. gfaABC1D ::GFP vector (Construct: Bryan Roth, Addgene plasmid #50473; AAV Serotype 5, UNC Vector Core, AAV vector with ITR2 where the human gfaABC1D promoter drives the expression of eGFP).

Techniques: Expressing