Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: NGF-induced PC12 cell differentiation toward sympathetic neurons and expressions of GAP-43 and TH. (A) Immunofluorescent stains of GAP-43 (red, upper row) and TH (red, lower row). Nuclei were stained blue by DAPI. Note that NGF induced significant neurite outgrowth. Scale bar, 10 μm. (B,C) Real-time PCR and western blotting results of GAP-43 and TH expressions in PC12 cells treated or untreated with NGF. ∗∗∗ p < 0.001 vs. respective control. ### p < 0.001 vs. respective control; n = 3 for each value.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Cell Differentiation, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: The suppressing effects of ivabradine (IVA) on the mRNA and protein expressions of HCN channel isoforms and GAP-43 and the neurite outgrowth. (A–D) Real-time PCR and western blotting results of the mRNA and protein expression levels respectively for HCN1-4 isoforms in NGF-treated PC12 cells relative to GADPH level. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. NGF treatment alone in the mRNA level. # p < 0.05, ## p < 0.01 vs. NGF treatment alone in the protein level. (E) Representative western blotting bands ( upper ) and the mRNA and protein semiquantitative values ( lower ) of GAP-43 in NGF-treated PC12 cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. NGF treatment alone in the mRNA level. # p < 0.05, ### p < 0.001 vs. NGF treatment alone in the protein level. (F) Immunofluorescent stains of GAP-43 protein (red), displaying that NGF induced, while ivabradine inhibited, the neurite outgrowth in differentiating PC12 cells. DAPI was used for cell nuclei staining (blue). Scale bar, 10 μm. (G,H) Quantitative morphological parameters of neurite outgrowth including the total neurite length and the maximal neurite length (longest process per cell). Results are calculated from three independent experiments and presented as mean ± SEM, 96 < n < 213, ∗ p < 0.05, ∗∗ p < 0.01 vs. NGF treatment alone.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: Knocking-down HCN2 and HCN4 expression with siRNAs inhibited GAP-43 expression and neurite outgrowth in PC12 cells. NGF-treated PC12 cells were transiently transfected with the negative control siRNA (NC-siRNA) or with the siRNA respectively for HCN1, HCN2, HCN3 or HCN4 using Lipofectamine ® RNAiMAX Reagent for 48 h before cells were harvested for detection. (A–D) The mRNA and protein expressions of HCN1-4 isoforms assayed by qPCR and western blotting in comparison to GADPH in NGF-treated PC12 cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. negative control siRNA (N.C.) in the mRNA level. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. N.C. in the protein level. (E) Representative western blotting results ( upper ) and the densitometric analysis of GAP-43 mRNA and protein expressions ( lower ) ( n = 3–6 independent experiments). ∗ p < 0.05, ∗∗∗ p < 0.001 vs. N.C. in the mRNA level. ### p < 0.001 vs. N.C. in the protein level. (F) Immunofluorescent stains of GAP-43 in PC12 cells transiently transfected with siRNA targeting HCN2 or HCN4. Scale bar, 10 μm. (G,H) Quantification of neurite morphological parameters (total length of neurite outgrowth and longest process per cell). Results are calculated from three independent experiments and presented as mean ± SEM, 50 < n < 84, ∗ p < 0.05, ∗∗ p < 0.01 vs. N.C.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Expressing, Transfection, Negative Control, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: Overexpression of HCN2 and HCN4 enhanced GAP-43 expression and neurite outgrowth in PC12 cells. Results were obtained 48 h after cell transfection with either empty plasmid pcDNA3.0/vector or pcDNA3.0/HCN2 or pcDNA3.0/HCN4. (A,B) Representative western blots ( upper ) and semiquantitative values ( lower ) of the mRNA and protein levels of HCN2 and HCN4 isoforms. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. control (pcDNA3.0/vector) in the mRNA level. # p < 0.05 vs. control (pcDNA3.0/vector) in the protein level, n = 4−6 independent experiments. (C) qPCR and western blotting results showing the effects of HCN2 or HCN4 overexpression on the mRNA and protein expressions of GAP-43. ∗∗∗ p < 0.001 vs. empty plasmid in the mRNA level. ### p < 0.001 vs. empty plasmid in the protein level, n = 4−6 independent experiments. (D) Immunofluorescent stains of GAP-43 (red) of PC12 cells transiently transfected respectively with pcDNA3.0/vector, pcDNA3.0/HCN2 or pcDNA3.0/HCN4 plasmids. Scale bar, 10 μm. (E,F) Quantification of neurite morphological parameters including total neurite outgrowth and longest process per cell. Results are calculated from three independent experiments and presented as mean ± SEM, 50 < n < 90, ∗ p < 0.05 vs. empty vector.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: Scientific Reports

Article Title: The BEACH Protein LRBA Promotes the Localization of the Heterotrimeric G-protein G olf to Olfactory Cilia

doi: 10.1038/s41598-017-08543-4

Figure Lengend Snippet: Histological and subcellular distribution of LRBA in olfactory and vomeronasal tissues. ( A ) LacZ enzyme histochemistry of Lrba/gt gene-trap mice (age, 3 weeks) demonstrates expression of the LRBA gene throughout the OE (top). Larger magnification (middle) shows that the staining is predominantly localized to the OSNs. Respiratory epithelium (RE, bottom) is also stained, though weaker than OE. ( B ) β-galactosidase staining was also seen in VNO neuron somata and the microvillar layer of Lrba/gt mice (top, middle), and confirmed by LRBA-IF of adult WT tissue (bottom; SE, sensory epithelium; L, lumen). Scale bars in A , B : overviews 100 µm, close-ups 25 µm. ( C ) LacZ staining of the olfactory bulb required longer incubation (16 h) than of the OE and VNO (2 h). Scale bar in C : 100 µm. ( D – K ) Triple immunostaining for LRBA ( D , H ), OMP ( E , I ), GAP43 ( F , J ) and merged images ( G , K ) in different regions of the adult mouse OE of low ( D – G ) or high ( H–K ) thickness. LRBA-immunoreactivity (ir) is found in the apical somatic cytoplasm and in dendrites as well as dendritic knobs of virtually all OMP-ir mature OSNs. Large and small arrows point to select LRBA-/OMP-ir cell bodies/dendrites and dendritic knobs, respectively, but LRBA is lacking in the OMP-ir ciliary layer (small arrowheads in D – G ). GAP43-ir immature neurons contain no (large arrowheads in D – G ) or only small amounts (double arrows in D – K ) of LRBA-immunoreactivity in the apical cytoplasm of their cell bodies and in dendritic knobs. Scale bar in K for D – K : 10 µm. OE strata are designated in panels G and K: cl, cilial layer; scl, sustentacular cell layer; osnl, OSN layer.

Article Snippet: Antibodies used were rabbit anti-mLRBA-B (1:100), goat anti-OMP (1:300; Wako) as a marker for mature OSNs , and mouse anti-GAP43 (1:300; MerckMillipore) as a marker for immature OSNs , .

Techniques: Expressing, Staining, Incubation, Triple Immunostaining

Journal: Nature Communications

Article Title: Immature olfactory sensory neurons provide behaviourally relevant sensory input to the olfactory bulb

doi: 10.1038/s41467-022-33967-6

Figure Lengend Snippet: a Schematic of breeding strategy to generate mice expressing GCaMP6s in either immature or mature OSNs under control of the Gγ8 or OMP promoter, respectively, using the tetracycline transactivator system. b MIP of confocal z-stack of coronal OE section from a Gγ8-GCaMP6s mouse showing lack of colocalization with OMP-stained OSNs. c Co-expression of OMP (OMP-ir) in Gγ8-GCaMP6s-expressing OSNs in the septal olfactory epithelium (OE) along the anterior-posterior (A-P) axis. Only 3.0 ± 1.2% of Gγ8-GCaMP6s-expressing neurons co-express OMP (mean ± s.d., n = 4118 Gγ8-GCaMP6s-expressing OSNs from 3 mice). Lines: median, symbols: values for individual mice. Inset shows location of analyzed OE sections. d In vivo 2-photon images of single optical sections showing baseline fluorescence of glomeruli in OMP-tdT-Gγ8-GCaMP6s mice. e Single time point from time series showing peak odorant response for the same optical section as in d. Odorant responses are for stimulation with 2-hexanone, isoamyl acetate, ethyl butyrate and hexanal. d , e Glomeruli from four different mice are shown. Source data are provided as a Source Data file.

Article Snippet: For histological analysis in 8-week-old MMZ- and saline-injected mice, OE sections (3 per antibody per mouse at 25%, 50% and 75% along the anterior-posterior axis) were stained for GAP43 as a marker of immature OSNs (anti-GAP43 primary antibody, 1:1000 for 48 h at 4 °C, NB300-143, Novus Biologicals; donkey anti-rabbit-Alexa Fluor 546 secondary antibody, 1:500 for 1 h at room temperature), or OMP (see above) as a marker of mature OSNs.

Techniques: Expressing, Control, Staining, In Vivo, Fluorescence

Journal: Nature Communications

Article Title: Immature olfactory sensory neurons provide behaviourally relevant sensory input to the olfactory bulb

doi: 10.1038/s41467-022-33967-6

Figure Lengend Snippet: a MIPs of 2-photon z-stacks of coronal OE sections stained for GAP43 or OMP. b OE width is significantly reduced 3 days post-MMZ compared to saline-injected mice (One-way ANOVA on Ranks. P < 0.001, Kruskal–Wallis statistic = 12.4. Dunn’s multiple comparisons tests. 3-day, P = 0.004, Z = 3.29; 5d, P = 0.071, Z = 2.37; 6d, P = 0.94, Z = 1.19; 7d, P = 0.68, Z = 1.37; vs. saline. n = 3 mice per group). c OE width increases linearly between 3 and 7 days post-MMZ (Linear regression. P < 0.001, R 2 = 0.80, n = 3 mice per group). d GAP43+ OSN linear density is significantly reduced at 3 and 5 days post-MMZ compared to saline-injected mice (One-way ANOVA on Ranks: P < 0.001, Kruskal–Wallis statistic = 13.4. Dunn’s multiple comparisons tests. 3d MMZ, P = 0.005, Z = 3.24; 5d MMZ, P = 0.048, Z = 2.51; 6d MMZ, P = 0.40, Z = 1.65; 7d MMZ, P = 1.00, Z = 0.82; vs. saline-injected mice. n = 3 mice per group). e GAP43+ OSN linear density increases linearly between 3 and 7 days post-MMZ (Linear regression. P < 0.001, R 2 = 0.88, n = 3 mice per group). f , g OMP+ OSNs are absent from the septal OE at 3–6 days post-MMZ and very sparse at 7 days post-MMZ. Note different y-axis scales in f and g . b – g Bars: mean per group, symbols: individual mice. h Widefield fluorescence images of lateral turbinates from OMP-stained coronal OE sections from mice that received either saline, or MMZ 3–7 d previously. n = 3 mice per group. i – j Confocal images from two mice injected with biotin-conjugated dextran at 5 days post-MMZ and perfused 24 h later. i MIP of coronal OE section stained with AF555-streptavidin (Dextran, cyan). j Single optical sections from different mice showing colocalization of tracer-labeled OSNs (Dextran, cyan) with GAP43 (yellow). Red asterisks: co-labeled cells in GAP43 images. All relevant statistical tests were two-tailed. Source data are provided as a Source Data file.

Article Snippet: For histological analysis in 8-week-old MMZ- and saline-injected mice, OE sections (3 per antibody per mouse at 25%, 50% and 75% along the anterior-posterior axis) were stained for GAP43 as a marker of immature OSNs (anti-GAP43 primary antibody, 1:1000 for 48 h at 4 °C, NB300-143, Novus Biologicals; donkey anti-rabbit-Alexa Fluor 546 secondary antibody, 1:500 for 1 h at room temperature), or OMP (see above) as a marker of mature OSNs.

Techniques: Staining, Saline, Injection, Fluorescence, Labeling, Two Tailed Test

Journal: Nature Communications

Article Title: Immature olfactory sensory neurons provide behaviourally relevant sensory input to the olfactory bulb

doi: 10.1038/s41467-022-33967-6

Figure Lengend Snippet: a Example single z-plane 2-photon images during single trial showing baseline fluorescence and odorant-evoked responses in OMP-GCaMP6s and Gγ8-GCaMP6s mice. Red arrows: ethyl butyrate-responsive glomeruli. b No effect of OSN maturity or reporter protein(s) on the density of detected glomeruli (Two-way ANOVA. Effect of OSN maturity: P = 0.99, F 1,24 = 2.93 × 10 −5 . Effect of reporter protein(s): P = 0.55, F 1,24 = 0.37. Interaction: P = 0.57, F 1,24 = 0.33. n = 10 OMP-sypGFP-tdT, 6 OMP-GCaMP6s, 7 Gγ8-sypGFP-tdT and 5 Gγ8-GCaMP6s mice). Lines: mean, symbols: individual mice. c A similar percentage of glomeruli respond to each of the 7 odorants in OMP-GCaMP6s vs. Gγ8-GCaMP6s mice (Wilcoxon signed rank test. P = 0.47, W = −10, n = 6 mice per group). Colored bars: median, symbols: individual mice. Gray shaded areas are to aid visualization only. d No significant difference in response amplitude between tested odorants in Gγ8-GCaMP6s mice (Nested one-way ANOVA. P = 0.080, F 6,30 = 2.12, n = 6 mice). e No significant difference in response amplitude between tested odorants in OMP-GCaMP6s mice (Nested one-way ANOVA. P = 0.073, F 6,31 = 2.17. n = 6 mice). d , e Lines: mean per mouse, symbols: individual glomeruli. Not all mice had glomeruli that responded to all odorants. f Example single z-plane 2-photon images showing immature OSNs in the OE responding to ethyl butyrate stimulation ex vivo. Top: imaged field of view showing medial OE surface of a Gγ8-GCaMP6s mouse in the hemi-head preparation. Lower: baseline and odorant-evoked fluorescence in boxed region in top image. Red arrows: ethyl butyrate-responsive immature OSNs. g Similar percentage of immature OSNs responded to each odorant (Friedman test. P = 0.15, Friedman statistic = 5.44, n = 3 mice). Lines: mean, symbols: individual mice. h Immature OSN response amplitudes were similar across odorants (Nested one-way ANOVA. P = 0.53, F 3,91 = 0.75, n = 6–13 responding OSNs per mouse for each odorant). Lines: mean per mouse, symbols: individual OSNs. All relevant statistical tests were two-tailed. Source data are provided as a Source Data file.

Article Snippet: For histological analysis in 8-week-old MMZ- and saline-injected mice, OE sections (3 per antibody per mouse at 25%, 50% and 75% along the anterior-posterior axis) were stained for GAP43 as a marker of immature OSNs (anti-GAP43 primary antibody, 1:1000 for 48 h at 4 °C, NB300-143, Novus Biologicals; donkey anti-rabbit-Alexa Fluor 546 secondary antibody, 1:500 for 1 h at room temperature), or OMP (see above) as a marker of mature OSNs.

Techniques: Fluorescence, Ex Vivo, Two Tailed Test

Journal: Nature Communications

Article Title: Immature olfactory sensory neurons provide behaviourally relevant sensory input to the olfactory bulb

doi: 10.1038/s41467-022-33967-6

Figure Lengend Snippet: a Schematic of breeding strategy to generate mice expressing ChIEF-Citrine in either immature or mature OSNs under control of the Gγ8 or OMP promoter, respectively, using the tetracycline transactivator system. b Schematic of relevant OB glomerular circuitry. Whole cell voltage-clamp recordings were made from STCs in either Gγ8-ChIEF-Citrine or OMP-ChIEF-Citrine mice. c Widefield fluorescence images showing ChIEF-Citrine-expressing axons in an OMP-ChIEF-Citrine and a Gγ8-ChIEF-Citrine mouse. d Integrated density of Citrine fluorescence per μm 2 of the glomerular layer in OMP-ChIEF-Citrine and Gγ8-ChIEF-Citrine mice ( n = 3 per group). Symbols: values for individual mice. e Widefield fluorescence image of an STC filled with AF594, showing an apical dendrite, a lateral dendrite, and a cut axon. Apical dendrites were visualized in 13 STCs from OMP-ChIEF-Citrine mice and 6 STCs from Gγ8-ChIEF-Citrine mice. f Example of an STC spike train evoked by step current injection, showing an irregular spiking pattern without a depolarizing envelope or rhythmic bursting. g , h Recordings from STCs made in normal ACSF containing 20 μM APV (control) and in ACSF containing 10 μM NBQX in addition to APV, demonstrating the presence of monosynaptic input from OSN axons. All traces are averages of 10 trials. g Example responses from two STCs elicited by 1 ms 100% intensity light pulse photoactivation of immature Gγ8-ChIEF-Citrine-expressing OSN axons. h Example responses from two STCs elicited by 1 ms 100 % intensity light pulse photoactivation of mature OMP-ChIEF-Citrine-expressing OSN axons. Source data are provided as a Source Data file.

Article Snippet: For histological analysis in 8-week-old MMZ- and saline-injected mice, OE sections (3 per antibody per mouse at 25%, 50% and 75% along the anterior-posterior axis) were stained for GAP43 as a marker of immature OSNs (anti-GAP43 primary antibody, 1:1000 for 48 h at 4 °C, NB300-143, Novus Biologicals; donkey anti-rabbit-Alexa Fluor 546 secondary antibody, 1:500 for 1 h at room temperature), or OMP (see above) as a marker of mature OSNs.

Techniques: Expressing, Control, Fluorescence, Injection

Journal: Materials today. Bio

Article Title: Ropivacaine microsphere-loaded electroconductive nerve dressings for long-acting analgesia and functional recovery following diabetic peripheral nerve injury.

doi: 10.1016/j.mtbio.2023.100712

Figure Lengend Snippet: Fig. 3. Axonal outgrowth on the ECH-MS/ROP composite. (A) Live/dead assay of the PC-12 cells cultured on each sample surface for 1 day. Live cells were stained in green and dead cells in red. Scale bar: 200 μm. (B) Quantification of live/dead assay (n ¼ 3). (C) The results of the CCK-8 assay displayed that cell viability exceeded 80% for each sample after seeding for 1, 3 and 7 days (n ¼ 5). (D) The cytoskeleton images showed the attachment of PC-12 cells cultured in each group for 3 days. Scale bar: 50 μm. (E) Quantification of cell spread area (n ¼ 10). (F) A heat map showing the RT-qPCR results on the axon-related gene expressions of PC-12 cells (n ¼ 3). (G) Column graph exhibiting the RT-qPCR results on the axon-related gene expressions in PC-12 cells (n ¼ 3). (H) IF images for the NF (green) and cell nuclei (blue) to observe the axonal extension of the PC-12 cells. Scale bar: 50 μm. (I) Quantification of the axonal lengths of the PC-12 cells on each sample (n ¼ 10). (J) WB analysis detected the protein expressions of NF and GAP43 in PC-12 cells cultured on each sample for 3 days. (K) Protein band intensity was quantified (n ¼ 3). (L) The axonal outgrowth of DRG cultured on each sample for 3 days and 7 days. Scale bar: 200 μm. (M) Analysis of the axon length and area of the DRG for each group (n ¼ 3). Statistical differences were determined by using One-way ANOVA with Bonferroni's multiple comparison tests (*P < 0.05, **P < 0.01, and ***P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Antibodies Species Type Dilution (IF/WB) Source Anti-GAPDH Rabbit Monoclonal lgG 1:1000 CST, America Anti-Neurofilament (NF) Rabbit Polyclonal lgG 1:200/1:1000 ProteinTech, China Anti-Growth-associated Rabbit Polyclonal lgG 1:2000 ProteinTech, China protein-43 (GAP43) Anti-Myelin basic protein Rabbit Polyclonal lgG 1:2000 ProteinTech, China (MBP) Anti-S100 beta (S100) Rabbit Polyclonal lgG 1:1000 ProteinTech, China Anti-MEK1/2 Mouse Monoclonal lgG 1:1000 CST, America Anti-Phospho-MEK1/2 Rabbit Monoclonal lgG 1:1000 CST, America (Ser217/221) Anti-p44/42 MAPK Rabbit Monoclonal lgG 1:1000 CST, America (Erk1/2) Anti-Phospho-MAPK Rabbit Monoclonal lgG 1:1000 CST, America (Erk1/2) (Thr202/ Ty204) PNI group only experienced nerve impairment without any treatment.

Techniques: Live Dead Assay, Cell Culture, Staining, CCK-8 Assay, Quantitative RT-PCR, Comparison