Journal: The Journal of Neuroscience

Article Title: MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons

doi: 10.1523/JNEUROSCI.3260-16.2017

Figure Lengend Snippet: Time-lapse analysis reveals a delayed entry of MAP7 into new branches. A, Bright-field (top), MAP7-EGFP (middle), and merged (bottom) images of rE14 DRG neurons transfected with MAP7-EGFP are shown in a sequence with 10 min intervals. EGFP tagged proteins are present in the main axon but lag behind the terminals (asterisks). At 10 min, a new branch begins to form (arrows in phase), but does not contain MAP7-EGFP. At 20 min, MAP7-EGFP begins to enter the new branch, which continues to grow. MAP7-EGFP signal at the base of the branch is also increased. n = 4. The middle two bright-field images were contrast enhanced in ImageJ. B, Live-cell analysis of filopodium formation along transfected axons. Phase, EGFP, and merged images are shown for filopodia (arrows) formed in axonal regions expressing EGFP or MAP7-EGFP. The EGFP signal enters into the filopodia from the axonal region containing EGFP, but not MAP7-EGFP. C, D, Quantification of the number of filopodia formed every 30 min (C) and the total life time before they retract (D) (n = 27 for EGFP and 13 for MAP7-EGFP). ns, not significant from t test (C) and Mann–Whitney test (D). Scale bars, 10 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various constructs by nucleofection (Lonza) using reagent P3 and the CU-133 program and then cultured at ∼30,000 cells on 18 mm glass coverslips coated with 10 μg/ml poly- d -lysine and 10 μg/ml laminin in F12 medium (with the N3 supplement, 40 m m glucose, and 25 ng/ml NGF).

Techniques: Transfection, Sequencing, Expressing, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons

doi: 10.1523/JNEUROSCI.3260-16.2017

Figure Lengend Snippet: MAP7 knockdown decreases axon branching in cultured rat DRG neurons. A, Western analysis of COS cells expressing MAP7-FLAG(M7) and control shRNA (sh-Ctrl) or MAP7-specific (sh-M7) shRNA using antibodies against FLAG for MAP7 and tubulin for loading control. B, C, Analysis of shRNA knockdown of endogenous MAP7 in rE17 DRG neurons transfected with control (ctrl) or MAP7 (M7) shRNA. Confocal sections of the soma immunostained for MAP7 (M7; green, B) and neurofilament (NF; red, B) as well as the merged images (M; B) are shown. Cells were selected based on mCherry expressed from the shRNA vector. The ratio in arbitrary units (a.u.) of fluorescence signals between MAP7 and NF is plotted for cells expressing control (Ctrl) or MAP7 shRNA (C). n = 41 for Ctrl and 47 for MAP7. Scale bar, 5 μm. D, E, Inverted fluorescent images of rE17 DRG neurons expressing control (Ctrl, D) or MAP7 (E) shRNA after grown overnight on laminin-coated coverslips and stained for mCherry expressed from the shRNA vector. Arrows point to interstitial branches. Scale bar, 100 μm. F–H, Quantification of the number (#) of branches per cell in rE17 DRG neurons (F) (total: EGFP: 5.8 ± 0.3; Ctrl shRNA: 5.5 ± 0.3; MAP7 shRNA: 3.3 ± 0.2; interstitial: EGFP: 2.8 ± 0.2; Ctrl shRNA: 3.3 ± 0.4; MAP7 shRNA: 1.6 ± 0.1; terminal: EGFP: 1.7 ± 0.2; Ctrl shRNA: 2.2 ± 0.4; MAP7 shRNA: 1.7 ± 0.1.), length of main axons (G), and number (#) of primary axons (H) (n = 139 for EGFP, 45 for Ctrl shRNA, and 159 for MAP7 shRNA). *p < 0.05; ***p < 0.005; ns, not significant from t test for all comparisons except in C (Mann–Whitney test).

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various constructs by nucleofection (Lonza) using reagent P3 and the CU-133 program and then cultured at ∼30,000 cells on 18 mm glass coverslips coated with 10 μg/ml poly- d -lysine and 10 μg/ml laminin in F12 medium (with the N3 supplement, 40 m m glucose, and 25 ng/ml NGF).

Techniques: Cell Culture, Western Blot, Expressing, shRNA, Transfection, Plasmid Preparation, Fluorescence, Staining, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons

doi: 10.1523/JNEUROSCI.3260-16.2017

Figure Lengend Snippet: MAP7 localizes to longer and more stable branches and colocalize with acetylated microtubules. A–C, A MAP7-EGFP-transfected rE14 DRG neurons is shown for MAP7-EGFP fluorescence (A, green) and antibody staining for α-tubulin (B, red) or acetylated tubulin (C, cyan). MAP7-EGFP is only found in longer branches (arrows) but absent from branches shorter than 5 μm (arrowheads). D, E, Quantification of the percentage of branches that contain MAP7 (D) and the fluorescent ratio in arbitrary units (a.u.) between acetylated tubulin (tub) and α-tubulin (E) in different branch length groups. Branches were divided into three groups based on length, <5 μm, 5–20 μm, and >20 μm. Each group in E was further divided into a MAP7-positive (+) or MAP7-negative (−) subgroup. *p < 0.05, **p < 0.01; ns, not significant (n = 37 branches) from Mann–Whitney test (D) and t test (E). p < 0.0001 from the Fisher's test for D. Scale bar, 20 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various constructs by nucleofection (Lonza) using reagent P3 and the CU-133 program and then cultured at ∼30,000 cells on 18 mm glass coverslips coated with 10 μg/ml poly- d -lysine and 10 μg/ml laminin in F12 medium (with the N3 supplement, 40 m m glucose, and 25 ng/ml NGF).

Techniques: Transfection, Fluorescence, Staining, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons

doi: 10.1523/JNEUROSCI.3260-16.2017

Figure Lengend Snippet: Increased branch formation in cultured Map7mshi neurons and rat DRG neurons expressing the NP fragment. A, B, Inverted fluorescent images of dissociated DRG neurons isolated from mE15.5 wild-type (WT, A) or Map7mshi (mshi, B) littermates and cultured for 24 h. Axons are visualized by immunostaining for neurofilament. C–E, Quantification of branch number (#) per neurons (C), main axon length (D), and primary axon number (#) (E) for dissociated mE15.5 DRG neurons with different genotypes (n = 93 for all genotypes). Homozygous neurons (mshi) have 7.9 ± 1.0 total branches per cell, heterozygous neurons (+/m) have 5.5 ± 0.6, and wild-type (WT) neurons have 3.3 ± 0.4. Interstitial branches per cell: WT: 2.3 ± 0.3; +/m: 3.4 ± 0.4; mshi: 4.6 ± 0.5; and terminal branches per cell: WT: 1.1 ± 0.2; +/m: 2.1 ± 0.4; mshi: 3.2 ± 0.6. F–G, rE14 DRG neurons cultured and transfected with NP-EGFP and visualized by EGFP (F) or staining for α-tubulin (G). H–J, Quantification of branches produced per neurons (H), total axon length (I), and number (#) of primary axon (J). n = 52 for EGFP and 23 for NP-EGFP. *p < 0.05; **p < 0.01; ns, not significant from t test for all except J (Mann–Whitney test). Scale bars, 20 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various constructs by nucleofection (Lonza) using reagent P3 and the CU-133 program and then cultured at ∼30,000 cells on 18 mm glass coverslips coated with 10 μg/ml poly- d -lysine and 10 μg/ml laminin in F12 medium (with the N3 supplement, 40 m m glucose, and 25 ng/ml NGF).

Techniques: Cell Culture, Expressing, Isolation, Immunostaining, Transfection, Staining, Produced, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons

doi: 10.1523/JNEUROSCI.3260-16.2017

Figure Lengend Snippet: Map7mshi mice have increased collateral branches and pain sensitivity. A–C, Schematic (A) and examples of DiI-labeled single DRG axons in E15.5 spinal cord of wild-type (WT, B) and MAP7mshi (mshi, C) mice. Arrows point to the newly formed collaterals. D, Comparison of the number of axons displaying at least one collateral branch from DiI labeling of DRG neurons from wild-type (WT), heterozygous (+/m), or homozygous (mshi) animals (n = 32 for WT, 50 for +/m, and 29 for mshi). *p < 0.05, Mann–Whitney test. p < 0.0001 from the Fisher's test. E, F, Analysis of forepaw withdrawal response time to a nociceptive thermal stimulus by 6-month-old heterozygous (+/m) and homozygous mutant (mshi) male mice (E) and assessment of forelimb motor functions using grip strength testing on the same animals (F) from left or right side of the body (n = 4 animals per condition). *p < 0.05; ****p < 0.0001; ns, not significant from t test. G, H, Neurofilament labeling of peripheral nerves in E15.5 forelimbs of heterozygous (+/m, G) and homozygous mutant (mshi, H) animals. Scale bars, 50 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various constructs by nucleofection (Lonza) using reagent P3 and the CU-133 program and then cultured at ∼30,000 cells on 18 mm glass coverslips coated with 10 μg/ml poly- d -lysine and 10 μg/ml laminin in F12 medium (with the N3 supplement, 40 m m glucose, and 25 ng/ml NGF).

Techniques: Labeling, MANN-WHITNEY, Mutagenesis

Journal: The Journal of Neuroscience

Article Title: MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons

doi: 10.1523/JNEUROSCI.3260-16.2017

Figure Lengend Snippet: MAP7 expression is upregulated in DRGs at the time of collateral branch formation. A, B, Inverted fluorescent images of dissociated DRG neurons that were isolated from rE14 (A) or rE17 (B) embryos and grown overnight on laminin-coated coverslips and then immunostained for neurofilament. C, Expression differences (fold change, mE15.5 vs mE12.5) of all known MAPs present in the microarray analysis of mouse DRGs. Blue markers indicate an increase in expression at mE15.5 and red markers indicate a decrease. D–I, RNA in situ hybridization analysis of MAP7 transcripts using the N-terminal probe are shown in the transverse sections of developing mouse embryos between mE12.5 and mE15.5. Low-magnification images are shown for both DRG and spinal cord (D, E) and high-magnification images are shown for individual DRGs (F–I). Scale bars, 100 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various constructs by nucleofection (Lonza) using reagent P3 and the CU-133 program and then cultured at ∼30,000 cells on 18 mm glass coverslips coated with 10 μg/ml poly- d -lysine and 10 μg/ml laminin in F12 medium (with the N3 supplement, 40 m m glucose, and 25 ng/ml NGF).

Techniques: Expressing, Isolation, Microarray, RNA In Situ Hybridization

Journal: The Journal of Neuroscience

Article Title: MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons

doi: 10.1523/JNEUROSCI.3260-16.2017

Figure Lengend Snippet: Overexpression of MAP7 increases branch formation in cultured rat DRG neurons. A, B, Inverted mCherry fluorescent images of dissociated rE14 DRG neurons expressing EGFP (A) or MAP7-EGFP (B). Neurons were cotransfected with pCAGGS-mCherry for visualization and cultured on laminin substrates for 24 h before staining. Arrows point to interstitial branches. C–E, Quantification of the number (#) of branches (C) as measured by counting the total number of tips per neuron in rE14 DRG neurons shown in A and B (EGFP: 0.55 ± 0.13; MAP7: 1.7 ± 0.4, p < 0.0001), length of main axons (D), and number (#) of primary axons (E). Branches were further divided into two groups: terminal branches arising from the distal 10% part of the axon (EGFP: 0.32 ± 0.11; MAP7: 0.36 ± 0.15, p > 0.05) and interstitial branches from the remaining 90% of the axon (EGFP: 0.23 ± 0.09; MAP7: 1.4 ± 0.3) (C). (n = 52 for EGFP and 32 for MAP7-EGFP). ***p < 0.001; ****p < 0.0001; ns: not significant from t test. Scale bar, 100 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various constructs by nucleofection (Lonza) using reagent P3 and the CU-133 program and then cultured at ∼30,000 cells on 18 mm glass coverslips coated with 10 μg/ml poly- d -lysine and 10 μg/ml laminin in F12 medium (with the N3 supplement, 40 m m glucose, and 25 ng/ml NGF).

Techniques: Over Expression, Cell Culture, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons

doi: 10.1523/JNEUROSCI.3260-16.2017

Figure Lengend Snippet: MAP7 is concentrated at branching sites. A–I, Dissociated rE14 DRG neurons expressing EGFP (A–C) or MAP7-EGFP (D–I) are shown by EGFP (green), antibody staining for α-tubulin (red), or merged images. MAP7-EGFP can be seen entering into a new branch (arrow). Higher magnification image (G–I) of the branch site (yellow box in F) shows the concentration of MAP7 at the branch site. J–L, rE17 DRG neurons were immunostained for endogenous MAP7 (J, cyan) and α-tubulin (K, red). The merged image is shown in L. MAP7 signal extends from the main axon into the branch (arrow). Scale bars, 20 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various constructs by nucleofection (Lonza) using reagent P3 and the CU-133 program and then cultured at ∼30,000 cells on 18 mm glass coverslips coated with 10 μg/ml poly- d -lysine and 10 μg/ml laminin in F12 medium (with the N3 supplement, 40 m m glucose, and 25 ng/ml NGF).

Techniques: Expressing, Staining, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Mechanisms Underlying Sensory Nerve-Predominant Damage by Methylmercury in the Peripheral Nervous System

doi: 10.3390/ijms252111672

Figure Lengend Snippet: Annexin V-fluorescein isothiocyanate (FITC)/PI flow cytometry analysis of DRG neurons after treatment with methylmercury for 24 h. ( A ) Fluorescence-activated cell sorting (FACS) using flow cytometry of cultured rat DRG neurons pretreated with or without necrostatin-1 (1 µM) for 1 h and then treated with methylmercury (0.5 µM) for 24 h. ( B ) Quantitative analysis of the flow cytometry data. Values are means ± standard error (S.E.) of three technical replicates. ** Significantly different from the control, p < 0.01; ## significantly different from the treatment with methylmercury alone, p < 0.01. The data in A are representative of several independent experiments.

Article Snippet: Briefly, primary DRG neurons were resected and isolated from 4-week-old male Wister rats (Sankyo Labo Service Corporation Inc., Tokyo, Japan) using a previous method [ ].

Techniques: Flow Cytometry, Fluorescence, FACS, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Mechanisms Underlying Sensory Nerve-Predominant Damage by Methylmercury in the Peripheral Nervous System

doi: 10.3390/ijms252111672

Figure Lengend Snippet: Apoptosis induced by methylmercury. ( A ) Representative images of the TUNEL assay ( left panels, 100× magnification) and the quantitative analysis ( right panels) of cultured DRG neurons ( upper panels), AHCs ( middle panels), and Schwann cells ( lower panels) after treatment with methylmercury (0.5, 1, and 3 µM) for 24 h. Values are means ± S.E. of three technical replicates. Significantly different from the corresponding control, * p < 0.05. ( B ) Activation of caspase 8 and caspase 3 by methylmercury in DRG neurons. The cells were treated with methylmercury (0.5, 1, and 3 µM) for 24 h, and the expression of caspase 8, cleaved caspase 8, caspase 3, and cleaved caspase 3 was determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The representative Western blot of at least two experiments is shown.

Article Snippet: Briefly, primary DRG neurons were resected and isolated from 4-week-old male Wister rats (Sankyo Labo Service Corporation Inc., Tokyo, Japan) using a previous method [ ].

Techniques: TUNEL Assay, Cell Culture, Control, Activation Assay, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Mechanisms Underlying Sensory Nerve-Predominant Damage by Methylmercury in the Peripheral Nervous System

doi: 10.3390/ijms252111672

Figure Lengend Snippet: Expression of signaling molecules in the downstream pathway of TNF-α-activated TNFR1 involved in the necroptosis of DRG neurons. ( A ) Expression of TNF-α, TNFR1, phosphorylated RIP3, RIP3, phosphorylated MLKL, and MLKL. The molecules were detected by Western blot analysis. GAPDH was used as an internal control. The data shown are representative of at least two experiments. ( B ) Quantitative analysis of the Western blot data.

Article Snippet: Briefly, primary DRG neurons were resected and isolated from 4-week-old male Wister rats (Sankyo Labo Service Corporation Inc., Tokyo, Japan) using a previous method [ ].

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Mechanisms Underlying Sensory Nerve-Predominant Damage by Methylmercury in the Peripheral Nervous System

doi: 10.3390/ijms252111672

Figure Lengend Snippet: A hypothesis regarding the pathogenesis of sensory nerve-predominant damage in the peripheral nervous system after exposure to methylmercury. (1) As DRG neurons are susceptible to methylmercury due to the higher accumulation of methylmercury in the peripheral nerve system , early exposure to methylmercury causes sensory nerve cell death in the sensory fibers. (2) The cell death triggers the infiltration of macrophages into the sensory fibers . The macrophages are stimulated by methylmercury and secrete TNF-α through the activation of the NF-κB pathway. (3) TNF-α induces apoptosis and necroptosis in sensory neurons by activating the caspase 8–caspase 3 pathway and the TNFR1-RIP1-RIP3-MLKL pathway, respectively. Although the possibility that that activation of the TNFR1-RIP1-RIP3-MLKL pathway was mediated by factors other than TNF-α such as TLR4 and Fas cannot be excluded, our data suggested that this pathway could be activated by TNF-α from macrophages infiltrating sensory fibers. Additionally, methylmercury causes necrosis in sensory neurons. The cell death of sensory neurons triggers macrophage infiltration in sensory fibers. These processes are cascaded and amplified, and methylmercury predominantly damages the sensory neurons.

Article Snippet: Briefly, primary DRG neurons were resected and isolated from 4-week-old male Wister rats (Sankyo Labo Service Corporation Inc., Tokyo, Japan) using a previous method [ ].

Techniques: Activation Assay, Amplification

Journal: Toxins

Article Title: The Venom of the Spider Selenocosmia Jiafu Contains Various Neurotoxins Acting on Voltage-Gated Ion Channels in Rat Dorsal Root Ganglion Neurons

doi: 10.3390/toxins6030988

Figure Lengend Snippet: Effects of S . jiafu venom on whole-cell Na v channel currents in rat dorsal root ganglion (DRG) neurons. ( A ) Tetrodotoxin-sensitive (TTX-S) Na + currents were induced by a 50-ms depolarized potential of −10 mV from a holding potential of −80 mV in large DRG neurons. TTX-S current amplitude was blocked to 64.0% ± 6.2% ( n = 5) by 100 μg/mLvenom. ( B ) Current-voltage (I-V) curves of TTX-S. For current-voltage curves, the currents were elicited by a series of 50-ms depolarizations from a holding potential of −80 mV, with a test potential ranging from −80 mV to +90 mV at increments of +10 mV. ( C ) Na + currents were evoked by a 50-ms depolarized potential of −10 mV from a holding potential of −80 mV in medium size DRG neurons. At 200 nM tetrodotoxin (TTX), the remnant tetrodotoxin-resistant (TTX-R) currents were inhibited by 46.5% ± 3.8% ( n = 5) in the presence of 100 μg/mL venom. ( D ) Current-voltage (I-V) curves of TTX-R. For current-voltage curves, the currents were elicited by a series of 50-ms depolarizations from a holding potential of −80 mV, with a test potential ranging from −80 mV to +50 mV at increments of +10 mV. The data were expressed as mean ± SE ( n = 5).

Article Snippet: Whole-cell voltage-clamp recordings of voltage-gated ion currents were made in rat DRG neurons which were acutely dissociated from 30-day old Sprague-Dawley rats and maintained in short-term primary culture according to the method described by Xiao and Liang [ ].

Techniques:

Journal: Toxins

Article Title: The Venom of the Spider Selenocosmia Jiafu Contains Various Neurotoxins Acting on Voltage-Gated Ion Channels in Rat Dorsal Root Ganglion Neurons

doi: 10.3390/toxins6030988

Figure Lengend Snippet: Effects of S . jiafu venom on whole-cell K v channel currents in rat DRG neurons. ( A ) K v currents were evoked by a 500-ms depolarized potential of 10 mV from a holding potential of −80 mV. Application of 100 μg/mL of venom led to a reduction of K + currents of 44.2% ± 5.3% ( n = 5); ( B ) Normalized current-voltage relation of K + currents before (filled circles) and after (open circles) venom treatment. Currents were elicited by a series of 500-ms depolarizations from a holding potential of −80 mV to +60 mV in 10-mV steps. The data were expressed as mean ± S.E ( n = 5).

Article Snippet: Whole-cell voltage-clamp recordings of voltage-gated ion currents were made in rat DRG neurons which were acutely dissociated from 30-day old Sprague-Dawley rats and maintained in short-term primary culture according to the method described by Xiao and Liang [ ].

Techniques:

Journal: Toxins

Article Title: The Venom of the Spider Selenocosmia Jiafu Contains Various Neurotoxins Acting on Voltage-Gated Ion Channels in Rat Dorsal Root Ganglion Neurons

doi: 10.3390/toxins6030988

Figure Lengend Snippet: Effects of S . jiafu venom on whole-cell Ca v channel currents in rat DRG neurons. ( A ) The application of 100 μg/mL of venom inhibited 65.4% ± 9.5% ( n = 5) of total Ca v currents evoked by a 150-ms depolarization to 10 mV from a holding potential of −90 mV. ( B ) Normalized current-voltage relationships of Ca v currents in the presence and absence of 100 μg/mL of venom. Currents were elicited by a series of 150-ms depolarizations from −80 mV to +90 mV in 5-mV steps with a holding potential of −90 mV. The data were expressed as mean ± SE ( n = 5). ( C ) The application of 100 μg/mL of venom inhibited 77.7% ± 9.9% ( n = 5) of LVA-Ca v currents evoked by a 150-ms depolarization to −20 mV from a holding potential of −90 mV. ( D ) The application of 50 μg/mL of venom inhibited 74.9% ± 2.8% ( n = 5) of HVA-Ca v currents elicited by a 150-ms depolarization to +10 mV from a holding potential of −40 mV.

Article Snippet: Whole-cell voltage-clamp recordings of voltage-gated ion currents were made in rat DRG neurons which were acutely dissociated from 30-day old Sprague-Dawley rats and maintained in short-term primary culture according to the method described by Xiao and Liang [ ].

Techniques: