Journal: Cell Reports

Article Title: A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

doi: 10.1016/j.celrep.2020.107968

Figure Lengend Snippet: Multiplexed Immunolabeling in Epoxy-Resin-Embedded Tissue after Etching and Antigen Retrieval (A) Multiple immunolabeling of an epoxy-resin-embedded section of the hippocampal CA1 area of an adult rat. Four rounds of triple labeling were performed using primary Abs raised in 3 different species (top row: mouse monoclonal IgG1; middle: rabbit polyclonal; bottom: Guinea pig polyclonal Abs). Reactions within one round were visualized with Alexa488-, Cy3-, and Cy5-coupled sAbs (all reactions are pseudocolored and shown in cyan). Boxed areas are shown at higher magnifications in panels (B 1 and B 2 ). (B 1 ) Immunolabeling for PV, Kv2.1, VGAT, and vGluT1 are present in different, nonoverlapping subcellular compartments. The vGluT1-positive terminals are only present around the PV+ IN soma but not the neighboring PC somata that are outlined by the Kv2.1 labeling. VGAT-positive terminals (cyan) surround the somata of the IN and the PCs. (B 2 ) Immunolabeling of PV, PSD95, CB1, and neuroligin-2 (NL-2). (C) Confocal images of a 200-nm-thick section of the mouse hilar region labeled for PSD95 (1 st and 3 rd rounds) and for VGAT (2 nd and 4 th rounds; both Guinea pig primary Abs). Circular ROIs were placed over PSD95-labeled glutamatergic synapses (yellow) and over VGAT-positive axons (cyan). The nonspecific background labeling (white ROI) was measured over the unlabeled neuropil. (D) Mean normalized integrated fluorescence values are plotted for 4 labeling rounds. Open symbols represent reactions from 3 mice, and the filled symbols are mean ± SD. The fluorescence in the inhibitory axons is 2.6% ± 2.4% in the 1 st and 4.6% ± 2.5% in the 3 rd rounds, similar to that measured over the excitatory synapses in the 2 nd (7.2% ± 1.1%) and 4 th (5.7% ± 2.9%) rounds. A total of 21–28 ROIs were analyzed in each condition. str. rad., stratum radiatum; str. pyr., stratum pyramidale.

Article Snippet: Mouse monoclonal IgG1 anti-Cav3.1 , NeuroMab , Cat#75-206; RRID: AB_2069421.

Techniques: Immunolabeling, Labeling, Fluorescence

Journal: Cell Reports

Article Title: A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

doi: 10.1016/j.celrep.2020.107968

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal IgG1 anti-Cav3.1 , NeuroMab , Cat#75-206; RRID: AB_2069421.

Techniques: Virus, Plasmid Preparation, Recombinant, Software, Microscopy

Journal: Theranostics

Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

doi: 10.7150/thno.62255

Figure Lengend Snippet: NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with Cav3.2 in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.

Article Snippet: Mice received intra-TG injection of 5'-cholesteryl-modified and 6-FAM-modified Cav3.2 siRNA (Cav3.2-siRNA, 5 μg) or a scrambled negative control siRNA (NC-siRNA) (RiboBio, Guangzhou) 2 d after the CFA injection. siRNA was mixed with polyethyleneimine (PEI, Fermentas Inc.) for 10 min before being delivered.

Techniques: Western Blot, Double Immunostaining, Two Tailed Test, Labeling, Injection, Expressing

Journal: Theranostics

Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

doi: 10.7150/thno.62255

Figure Lengend Snippet: The G βγ -dependent AMPK/PKA pathway mediates the NmbR response. A, time course of I T changes induced by 100 nM Nmb in TG neurons dialyzed with QEHA (10 μM). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. B, summary data indicate the effect of 100 nM Nmb on I T in TG neurons dialyzed with QEHA (10 μM, n = 6) or SKEE (10 μM, n = 7). ** p < 0.01 versus control, two-tailed t test. C, protein expression of G β in cells transduced with G β shRNA (G β -shRNA) or negative control shRNA (NC-shRNA). Blots depicted are representative of three independent experiments. # p < 0.05 versus NC-shRNA, two-tailed t test. D, summary data show the effect of Nmb (100 nM) on I T in TG neurons transduced with NC-shRNA ( n = 9) or G β -shRNA ( n = 8). * p < 0.05 versus NC-shRNA + control, one-way ANOVA followed by Bonferroni post hoc test. E, time course ( left panel ) and summary data ( right panel ) show the effect of Nmb (100 nM) on I T in cells pre-incubated with KT-5720 (1 μM, n = 7). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. ** p < 0.01 versus control, two-tailed t test. F, exemplary current traces and summary of results show the effect of forskolin (10 µM) on I T in TG neurons in the absence ( n = 7) or presence of KT-5720 (1 μM, n = 6). ** p < 0.01 versus control, two-tailed t test. G, representative immunoblot and summary data show that treatment of TG cells with 100 nM Nmb did not affect the protein expression level of Cav3.2. GAPDH was used as the loading control. H, pretreatment of TG cells with 0.5 µM BIM23042, but not 1 μM KT-5720, attenuates the increased expression level of phosphorylated AMPK ( p -AMPK) induced by 100 nM Nmb. GAPDH was used as the loading control. Blots depicted are representative of three independent experiments. * p < 0.05 versus control, two-tailed t test. I, summary data show the effect of 100 nM Nmb on PKA activity in TG cells pretreated with 0.5 µM BIM23042 or 10 µM compound C. All experiments were performed in triplicate with similar results. # p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. J, time course ( left panel ) and summary of results ( right panel ) indicate the effect of 100 nM Nmb on I T in TG neurons pretreated with compound C (10 µM, n = 7). Inset s show the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus control, two-tailed t test.

Article Snippet: Mice received intra-TG injection of 5'-cholesteryl-modified and 6-FAM-modified Cav3.2 siRNA (Cav3.2-siRNA, 5 μg) or a scrambled negative control siRNA (NC-siRNA) (RiboBio, Guangzhou) 2 d after the CFA injection. siRNA was mixed with polyethyleneimine (PEI, Fermentas Inc.) for 10 min before being delivered.

Techniques: Two Tailed Test, Expressing, Transduction, shRNA, Negative Control, Incubation, Western Blot, Activity Assay

Journal: Theranostics

Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

doi: 10.7150/thno.62255

Figure Lengend Snippet: Activation of NmbR stimulates recombinant Cav3.2 channels heterologously expressed in HEK293 cells. A, western blot analysis of NmbR in HEK293 cells transiently transfected with NmbR cDNA. Blots depicted are representative of three independent experiments. B, membrane localization of NmbR in transfected HEK293 cells. Alphabets a through c in the diagram indicate the differential interference contrast (DIC, a ), the EGFP fluorescent signals of NmbR ( b ), and the merged image ( c ), respectively. C, exemplary current traces show the effect of Nmb (100 nM) on Cav3.1 (α1G), Cav3.2 (α1H) and Cav3.3 (α1I) channel currents. Currents were elicited by a 100 ms depolarizing step pulse from the holding potential of -110 mV to -30 mV. D, summary data show the effect of 100 nM Nmb on Cav3.1 ( n = 10), Cav3.2 ( n = 9) and Cav3.3 ( n = 7) channel currents. ** p < 0.01 versus control, two-tailed t test. E, dose-dependent effects of Nmb on Cav3.2 channel currents. Solid line represents the sigmoidal dose-response fits. Numbers in parentheses denote n cells tested at each concentration. F, summary data show the effect of Nmb (100 nM) on Cav3.2 channel currents in cells pre-incubated with 10 µM compound C ( n = 8), in cells pretreated with 1 µM KT-5720 ( n = 9), and in cells dialyzed with 10 μM PKC 19-36 ( n = 9), respectively. ** p < 0.01 and *** p < 0.001 versus control, two-tailed t test.

Article Snippet: Mice received intra-TG injection of 5'-cholesteryl-modified and 6-FAM-modified Cav3.2 siRNA (Cav3.2-siRNA, 5 μg) or a scrambled negative control siRNA (NC-siRNA) (RiboBio, Guangzhou) 2 d after the CFA injection. siRNA was mixed with polyethyleneimine (PEI, Fermentas Inc.) for 10 min before being delivered.

Techniques: Activation Assay, Recombinant, Western Blot, Transfection, Two Tailed Test, Concentration Assay, Incubation

Journal: Theranostics

Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

doi: 10.7150/thno.62255

Figure Lengend Snippet: Involvement of peripheral NmbR in mechanical pain hypersensitivity. A, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces mechanical hypersensitivity ( n = 6 - 8 mice per group). * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. BL, baseline. B, the total contact time assessed by orofacial operant test was significantly decreased 1 h after intra-TG injection of 1 nmol Nmb, compared to vehicle group. * p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. C, pretreatment of BIM23042 (5 nmol), KT-5720 (2 nmol), Z941 (2 nmol) or TTA-P2 (1 nmol) significantly attenuates 1 nmol Nmb-induced mechanical hypersensitivity ( n = 8 - 10 mice per group). * p < 0.05 versus vehicle at 1 h, + p < 0.05 versus Z941 + vehicle, # p < 0.05 versus TTA-P2 + vehicle, one-way ANOVA followed by Bonferroni post hoc test. D, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces heat hypersensitivity. * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. E, intra-TG administration of BIM23042 (5 nmol) prevented 1 nmol Nmb-induced heat hypersensitivity (n = 6 - 8 mice per group). * p < 0.05 versus vehicle at 1 h, one-way ANOVA followed by Bonferroni post hoc test. F, rofecoxib (1 mg/kg administrated subcutaneously) reduced the mechanical allodynia produced by CFA. Behavioral analysis was performed at 2 d after CFA injection, and the effect of rofecoxib was assessed at 1 h after administration (n = 7 - 9 mice per group). ** p < 0.01 versus basline (BL) in vehicle group, ## p < 0.01 versus basline (BL) in rofecoxib group, ++ p < 0.01 versus vehicle at 1 h. G, intra-TG injection of BIM23042 (5 nmol) 2 d after CFA injection significantly suppresses the mechanical hypersensitivity in CFA-treated group ( n = 7 - 9 mice per group). * p < 0.05 and ** p < 0.01 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. H-I, intra-TG administration of Cav3.2 siRNA resulted in a significant decrease of Cav3.2 protein abundance in mouse TGs ( H ), while the expression level of Cav3.1 or Cav3.3 ( I ) remained unchanged. The blots shown are representative of three independent experiments. # p < 0.05 versus NC-siRNA, two-tailed t test. J, siRNA knock-down of Cav3.2 attenuates the BIM23042-induced alleviation of mechanical allodynia in CFA-treated mice ( n = 8 - 10 mice per group). * p < 0.01 and ** p < 0.01 versus NC-siRNA in CFA-treated mice; ++ p < 0.01 versus CFA 2 d; # p < 0.05 versus 0 h point in NC-siRNA-treated mice, two-way ANOVA followed by Bonferroni post hoc test. The black arrow indicates the intra-TG injection of 5 nmol BIM23042.

Article Snippet: Mice received intra-TG injection of 5'-cholesteryl-modified and 6-FAM-modified Cav3.2 siRNA (Cav3.2-siRNA, 5 μg) or a scrambled negative control siRNA (NC-siRNA) (RiboBio, Guangzhou) 2 d after the CFA injection. siRNA was mixed with polyethyleneimine (PEI, Fermentas Inc.) for 10 min before being delivered.

Techniques: Injection, Produced, Expressing, Two Tailed Test

Journal: Theranostics

Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

doi: 10.7150/thno.62255

Figure Lengend Snippet: Proposed mechanisms of NmbR signalling on Cav3.2 channels in TG neurons. The binding of Nmb triggers NmbR to activate G q protein and releases the G βγ subunits. The released G βγ dimer then stimulates AMPK activity, induces the activation of PKA and subsequently phosphorylates Cav3.2 channels to increase I T . Neither PLC/PKC nor direct binding of G βγ to Cav3.2 channels is required for T-type channel response mediated by NmbR.

Article Snippet: Mice received intra-TG injection of 5'-cholesteryl-modified and 6-FAM-modified Cav3.2 siRNA (Cav3.2-siRNA, 5 μg) or a scrambled negative control siRNA (NC-siRNA) (RiboBio, Guangzhou) 2 d after the CFA injection. siRNA was mixed with polyethyleneimine (PEI, Fermentas Inc.) for 10 min before being delivered.

Techniques: Binding Assay, Activity Assay, Activation Assay