custom-designed probes  (Twist Bioscience)

Journal: Pain

Article Title: Activation of GABAergic neurons in the dorsal raphe nucleus alleviates hyperalgesia induced by ovarian hormone withdrawal

doi: 10.1097/j.pain.0000000000003362

Figure Lengend Snippet: Decreased activity of DRN GABA neurons was involved in the hyperalgesia induced by ovarian hormone withdrawal. (A) Schematic representation of the ovariectomy surgery. (B) Quantification of the mechanical pain threshold in response to Von Frey stimulation in Sham (n = 8) and OVX (n = 10) mice. 1W, 2W, 3W, 4W: 1, 2, 3, 4 weeks after surgery. ** P < 0.01 and *** P < 0.001, by 2-way ANOVA followed by Sidak multiple comparisons test. (C and D) Quantification of the withdrawal latency in response to thermal stimulation (C) and scores in response to acetone stimulation (D) in Sham (n = 11) and OVX (n = 12) mice. Ns: no significance, by unpaired t test. (E and F) Representative images (E) and bar graph (F) showing c-fos cells in the DRN of Sham (n = 6 mice) and OVX (n = 6 mice) mice. Scale bar: 100 μm. ** P < 0.01, by unpaired t test. (G and H) Representative images (G) and bar graph (H) showing c-fos and Vgat -colabeled cells (arrowheads) in the DRN of Sham (n = 6 mice) and OVX (n = 6 mice) mice. Scale bars: 100 μm and 50 μm. ** P < 0.01, unpaired t test. (I) Representative image of GCaMP6f virus injection and fiber implantation in the DRN (upper) and schematic of fiber photometry recording of Ca 2+ activity in DRN GABA neurons in response to noxious stimulation (lower). Scale bar: 500 μm. (J–L) Heatmap of individual trials (J), averaged GCaMP6f responses (K), and quantification of the area under the curve (AUC) (L) of GCaMP6f signal in response to pinprick from DRN GABA neurons in Sham (6 mice) and OVX (6 mice) mice. * P < 0.05, by unpaired t test. (M–O) Heatmap of individual trials (M), averaged GCaMP6f responses (N), and quantification of the AUC (O) of GCaMP6f signal in response to electric shock from DRN GABA neurons in Sham (6 mice) and OVX (6 mice) mice. * P < 0.05, by Mann–Whitney test. All data are presented as mean ± SEM. DRN, dorsal raphe nucleus; OVX, ovariectomized.

Article Snippet: In situ hybridization was performed according to the manufacturer's instructions from the RNAscope multiplex fluorescent manual assay kit (323100, Advanced Cell Diagnostics, ACD, CA) with the custom-designed probe for Vgat (319191, Mm-Slc32a1-C2, ACD), ERα (478201-C3, Esr1-C3 , ACD), and ERβ (316121-C4, Esr2-C3, ACD).

Techniques: Activity Assay, Virus, Injection, MANN-WHITNEY

Journal: Pain

Article Title: Activation of GABAergic neurons in the dorsal raphe nucleus alleviates hyperalgesia induced by ovarian hormone withdrawal

doi: 10.1097/j.pain.0000000000003362

Figure Lengend Snippet: Activation of DRN GABA neurons alleviated mechanical hyperalgesia induced by ovarian hormone withdrawal, whereas inhibition or ablation of DRN GABA neurons promoted hypersensitivity to pain. (A) Schematic and the representative image of DRN injection of ChR2 and eYFP viruses with the optical fiber implanted in the DRN. Scale bar: 500 μm. (B) Bar graphs showing the mechanical pain threshold of OVX mice injected with eYFP (n = 8) and ChR2 (n = 6) viruses in baseline and test phases. **** P < 0.0001, by 2-way ANOVA, followed by Sidak multiple comparisons test. (C) Schematic representation of the RTPP test paired with Von Frey filament stimulation. (D) Representative traces of eYFP and ChR2 mice during the RTPP test paired with Von Frey filament stimulation. (E) Bar graphs showing the time spent in the stimulation chamber of eYFP (n = 6) and ChR2 (n = 6) mice during the RTPP test paired with Von Frey filament stimulation. * P < 0.05 and ** P < 0.01, by 2-way ANOVA, followed by Sidak multiple comparisons test. (F) Schematic and representative image of eNpHR and eYFP viruses and optical fiber implantation in the DRN. Scale bar: 500 μm. (G) Bar graphs showing the mechanical pain threshold of Sham mice injected with eYFP (n = 5) and eNpHR (n = 6) viruses in baseline and test phases. ** P < 0.01 and **** P < 0.0001, by 2-way ANOVA, followed by Sidak multiple comparisons test. (H and I) Representative traces (H) and bar graphs (I) showing the time spent in the stimulation chamber of eYFP (n = 5) and eNpHR (n = 5) mice during RTPA test paired with Von Frey filament stimulation. *** P < 0.001, by 2-way ANOVA, followed by Sidak multiple comparisons test. (J) Schematic of the injection of Caspase3 virus into the DRN to ablate GABAergic neurons. (K) Representative images showing Vgat -positive cells in the DRN of control (Con) and Caspase3 mice. Scale bar: 100 μm. (L and M) Bar graphs showing the fluorescence area (L) and number of Vgat + cells (M) in the DRN of Con (7 slices/3 mice) and Caspase3 (7 slices/3 mice) mice. *** P < 0.001 and * P < 0.05, by unpaired t test. (N) Quantification of mechanical pain threshold of Con (n = 5) and Caspase3 (n = 6) mice. ** P < 0.01 and **** P < 0.0001, by 2-way ANOVA, followed by Sidak multiple comparisons test. All data are presented as mean ± SEM. DRN, dorsal raphe nucleus; OVX, ovariectomized; RTPP, real-time place preference.

Article Snippet: In situ hybridization was performed according to the manufacturer's instructions from the RNAscope multiplex fluorescent manual assay kit (323100, Advanced Cell Diagnostics, ACD, CA) with the custom-designed probe for Vgat (319191, Mm-Slc32a1-C2, ACD), ERα (478201-C3, Esr1-C3 , ACD), and ERβ (316121-C4, Esr2-C3, ACD).

Techniques: Activation Assay, Inhibition, Injection, Virus, Control, Fluorescence