optogenetic inhibition in vivo paav ef1a dio enphr eyfp viruses  (Addgene inc)

Journal: The Journal of Physiology

Article Title: Cathodal weak direct current decreases epileptic excitability with reduced neuronal activity and enhanced delta oscillations

doi: 10.1113/jp287969

Figure Lengend Snippet: Figure 3. Decreased excitatory neuronal firing by cathodal weak direct current stimulation in rats with KA-induced seizures A, In vivo set-up (left upper panel) and schematic location of the unit spike recording electrode at CA1 stratum pyramidale. a, The trough; b and c, two peaks of the spike; the distance between b and c on the x-axis represents the peak-to-peak spike width; the distance between a and the higher peak on the y-axis indicates the spike amplitude (middle panel). Waveform presented with mean values of the spike width, amplitude and slope (left lower panel). Autocorrelation and spike feature distribution based on spike width, amplitude and firing frequency (right panel). B, In vivo set-up and comparison of the basal firing frequencies of the unit spikes between rats with KA-induced seizures (n = 12) and naive controls (n = 6). C, Comparison of unit spike number changes between anodal tDCS 0.5 mA (n = 3), anodal tDCS 1 mA (n = 3), cathodal tDCS 0.5 mA (n = 3) and cathodal tDCS 1 mA (n = 14) in KA-induced seizure rats. D, Experimental timeline and the comparison of the changes in unit spike numbers between ctDCS-treated and sham-treated KA rats over time. E, Comparison of unit spike frequencies across time in ctDCS-treated KA rats (n = 14, left panel). Unit spike raster across pre-ctDCS, ctDCS and post-ctDCS periods in all animals (t1–t14, right panel). F, Comparison of unit spike frequencies across time in sham-stimulated KA rats (n = 14, left panel). Unit spike raster across pre-sham, sham and post-sham periods in all animals (s1–s14, right panel). P60–90 was not recorded in animal s1. G, Upper panel, in vivo set-up and experimental timeline. Aligned unit spike waveforms detected from animals with KA-induced seizures treated with CaMKII opto-inhibition and ctDCS. Left lower panel, viral expression in CA1 injected with pAAV-CaMKIIa-eNpHR3.0-EYFP, shown in immunofluorescent staining of DAPI (blue) and EYFP virus (green). Right lower panel, comparison of the unit spike number changes at pre-optostim, optostim and post-optostim periods of optogenetic inhibition to CaMKIIa-expressed excitatory neurons. H, Upper panel, in vivo set-up and schematic location of optofibre and unit recording electrode in mice with KA-induced seizure. Aligned unit spike waveforms detected from animals with KA-induced seizures treated with VGAT opto-inhibition and ctDCS. Left lower panel, viral expression in VGAT-cre mice injected with pAAV-Ef1a-DIO eNpHR-EYFP, shown in immunofluorescent staining of NeuN (red) and EYFP virus (green). Right lower panel, comparison of the unit spike number changes at pre-optostim, optostim and post-optostim periods of optogenetic inhibition to VGAT-Cre-expressed GABAergic inhibitory neurons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. n, animal number. Colour curves with bands, mean ± SD in C and D. Bars, median ± IQR (interquartile range) in B and E–H. amp, amplitude; ctDCS, cathodal transcranial direct current stimulation; CTL, naive control rats; KA, kainic acid; sp, stratum pyramidale; stim, stimulation; P0–30, 0–30 min; P30–60, 30–60 min; and P60–90, 60–90 min after stimulation.

Article Snippet: Optogenetic inhibition in vivo pAAV-Ef1a-DIO eNpHR-EYFP viruses (Addgene, 26966, MA, USA) were transfected into Vgat-ires-cre mice (Slc32a1tm2(cre)Lowl/J mice, The Jackson Laboratory, 016962, ME, USA), whereas pAAV-CaMKIIa-eNpHR3.0-EYFP viruses (Addgene, 26971) were unilaterally injected into C57BL/6J male mice, 6–10 weeks old (NCKU animal centre), at CA1 (AP: −3, ML: 2.5, DV: −1.6 mm) under anaesthesia with 2%–2.5% isoflurane inhalation and pre-surgical analgesia with ketoprofen (3 mg/kg, intraperitoneal).

Techniques: In Vivo, Comparison, Inhibition, Expressing, Injection, Staining, Virus, Control

Journal: Molecular brain

Article Title: Distinct neural responses of ventromedial prefrontal cortex-projecting nucleus reuniens neurons during aversive memory extinction.

doi: 10.1186/s13041-025-01185-y

Figure Lengend Snippet: Fig. 4 Optogenetic manipulation of vmPFC-projecting neurons in the RE or MD. (A) Schematic of retroAAV-Cre injection into the vmPFC, combined with AAV-Flex-Jaws-GFP or AAV-DIO-GFP injection into the RE or MD. (B) Representative images showing Jaws-GFP expression. White bar = 200 μm. (C, D) Inhibition of vmPFC-projecting (C) RE (Extinction, interaction, F(5, 95) = 1.29, p = 0.28; main effect of group, F (1, 19) = 0.024, p = 0.88. Extinction retrieval, interaction, F (2, 38) = 2.25, p = 0.12; main effect of group, F (1, 19) = 0.0023, p = 0.96) or (D) MD (Extinction, interaction, F(5, 70) = 0.84, p = 0.52; main effect of group, F (1, 14) = 0.47, p = 0.50. Extinction retrieval, interaction, F (2, 28) = 0.072, p = 0.93; main effect of group, F () = 0.066, p = 0.80) neurons didn’t affect freezing during extinction and retrieval. (E) Schematic of retroAAV-Cre injection into the vmPFC, combined with AAV injection encoding Cre-dependent ChrimsonR or control fluorophore into the RE or MD. (F) Representative images showing ChrimsonR-tdTomato expression. White bar = 200 μm. (G, H) 10 Hz stimulation of vmPFC-projecting (G) RE (Extinction, interaction, F(5, 115) = 0.63, p = 0.68; main effect of group, F (1, 23) = 0.10, p = 0.75. Extinction retrieval, interaction, F (2, 48) = 0.95, p = 0.39; main effect of group, F (1, 23) = 0.99, p = 0.33) or (H) MD (Extinction, interaction, F(5,80) = 0.50, p = 0.77; main effect of group, F (1, 16) = 0.0085, p = 0.93. Extinction retrieval, interaction, F (2, 32) = 1.79, p = 0.18, main effect of group: F (1, 16) = 0.31, p = 0.58) neurons had no effect on freezing during extinction and retrieval. All error bars indicate SEM across subjects. Numbers in brackets represent sample size. Two-way repeated measures ANOVA

Article Snippet: For optogenetic inhibition experiments, 300 nL of retroAAV-hSyn-Cre (Addgene, 105553, titer: 2.1 × 10e13 vg/ml) was injected bilaterally into the vmPFC, and 300 nL of AAV5-hSyn-Flex-Jaws-KGC-GFP-ER2 (UNC Vector Core, 6.1 × 10e12 vg/ml) or AAV5-hSyn-DIOEGFP (Addgene, 50457, 6.5 × 10e12 vg/ml) was injected bilaterally into the RE (AP: +1.50 mm, ML: ±1.10 mm, DV: -4.50 mm, angle 10°) or MD (AP: -1.70 mm, ML: ±0.30 mm, DV: -3.30 mm).

Techniques: Injection, Expressing, Inhibition, Control