aav2 vector backbone  (Addgene inc)

Journal: bioRxiv

Article Title: Auditory regulation of hippocampal locomotion circuits by a non-canonical reticular-limbic pathway

doi: 10.1101/2025.03.26.645464

Figure Lengend Snippet: A) Schematic of bilateral injections into the pontine reticular nucleus (PRN, red) with a retrograde optogenetic construct (AAVrg-CAG-hChR2-H134R-tdTomato). Fluorescent image showing tdTomato indicating channelrhodopsin 2 (ChR2) expression in axonal terminals in the PRN. B) Left : Fluorescence image showing tdTomato expressing DCN neurons, indicating that they project to the PRN. Right: Schematic drawing of bilateral optic fiber implants into the most dorsal part of the DCN. C) Light (473 nm) stimuli mimicking a broadband noise (see methods). D) Mean PSD plot of LFP signal (0-15Hz) recorded as mice (n=3) run on a treadmill during silence (gray), blue light stimulation (blue), and noise (red). E) Line graph showing individual theta 1 frequency response of the dHPC during silence (gray), optogenetic stimulation (blue), and noise stimuli (red). The mean theta 1 frequency was significantly different between silence (circle) and optogenetic stimulation (square) and between silence and noise stimuli (triangle), p=0.0392 and p=0.0303, respectively (n=3). F) Bar graph showing decreased mean theta power during noise stimuli (red) and optogenetic stimulation (blue) compared to during silence (gray), p=0.0052 and p=0.0353, respectively, (n=3). One-way repeated measures ANOVA, error bars show SEM, *p<0.05, **p<0.01. G) Schematic of control experiment with retrograde constructs (AAVrg-CAG-mCherry) injected into the PRN and bilateral optic fiber implants into the most dorsal part of the DCN. H) Line graph showing no change in theta due to light stimulation silence (gray), and light noise (blue). F) Bar graph showing no difference in mean theta power during silence (gray) and light stimulation (blue) in mice not expressing ChR2. T-test, error bars show SEM, *p<0.05, **p<0.01.

Article Snippet: For optogenetic stimulation of the dorsal cochlear nucleus (DCN) neurons projecting through the pontine reticular nucleus (non-canonical pathway), an AAV retrograde vector (AAVrg-CAG-hChR2-H134R-tdTomato, Addgene, 100µL at titer ≥7×10¹² vg/mL, dilution 1:3 in saline) was injected (0.75 μl per hemisphere) into the pontine reticular nucleus of C57BL/6 mice at the following coordinates: AP: -5.2 mm, ML: ±0.7 mm, DV: -4.0 and -4.5 mm [ ], to be able to excite channelrhodopsin 2 (ChR2)-expressing DCN neurons with blue light to excite the DCN – pontine reticular nucleus pathway.

Techniques: Construct, Expressing, Fluorescence, Control, Injection

Journal: bioRxiv

Article Title: Auditory regulation of hippocampal locomotion circuits by a non-canonical reticular-limbic pathway

doi: 10.1101/2025.03.26.645464

Figure Lengend Snippet: A) Schematic drawing of the angled (12 degrees) injection of cre-dependent chemogenetic construct (pAAV9.hSyn.DIO.hM4D(Gi).mCherry) into the MS of Chat-cre mice (n=5). Right, Fluorescent image of mCherry expression in Chat+ neurons of the MS. B) Experimental schematic of silence and sound sessions after administration of saline and CNO. C) Mean PSD plot of the LFP (0-15Hz) of the dHPC during treadmill running upon saline and CNO injection (0.5mg/kg, i.p.) during silence and noise. D) Line graph showing mean theta 1 frequency is significantly different between silence (gray and blue) and noise stimuli (red and pink) independent of saline or CNO injection *p<0.05, **p<0.01. E) Bar graph shows that the mean power was not significantly different when inhibiting MS Chat+ neurons independently of stimuli or treatment. F) Left; Schematic drawing of optic fiber inserted with a 12-degree angle into the MS of PV-cre mice (n=4) previously injected with a cre-dependent optogenetic construct for channelrhodopsin expression (pAAV9-EF1a-double floxed-hChR2(H134R)-mCherry). Right: Fluorescent image showing mCherry expression by PV+ MS neurons. G) Mean PSD plot of the dHPC LFP signal (0-15Hz) recorded during silence (gray), noise (red), light (473 nm, light noise, see methods, blue), and blue light during noise stimuli (pink). H) Mean frequency of theta frequency was significantly different between silence and noise (red), light stimulation (blue), and light+noise stimuli (pink). I) Mean power was higher for silent recording than noise (p=0.0411 Friedman test of multiple comparisons). Error bars show SEM, *p<0.05.

Article Snippet: For optogenetic stimulation of the dorsal cochlear nucleus (DCN) neurons projecting through the pontine reticular nucleus (non-canonical pathway), an AAV retrograde vector (AAVrg-CAG-hChR2-H134R-tdTomato, Addgene, 100µL at titer ≥7×10¹² vg/mL, dilution 1:3 in saline) was injected (0.75 μl per hemisphere) into the pontine reticular nucleus of C57BL/6 mice at the following coordinates: AP: -5.2 mm, ML: ±0.7 mm, DV: -4.0 and -4.5 mm [ ], to be able to excite channelrhodopsin 2 (ChR2)-expressing DCN neurons with blue light to excite the DCN – pontine reticular nucleus pathway.

Techniques: Injection, Construct, Expressing, Saline

Journal: Brain structure & function

Article Title: Dual medial prefrontal cortex and hippocampus projecting neurons in the paraventricular nucleus of the thalamus

doi: 10.1007/s00429-022-02478-x

Figure Lengend Snippet: A) Paired bilateral injections of retrograde AAV-CAG-tdTomato (magenta) and retrograde AAV-CAG-GFP (green) delivered in mPFC and vHC for florescence protein expression of neurons in PVT. B) mPFC injection spread was confined to PL/IL regions, primarily layers V/VI (n = 4). Epifluorescence capture (4X) on the left depict the injection spread in PL/IL in one case (NCL 235). Retrograde labeling can be seen locally and in other frontal regions (VO, LO, ACC). On the right, a schematic representation of injection sites for each case across brain levels are illustrated. C) vHC injection spread was confined to CA1 molecular layers (n = 4). Epifluorescence capture (4X) on the left depict the injection sites in vCA1 in one case (NCL 235). Retrograde labeling can be seen locally in CA1, Sub and EC. On the right, a schematic representation of injection spread in vCA1 for each case across brain levels are illustrated. Abbreviations: AAV, adeno-associated virus; ACC, anterior cingulate cortex; CA1, CA1 subfield of hippocampus; CAG, chicken beta-Actin promoter; DG, dentate gryus; GFP, green fluorescent protein; HC, hippocampus; IL, infralimbic cortex; LO, lateral orbital cortex; mPFC, medial prefrontal cortex; PL, prelimbic cortex; rad, stratum radiatum; slm, stratum lacunosum moleculare; tdTomato, tandem dimer Tomato; vCA1, ventral CA1; VO, ventral orbital cortex.

Article Snippet: Retrograde AAV vectors AAV-CAG-tdTomato (59462-AAVrg; Addgene, MA) and AAV-CAG-GFP (Addgene, MA; 37825-AAVrg) were delivered bilaterally in mPFC (AP +3.0 mm, ML +/− 1.2mm, DV −5.0mm, 50nL; −4.4mm, 150 nL; and −3.8mm, 200 nL) and vHC (AP −5.6mm, ML+/− 5.85mm, DV −7.2 mm,100 nL; 6.8 mm, 200 nL , and 6.2 mm, 200 nL), respectively (see ).

Techniques: Expressing, Injection, Labeling, Virus