aav9 th cre  (Addgene inc)

Journal: eLife

Article Title: Optical dopamine monitoring with dLight1 reveals mesolimbic phenotypes in a mouse model of neurofibromatosis type 1

doi: 10.7554/eLife.48983

Figure Lengend Snippet: ( A ) Illustration showing location of stereotaxic injection of the AAV9-hSyn-dLight1.2 viral vector and photometry fiber implantation ( left ). Representative histological image ( right , scale: 300 μm) showing the fiber tip location and expression of dLight1.2 (stained for GFP, green) and dopaminergic terminal tyrosine hydroxylase (TH, Red). ( B ) Schematic of fiber photometry system used for dLight1.2 (490 nm) and isosbestic (405 nm; reference signal) excitation and emission signal detection in freely moving mice. ( C ) Representative dLight1.2 traces in Nf1 +/+ and Nf1 +/- mice. ( D ) Representative trace and analysis features for baseline peak detection. ( E ) Peak analysis of baseline dLight1.2 recordings revealed that Nf1 +/- mice (n = 33) exhibit reduced transient frequency (unpaired t-test; t 50 = 3.06, p=0.004) but not median fluorescence (unpaired t-test; t 50 = 1.01, p=0.32), transient amplitude (unpaired t-test; t 50 = 0.83, p=0.41), or full width at half maximal amplitude (FWHM; unpaired t-test; t 50 = 0.43, p=0.67) when compared to Nf1 +/+ littermates (n = 19). ( F ) 4X differential interference contrast (DIC) image ( left ) of an acute horizontal midbrain slice containing the ventral tegmental area (VTA) and 4X epifluorescence image ( right ) with GFP-labeled catecholaminergic neurons following systemic delivery of AAV-PHP.eB- Th -GFP (1 × 10 11 v.g./mouse). ( G ) Representative traces showing spontaneous whole-cell firing of putative VTA dopaminergic neurons ( left ). Spontaneous firing rates ( right ) were lower (unpaired t-test; t 28 = 2.58, p=0.0 w) in Nf1 +/- putative dopaminergic neurons (n = 18) compared to Nf1 +/+ neurons (n = 12). ( H ) Representative electrophysiological traces ( left ) showing evoked firing by a 1 pA/ms ramp current from −60 mV in Nf1 +/+ and Nf1 +/- putative dopaminergic neurons. Rheobase ( right ; unpaired t-test; t 48 = 4.05, p<0.001) but not action potential threshold (I; t 48 = 1.93, p=0.06) or width (J; t 48 = 0.39, p=0.70) was increased in Nf1 +/- (n = 29) putative dopaminergic neurons compared to Nf1 +/+ (n = 21). *denotes p<0.05 vs Nf1 +/+ . Data presented as mean ± SEM.

Article Snippet: For optogenetic inhibition of VTA non-Th neurons, 500 nL of a 1:4 mixture of AAV9- Th -PI-Cre-SV40 (gift of James M. Wilson, Addgene viral prep # 107788-AAV9) and AAV-DJ-Ef1α-DO-eNpHR3.0-eYFP-WPRE-pA (gift of Bernardo Sabatini, Addgene # 37087) or AAV-DJ-Ef1α-DO-mCherry-WPRE-pA (gift of Bernardo Sabatini, Addgene # 37119) was injected bilaterally into the VTA (antero-posterior: −3.3 mm, medio-lateral: ± 0.5 mm, dorso-ventral: −4.2 mm relative to Bregma), followed by implantation of 5 mm long, 300 μm mono fiber-optic cannulae (MFC_300/330–0.48_5 mm_ZF1.25_FLT; antero-posterior: −3.3 mm, medio-lateral: ± 1.84 mm, dorso-ventral: −3.59 mm relative to Bregma) at angle of twenty degrees.

Techniques: Injection, Plasmid Preparation, Expressing, Staining, Fluorescence, Labeling

Journal: eLife

Article Title: Optical dopamine monitoring with dLight1 reveals mesolimbic phenotypes in a mouse model of neurofibromatosis type 1

doi: 10.7554/eLife.48983

Figure Lengend Snippet: ( A ) Illustration showing location of stereotaxic injection of the AAV5-hSyn-ChR2-eYFP viral vector and optical fiber in the ventral superior colliculus (vSC), as well as the location of the stereotaxic injection of the AAV9-hSyn-dLight1.2 viral vector and photometry fiber implantation in the LNAc. ( B ) Average fluorescence trace from individual mice showing optical LNAc dopamine signals evoked by activation of ChR2 in the ipsilateral vSC via two seconds of 20 Hz, 5 mW, 5 ms pulse-width, 473 nm laser stimulation. ( C ) Additional images showing Th -Off-NpHR-eYFP (cyan) and tyrosine hydroxylase (red)-labeled neurons in the VTA (scale = 50 μm).

Article Snippet: For optogenetic inhibition of VTA non-Th neurons, 500 nL of a 1:4 mixture of AAV9- Th -PI-Cre-SV40 (gift of James M. Wilson, Addgene viral prep # 107788-AAV9) and AAV-DJ-Ef1α-DO-eNpHR3.0-eYFP-WPRE-pA (gift of Bernardo Sabatini, Addgene # 37087) or AAV-DJ-Ef1α-DO-mCherry-WPRE-pA (gift of Bernardo Sabatini, Addgene # 37119) was injected bilaterally into the VTA (antero-posterior: −3.3 mm, medio-lateral: ± 0.5 mm, dorso-ventral: −4.2 mm relative to Bregma), followed by implantation of 5 mm long, 300 μm mono fiber-optic cannulae (MFC_300/330–0.48_5 mm_ZF1.25_FLT; antero-posterior: −3.3 mm, medio-lateral: ± 1.84 mm, dorso-ventral: −3.59 mm relative to Bregma) at angle of twenty degrees.

Techniques: Injection, Plasmid Preparation, Fluorescence, Activation Assay, Labeling

Journal: eLife

Article Title: Optical dopamine monitoring with dLight1 reveals mesolimbic phenotypes in a mouse model of neurofibromatosis type 1

doi: 10.7554/eLife.48983

Figure Lengend Snippet:

Article Snippet: For optogenetic inhibition of VTA non-Th neurons, 500 nL of a 1:4 mixture of AAV9- Th -PI-Cre-SV40 (gift of James M. Wilson, Addgene viral prep # 107788-AAV9) and AAV-DJ-Ef1α-DO-eNpHR3.0-eYFP-WPRE-pA (gift of Bernardo Sabatini, Addgene # 37087) or AAV-DJ-Ef1α-DO-mCherry-WPRE-pA (gift of Bernardo Sabatini, Addgene # 37119) was injected bilaterally into the VTA (antero-posterior: −3.3 mm, medio-lateral: ± 0.5 mm, dorso-ventral: −4.2 mm relative to Bregma), followed by implantation of 5 mm long, 300 μm mono fiber-optic cannulae (MFC_300/330–0.48_5 mm_ZF1.25_FLT; antero-posterior: −3.3 mm, medio-lateral: ± 1.84 mm, dorso-ventral: −3.59 mm relative to Bregma) at angle of twenty degrees.

Techniques: Recombinant, Plasmid Preparation, Produced, Software, Control, Refractive Index