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pAAV-CaMKIIa-hChR2(H134R)-mCherry in Mouse (AAV9)

Addgene #26975-AAV9

Data Submitted By
Stylianos Kouvaros and Thanos Tzounopoulos
Submission Date
July 29, 2019
Publication Date
July 29, 2019 (modified May 16, 2023)
Abstract
Not Provided
Under 40X in Layer 2,3 of auditory cortex you can see the virus expression after photostimulating with green LED. Note that this image is coming from the previous one (at 4X magnification) in which the virus was undetectable in Layer 2,3)
Caption: Under 40X in Layer 2,3 of auditory cortex you can see the virus expression after photostimulating with green LED. Note that this image is coming from the previous one (at 4X magnification) in which the virus was undetectable in Layer 2,3)
In this image you can see an NMDA EPSC recorded from a SST interneuron (at +40 mV_VClamp) in Layer 2,3 of auditory cortex from the slice shown above. Note the small responses (around 40-50 pA) due to the insufficient virus expression.
Caption: In this image you can see an NMDA EPSC recorded from a SST interneuron (at +40 mV_VClamp) in Layer 2,3 of auditory cortex from the slice shown above. Note the small responses (around 40-50 pA) due to the insufficient virus expression.
In this image you can see the intense expression of the virus in Layers 4,5,6 of auditory cortex and hippocampus under green LED in 4X magnification. Despite the fact that under 4X we cannot see the virus expression, under 40X is apparent. See the next image....
Caption: In this image you can see the intense expression of the virus in Layers 4,5,6 of auditory cortex and hippocampus under green LED in 4X magnification. Despite the fact that under 4X we cannot see the virus expression, under 40X is apparent. See the next image....

Any further use of this image requires permission from the lab who submitted the data.

Vector Used
pAAV-CaMKIIa-hChR2(H134R)-mCherry
Addgene #26975-AAV9

Virus & Injection

Virus

Virus Name
pAAV-CaMKIIa-hChR2(H134R)-mCherry
Serotype
AAV9
Source
Addgene
Catalog Number
26975-AAV9
Promoter
CaMKII
Cargo Type
Optogenetics
Injection Titer / Dose
1.00E+13 GC/ml
Injection Volume
700 nl
Injection Rate
100 ml/min
Injector Material
Not Provided
Injection Site / Route
Auditory cortex, NeuroNames: 1556
Additional Details
I injected in auditory cortex targeting the excitatory axons of principal neurons (0. 5 to 1 mm deep from pial).
View in Addgene Catalog

Other Details

Species
Mouse
Strain or Cell Line
FVB-Tg(GadGFP)45704Swn/J
Source
Jackson Labs
Stock Number
3718
Age at Injection
21-26 days old
Time After Injection
3 weeks
Detection Method
Direct fluorescence
Assay & Results
I ended up injecting too much volume of the virus from the original stock (without dilluting with 1XPBS) and to expect too many days (2 weeks and more) to get enough expression.
Functional Testing
I recorded AMPA and NMDA EPSCs in SST and PV interneurons which were patched in voltage clamp.
Other Information
I did not see any toxicity but the virus expression was not enough
Tips & Comments
I would recommend to inject over 700 nl of the virus in the original stock (1E13) and to be very precise in the injection site. Obviously the virus does not spread a lot.