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

Addgene #26969-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
Here you can observe the NMDA EPSC from a principal neuron in L2,3 after the photoactivation of the adjacent excitatory axons with the blue LED. The neuron is in Voltage Clamp at +40 mV and you can see an average response pre and after a drug. Note that to keep a principal neuron at +40 mV for about an hour (duration of the experiment) is too difficult. Declaring that there is no or less toxicity from the virus.
Caption: Here you can observe the NMDA EPSC from a principal neuron in L2,3 after the photoactivation of the adjacent excitatory axons with the blue LED. The neuron is in Voltage Clamp at +40 mV and you can see an average response pre and after a drug. Note that to keep a principal neuron at +40 mV for about an hour (duration of the experiment) is too difficult. Declaring that there is no or less toxicity from the virus.
Image taken under 40X magnification from an Olympus BX51XI from the Layer 2,3 of left auditory cortex after turning the blue LED (M470L3-C1) in full intensity. You can observe the green axons in that case.
Caption: Image taken under 40X magnification from an Olympus BX51XI from the Layer 2,3 of left auditory cortex after turning the blue LED (M470L3-C1) in full intensity. You can observe the green axons in that case.

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

Vector Used
pAAV-CaMKIIa-hChR2(H134R)-EYFP
Addgene #26969-AAV9

Virus & Injection

Virus

Virus Name
pAAV-CaMKIIa-hChR2(H134R)-EYFP
Serotype
AAV9
Source
Addgene
Catalog Number
26969-AAV9
Promoter
CaMKII
Cargo Type
Optogenetics
Injection Titer / Dose
4.48E+13 GC/mL
Injection Volume
400 nL
Injection Rate
100 nL/min
Injector Material
Not Provided
Injection Site / Route
Auditory cortex, NeuroNames ID: 1556
Additional Details
I injected 400 nl of diluted virus (3 μl of virus with 3 μl of 1X PBS) in the auditory cortex (0. 5-1 mm deep) to activate the principal neurons.
View in Addgene Catalog

Other Details

Species
Mouse
Strain or Cell Line
ICR (CD-1)
Source
Envigo
Age at Injection
21-26 days old
Time After Injection
1 week
Detection Method
Direct fluorescence
Assay & Results
Only principal neurons axons and cell bodies.I could detect the fluorescence under blue LED (axons mostly and less neurons).
Functional Testing
The tool operated perfectly under photoactivation with blue LED light. If you record from principal neurons that express the virus, try to avoid direct photostimulation of this neuron. Move the lens through which you photoactivate the axons adjacent the recorded neuron. Also, I was able to get responses (AMPA or NMDA EPSCs) in Voltage Clamp (with range 50 to 2000 pA) depending the intensity of LED activation in principal neurons, Somatostatin and Parvalbumin interneurons.
Other Information
No inflammation observed (try to use thin glass pipette for virus injection to cause minimal brain damage). Cells were fine. No critical toxicity observed
Tips & Comments
My only tip that I say to myself is to conduct all the experiments with the same virus. Try to finish all your experimental series before you need to order another vial of new virus.