pAAV-CaMKIIa-hChR2(H134R)-EYFP in Mouse (AAV9)
Addgene #26969-AAV9

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.

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.

Virus & Injection

Primary Virus
Promoter
CaMKIIa
Injected Titer
4.48E+13 GC/ml
Injected Volume
400 nl
Injection Rate
0. 1 μl/min
Injection Site
Brain parenchyma
Brain Region
Forebrain: Cerebral cortex (neocortex)
Injection Site Comments
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

Subject

Species / Strain
Mouse ICR (CD-1)
Stock Number
Envigo
Age at Injection
21-26 days old

Data Collection

Time After Injection
1 week
Detection Method
Direct fluorescence, I could detect the fluorescence under blue LED (axons mostly and less neurons). 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.
Expression Site
Only principal neurons axons and cell bodies
Genetic Tool Outcome
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
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