AAV-EF1a-BbTagBY in Rat (AAV9)
Addgene #45185-AAV9

This is an image taken at 63X on a Zeiss AxioImager microscope. The tissue is paraformaldehyde-fixed, frozen-sectioned, then mounted onto a glass slide and coverslipped with Prolong Glass antifade mountant from Thermo-Fisher. The cyan neuron mainly expresses mTFP (via the other virus that was co-injected), while the yellow axons originate from a neuron mainly expressing EYFP. This image was taken in the superior colliculus of a VGAT-Cre rat; neurons expressing fluorescent protein are believed to be GABAergic. The inset shows an EYFP-expressing axon making presumptive contact with a dendrite of the mTFP-expressing cell.

Caption: This is an image taken at 63X on a Zeiss AxioImager microscope. The tissue is paraformaldehyde-fixed, frozen-sectioned, then mounted onto a glass slide and coverslipped with Prolong Glass antifade mountant from Thermo-Fisher. The cyan neuron mainly expresses mTFP (via the other virus that was co-injected), while the yellow axons originate from a neuron mainly expressing EYFP. This image was taken in the superior colliculus of a VGAT-Cre rat; neurons expressing fluorescent protein are believed to be GABAergic. The inset shows an EYFP-expressing axon making presumptive contact with a dendrite of the mTFP-expressing cell.

Image attribution: Taken by Nichole Beebe

Viruses & Injection

Primary Virus
Promoter
EF1a
Injected Titer
3.40E+13 GC/ml
Injected Volume
0.03 μl to 0.15 μl (multiple volumes were compared across several cases)
Injection Rate
0. 01 μl per minute
Injection Site
Brain parenchyma
Brain Region
Midbrain: Tectum
Injection Site Comments
We were attempting to achieve Brainbow labeling of GABAergic cells in the inferior colliculus of VGAT-Cre rats in order to reconstruct the projections of these neurons. In one case, an injection was accidentally placed in caudal superior colliculus
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Secondary Virus
AAV-EF1a-BbChT (AAV9)
Promoter
EF1a
Injected Titer
1.7E13 GC/ml GC/ml
Injected Volume
The two viruses were mixed and co-injected.
Injection Rate
0. 01 μl per minute
Injection Site
Brain parenchyma
Brain Region
Midbrain: Tectum
Injection Site Comments
We co-injected the two viruses in an attempt to achieve Brainbow labeling in GABAergic cells on the rat inferior colliculus. The viruses were mixed before being aliquoted and were injected in the same syringe
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Subject

Species / Strain
Rat VGAT-Cre rat on a Long-Evans background: LEH-Vgat-Cretm1sage. Rats were supplied by Horizon Discovery Group.
Stock Number
TGRL9310
Age at Injection
approximately 3-6 months

Data Collection

Time After Injection
8 weeks
Detection Method
Direct fluorescence
Expression Site
Based on location and morphology, we felt confident that fluorescent cells in the inferior and superior colliculus were GABAergic, which was the cell class expressing Cre-recombinase in the animal model. In each case where an injection was made, fluorescent cells were present in the injected nucleus
Genetic Tool Outcome
The tool operated correctly. Cre-based fluorescent protein expression should have been restricted to GABAergic cells in the midbrain of this animal model, and that appeared to be true
Other Information
With greater injection volumes (ex. Three 0. 05 ul injections), more of an effect of inflammation or toxicity seemed present. This seemed less apparent with our smallest volume (0. 03 ul)
Tips & Comments
For the Brainbow viruses, we have decided to try to minimize our injection volume and maximize our survival time. The best success in fluorescent protein expression was achieved with an injection of 0. 03 ul and a survival time of 8 weeks