These Ai85(TITL-iGluSnFr)-D mice (also called Ai85D) are a Cre/Tet-dependent, fluorescent glutamate indicator line, created by targeted insertion of floxed-STOP-iGluSnFr at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Ai85D mice harbor the TIGRE-Ins-TRE-LSL-iGluSnFr conditional allele, designed with a modified Tet response element (TRE or tetO) promoter and loxP-flanked STOP cassette upstream of the intensity-based glutamate-sensing fluorescent reporter (iGluSnFr; see detailed description below). When bred with mice expressing Cre recombinase, and either the tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), iGluSnFr is expressed in cells/tissues where the expression patterns of the individual promoters driving Cre and tTA/rtTA overlap, and expression can be regulated with tetracycline or its analog doxycycline (dox).

Specifically, the donating investigator reports Ai85D mice have no reported levels of iGluSnFr expression (GFP fluorescence) in the absence of Cre and tTA (iGluSnFr+/tTA-/Cre-), in Cre-negative double transgenic mice (iGluSnFr+/tTA+/Cre-) or in tTA-negative double transgenic mice (iGluSnFr+/tTA-/Cre+).

When Ai85D mice are bred with tTA-driver and Cre-driver lines to create triple transgenic animals (iGluSnFr+/tTA+/Cre+), cells expressing both tTA and Cre exhibit low GFP fluorescence in the absence of glutamate binding. Following glutamate binding (such as excitatory synaptic activity), robust GFP fluorescence is observed in these cells. The donating investigator has not tested iGluSnFr expression/function in cells other than brain.

Heterozygous Ai85D mice are viable and fertile with no reported gross physical or behavioral abnormalities. In 2017, breeding homozygous mice together at The Jackson Laboratory successfully identified that homozygous Ai85D mice are viable and fertile.

For characterization information, see images at the Allen Institute for Brain Science website (Ai85(TITL-iGluSnFr) images).

The intensity-based glutamate-sensing fluorescent reporter iGluSnFr optimized for in vivo imaging with fast kinetics, single-wavelength glutamate sensitivity and maximum fluorescence response (Marvin et al. 2013 Nat Methods 10:162). Designed by Dr. Loren Looger (Janelia Farm, HHMI), the iGluSnFr fusion gene is constructed from the Escherichia coli gltI gene (YbeJ) and circularly permutated green fluorescent protein (cpGFP).
The iGluSnFr fusion gene has an IgG K-leader sequence (to direct the protein to the secretory pathway) at the N-terminal end, and a c-Myc epitope tag and a 49 aa PDGR sequence (to anchor the protein to the plasma membrane [displaying it on the extracellular side]) at the C-terminal end. Because iGluSnFr can directly access the synaptic cleft, it allows extremely rapid detection of glutamate transients with high spatial resolution; enabling direct visualization of excitatory synaptic release (as opposed to calcium imaging). iGluSnFr is sensitive to glutamate peaks just prior to calcium peaks and action potential. iGluSnFr has single-photon and two-photon fluorescence excitation with ~470-485 nm and ~910-920 nm light, respectively. The pK_a of iGluSnFr is 6.5 in the glutamate-bound and 7.0 in the ligand-free state. The purified iGluSnFr protein has no affinity to a panel of other amino acids or other neurotransmitters, excepting that some in vitro affinity to aspartate is reported. In the absence of glutamate binding, dim GFP fluorescence is expected (presumably due to distortion of the cpGFP β-barrel). Upon glutamate binding, the correct conformation is restored; resulting in bright and photostable fluorescence.