Inducible gene targeting in mouse retinal neurons expressing Grm6

Program Number: 2219 Poster Board Number: A0395 Presentation Time: 3:45 PM–5:30 PM Cx36-independent rod pathways mediate visual contrast sensitivity to high temporal frequencies Rose Pasquale1, 2, Yumiko Umino2, Eduardo C. Solessio2. 1Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY; 2Ophthalmology, Center for Vision Research, SUNY Upstate Medical University, Syracuse, NY. Purpose: In the retina, rod signals are transmitted along multiple pathways, each connecting to underlying cone circuitry. Rod signals spread to cone pathways via Cx36 gap junction electrical synapses (Cx36-dependent rod pathways) as well as by direct chemical synapses from rods to cone bipolar cells (Cx36-independent rod pathways), with the contribution of the latter to visual function still unclear. Our goal is to dissect the contributions of Cx36-dependent and independent rod pathways to temporal contrast sensitivity (TCS). Methods: We utilized transgenic mouse lines (3-6 months) to separate retinal pathways and used a novel operant behavior assay developed in our lab to measure the TCS of mice in response to fullfield flicker applied over a wide range of temporal frequencies and background intensities. Immunohistochemical and electroretinogram (ERG) analyses were performed to assess retinal integrity. Statistical significance was tested by two-way ANOVA. Results: Operant behavior results show that mice with non-functional cones (GNAT2cpfl3) have normal TCS at low mesopic backgrounds (100R*/rod/s). At higher mesopic backgrounds (2000R*/rod/s), GNAT2cpfl3 mice exhibited a significant reduction in sensitivity to low temporal frequencies (1.5-12 Hz) (n=5+/-SEM) while their sensitivity to high temporal frequencies (15-42 Hz) remained normal. TCS to high frequencies also remained normal in Cx36-/-::GNAT2cpfl3 double mutant mice with isolated Cx36-independent rod pathways (n=4+/SEM) suggesting that Cx36-independent rod pathways function to relay fast temporal information at 2000R*/rod/s. Control studies show that TCS to high frequency flicker is negligible (n=5+/-SEM) when presented in high (8000R*/rod/s) background levels suggesting that the observed responses are driven largely by rods and not by remaining cone function in GNAT2cpfl3 mice. ERG recordings indicate that the mouse lines used for these studies did not exhibit overt remodeling (n=9+/-SEM). Conclusions: Our results suggest that, at high mesopic light levels (2000R*/rod/s): 1) Cone pathways contribute to the detection of low temporal frequency flicker; and 2) the poorly understood Cx36independent rod pathways relay high temporal frequencies. Together, these data are consistent with the concept that rod pathways play a dynamic role in mammalian visual function. Commercial Relationships: Rose Pasquale, None; Yumiko Umino, None; Eduardo C. Solessio, None Support: RO1 EY026216 Program Number: 2220 Poster Board Number: A0396 Presentation Time: 3:45 PM–5:30 PM Inducible gene targeting in mouse retinal neurons expressing Grm6 Yu-Jiun Chen1, Hoon Shim2, Crystal S. Shin1, Ghanashyam Acharya1, Ching-Kang J. Chen1, 3. 1Ophthalmology, Baylor College of Medicine, Houston, TX; 2Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA; 3Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX. Purpose: The purpose of the study is to generate mouse lines for inducible gene targeting in depolarizing bipolar cells (DBCs). Methods: Three engineered transgenic constructs with iCre, mCherry, and ERT2-Cre-ERT2 (ECE) cDNAs downstream of a 10 kb mouse Grm6 promoter fragment were made, respectively. The iCre and mCherry constructs were mixed and injected pronuclearly into embryos of C57BL/6 and Balb/c mixed background and one founder line, MCV11F, was established. The ECE construct was injected alone and one founder line, T1, was established. Cre activity was revealed by commercial Z/EP or Ai9 reporter lines. Cre induction in the T1/Ai9 line was done by intraperitoneal injections of varying doses of tamoxifen (TAM) or by topical application of 4-hydroxytamoxifen (4-HT) loaded nanowafers on the cornea. Results: The MCV11F line has robust mCherry and Cre recombinase expression in all adult DBCs. However, in the Z/EP background, we found GFP expression throughout the entire retina, indicating that Cre was expressed earlier during development. Without induction, the T1/Ai9 line displays Cre activity in ~50 neurons in young adults and ~100 in mice over one year of age. Intraperitoneal delivery of 500 nmol per gram body weight per day of TAM over five days is sufficient to induce Cre activity in >90% of DBCs. By titrating the amount of TAM injected, a single delivery of 10-25 nmol per gram body weight could induce tdTomato expression in ~300 intermittently dispersed neurons per retina. To induce Cre activity in the T1 mice unilaterally, we employed a nanowafer delivery system of 4-HT at the dose of (5 μg/wafer/day). This could also induce reporter expression between 200-1000 DBCs in the treated eye. Under inductive conditions where 100 to 1000 DBCs expressed tdTomato, examination of the axonal stratification levels of ~200 cells in the inner plexiform layer (IPL) showed that rod bipolar cells (RBCs) and all known cone ON bipolar cells except the type-9 (CB9) could be labeled. Interestingly, we encounter a few cells whose axons stratify to the OFF center, suggesting that certain hyperpolarizing bipolar cells may also express GRM6. Conclusions: Despite being a good marker line, the precarious Cre expression during development of MCV11F mice restricts its use. The T1 line is more suitable for inducible gene targeting in DBCs. Its utility may be expanded by further exploration of the type, amount, and routes of reagents used for induction. Commercial Relationships: Yu-Jiun Chen, None; Hoon Shim, None; Crystal S. Shin, None; Ghanashyam Acharya, None; Ching-Kang J. Chen, None Support: NIH Grants EY013811, EY022228, EY002520, unrestrcted grant from Research to Prevent Blindness, Retina Research Foundation