Elsevier

Neuroscience

Volume 307, 29 October 2015, Pages 319-337
Neuroscience

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines

https://doi.org/10.1016/j.neuroscience.2015.08.060Get rights and content

Highlights

  • TH-RFP retinas only express fluorescently labeled type 1 and 2 dopamine (DA) amacrine cells.

  • TH-BAC-tdTomato retinas mainly express fluorescently labeled AII amacrine cells, and a few type 1 DA amacrine cells.

  • TH-tdTomato retinas express fluorescently labeled GABA-containing amacrine cells, including polyaxonal amacrine cells.

  • DAT-tdTomato retinas express fluorescently labeled two monostratified and two bistratified amacrine cell types.

Abstract

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing.

Introduction

Transgenic mouse models are one of the major tools currently used for investigations of the intricate anatomical, biophysical and functional properties of neuronal cell populations and their networks. For instance, studies using mouse models have contributed to the identification and manipulation of the connectivity, interactions and activity of neurons within a network, which has advanced our understanding of their functional role in neuronal processing in multiple structures, including the retina, cortex, hippocampus, olfactory bulb and cerebellum (Ramirez et al., 2013, Hammen et al., 2014, Pohlkamp et al., 2014, Robinson et al., 2014, Sternson and Roth, 2014, Zhu and Roth, 2014, Zhu et al., 2014a). The use of transgenic mouse models has been especially powerful for studies of intrinsic retinal circuitry and function, including those of amacrine and ganglion cell subtypes, as well as the understanding of the functional connectivity of retinal ganglion cells with central visual nuclei (Kim et al., 2010, Kay et al., 2011a, Rivlin-Etzion et al., 2011, Yamagata and Sanes, 2012, Zhang et al., 2012, Dhande et al., 2013, Farrow et al., 2013, Yonehara et al., 2013, Vlasits et al., 2014, Zhu et al., 2014b).

Amacrine cells are classified based on their general morphology, including somal size, the stratification pattern of their processes in the inner plexiform layer (IPL), and their neurochemical expression (Ramón y Cajal, 1893, Boycott and Wässle, 1974, Brecha et al., 1979, Karten and Brecha, 1980, Kolb et al., 1981, Vaughn et al., 1981, Masland, 1988, Casini and Brecha, 1991). Based on these criteria, 30–40 different amacrine cell types have been identified in the mammalian retina (Kolb and Nelson, 1981, Xin and Bloomfield, 1997, MacNeil and Masland, 1998, MacNeil et al., 1999, Masland, 2001, Badea and Nathans, 2004). Furthermore, amacrine cells form multiple microcircuits in the inner retina to process visual information (Masland, 2001, Wässle, 2004).

One well-studied wide-field amacrine cell type is the tyrosine hydroxylase (TH) or dopamine (DA) amacrine cell, which is also known as a type 1 DA amacrine cell. This amacrine cell synthesizes and releases DA, in addition to the inhibitory neurotransmitter GABA (Wulle and Wagner, 1990, Hirasawa et al., 2009, Hirasawa et al., 2012, Contini et al., 2010). The morphology of type 1 DA amacrine cells have been thoroughly characterized in retinas of multiple species, including amphibians, rodents, lagomorphs, felines and primates using antibodies against TH, the rate-limiting enzyme for the synthesis of DA (Brecha et al., 1984, Versaux-Botteri et al., 1984, Oyster et al., 1985, Mariani and Hokoç, 1988, Nguyen-Legros, 1988, Dacey, 1990, Tauchi et al., 1990, Zhu and Straznicky, 1990). Physiological studies indicate multiple retinal functions for DA, including a role in light/dark adaptation of retinal circuits and serving as the output of the retinal circadian clock. Moreover, low levels of DA are often concomitant with retinal disease including diabetic retinopathy (Teakle et al., 1993, Witkovsky, 2004, Aung et al., 2014).

The identification of type 1 DA amacrine cells in transgenic lines have been of great value in determining the circuits they participate in within the inner retina. For example, transgenic labeling of type 1 DA amacrine cells have aided in defining the connectivity between these cells and AII and GABA-containing amacrine cells, ON-cone bipolar cells, and melanopsin-containing ganglion cells (Gustincich et al., 1997, Feigenspan et al., 2000, Zhang et al., 2007, Zhang et al., 2012, Contini et al., 2010, Van Hook et al., 2012, Newkirk et al., 2013). Other studies that used isolated type 1 DA amacrine cells, labeled by human placental alkaline phosphatase (hPLAP), report these cells have transient A-type K+, Ca2+ and TTX-sensitive Na+ currents, and rhythmic spontaneous action potentials (Feigenspan et al., 1998, Xiao et al., 2004). More recently, type 1 DA amacrine cells identified by green fluorescent protein (GFP) in retinal whole mounts of a DA receptor 2 transgenic mouse line were used to characterize their spontaneous spike properties and light responses (Newkirk et al., 2013).

Despite many advances in understanding type 1 DA amacrine cells, their functional influence on other cells in the retinal network has not been completely established. Studies addressing this topic would be greatly facilitated by using a mutant mouse line with Cre recombinase activity exclusively in type 1 DA amacrine cells to genetically manipulate these cells. In this study, we have evaluated three catecholamine-related transgenic mouse lines expressing Cre, and compared them to a previously published TH-red fluorescent protein (RFP) mouse line with DA amacrine cells expressing RFP (Zhang et al., 2004). Cre expression under the control of TH or dopamine transporter (DAT) regulatory elements is expected to provide specific labeling of the type 1 DA amacrine cells in the retina, as in the rest of the central nervous system (Gelman et al., 2003, Lindeberg et al., 2004, Zhuang et al., 2005, Bäckman et al., 2006). However, our studies of the cellular localization of Cre reporter expression in the retinas of these mouse lines show that there is a surprisingly small percentage or a lack of type 1 DA amacrine cells labeled in these retinas. In contrast, these retinas contained ectopic yet reliably identifiable Cre-dependent fluorescently labeled cell types in the inner retina, including AII amacrine cells, polyaxonal amacrine cells, monostratified and bistratified amacrine cells, displaced amacrine cells, and a few ganglion cells. These Cre-expressing mouse lines will be of value to other investigations of the aforementioned amacrine cells.

Section snippets

Experimental procedures

Animal care and all experiments were carried out in accordance with the guidelines for the welfare of experimental animals issued by the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals and the University of California Los Angeles (UCLA) Animal Research Committee.

TH-RFP

As reported previously, TH-RFP retinas express RFP in sparsely distributed somata located in the proximal inner nuclear layer (INL) with numerous processes that ramify in the OFF layer of the IPL (Fig. 1A) (Zhang et al., 2004). A few varicose processes also ramified in the middle of the IPL (not shown) and in the outer plexiform layer (OPL) (Fig. 1A, arrow). There was a lower level of RFP expression in small caliber processes (Fig. 1A, arrowheads) and small somal diameter amacrine cells (not

Discussion

This study has evaluated Cre-mediated fluorescence in the retinas of three Cre transgenic mouse lines crossed with a tdTomato reporter line (Ai14) (Savitt et al., 2005, Bäckman et al., 2006, Gong et al., 2007, Madisen et al., 2010). These Cre-expressing retinas either lacked or had a low percentage of type 1 DA amacrine cells. In comparison, the previously characterized TH-RFP, TH-PLAP and TH-GFP mouse lines, only have labeled type 1 and 2 DA amacrine cells (Gustincich et al., 1997, Matsushita

Conclusion

Retinas of the three Cre lines characterized in this report can serve as a platform for future investigations of amacrine cells. The TH-BAC-tdTomato retina can be better used to study AII amacrine cells. Retinas of TH-tdTomato mouse line can be used for studying polyaxonal wide-field amacrine cells, including their synaptic partners, intrinsic properties, and specific functions in visual processing. The DAT-tdTomato retina contains multiple types of labeled wide-field bistratified and

Acknowledgments

We thank Dr. Arlene Hirano for her comments and discussion on this project and manuscript.

Support and grant information: Support for these studies was from NIH R01 EY04067 (N.C.B.), P30 DK41301 (Imaging and Stem Cell Biology Core; CURE:DDRC), a VA Merit Review (N.C.B.), NIH T32 EY007026 (H.E.V.), Whitcome Summer Undergraduate Research Fellowship (C.N.H.) and NIH R01 EY09256 (D.G.M.). N.C.B. is a VA Career Research Scientist.

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