Leaky expression of channelrhodopsin-2 (ChR2) in Ai32 mouse lines

Optogenetics enables the selective activation of genetically-targeted neuronal populations using light-sensitive ion channels. Genetic strategies using Cre-dependent mouse strains, especially the Ai32 line expressing Channelrhodopsin (ChR2)-EYFP fusion protein, have been a popular means to drive opsin expression in a cell-type specific manner. Here we report a low level of leaky ‘off-target’ (Cre-independent) ChR2-EYFP expression in Ai32/Ai32 homozygous mice throughout the nervous system. This leaky off-target expression was characterized in multiple prevalent nervous system regions using anti-EYFP immunostaining. Expression of full-length ChR2-EYFP protein was confirmed using immunoprecipitation followed by Western blotting. Notably, light stimulation of these ChR2-EYFP expressing neurons in the spinal cord dorsal horn did not induce detectable photocurrents in juvenile 4-week old mice. Given the wide use of the Ai32 line by many labs, our results suggest researchers should be vigilant of possible off-target ChR2-EYFP expression in their region of interest, especially when generating Ai32/Ai32 homozygotes to drive high levels of ChR2-EYFP expression in adult mice.


Introduction
Optogenetics has revolutionized neuroscience by allowing for selective activation of genetically-targeted neuronal populations using light. In this technique, target cells are driven to express light-sensitive opsins, which are commonly ion channels. For example, Channelrhodopsin-2 (ChR2) is a blue light-gated non-specific cation channel that drives neuronal activation [1]. Blue light illumination in ChR2 expressing animals allows for specific, temporally precise control of neuronal activity in a wide range of in vitro and in vivo contexts [2].
Genetic strategies using Cre-dependent mouse strains have been a popular means to drive opsin expression in a cell-type specific manner. For example, using a Rosa knock-in loxP-STOP-loxP strategy that allows for high-level, specific transgene expression [3], Madisen et al engineered multiple mouse lines for Cre-dependent, robust expression of opsins [4]. These a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 lines are very useful reagents for the neuroscience research community. One of these lines, the Ai32 line expressing a ChR2-EYFP fusion protein, has been widely used for cell-type specific expression of ChR2. Following the cassette design for the Rosa knock-in allele, the Ai32 uses the CAG promoter and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to drive high levels of ChR2-EYFP expression [3,4]. In the original paper, the authors reported that, while other lines showed some background (Cre-independent) expression of opsin mRNA, the Ai32 line does not express ChR2-EYFP in the absence of Cre recombinase.
In our research, we occasionally noticed ChR2-EYFP expression in Cre-positive Ai32 mice in a manner that was not predicated based on Cre-line recombination pattern. We therefore sought to examine the possibility of off-target, or "leaky", expression of ChR2-EYFP in Ai32/ Ai32 homozygous mice in the absence of Cre. We performed immunostaining for EYFP of Ai32/Ai32 mice and indeed found leaky ChR2-EYFP expression throughout the nervous system. We also confirmed that this EYFP signal corresponds to full length ChR2-EYFP protein using immunoprecipitation. Our results suggest that the strong gene expression driven by this Rosa cassette can result in background off-target expression of ChR2-EYFP, especially in Ai32/Ai32 homozygous mice. Nevertheless, light stimulation of these ChR2-EYFP expressing neurons in dorsal horn of the spinal cord slices did not induce detectable photocurrents in 4-week old juvenile mice. In short, our study clearly showed background expression of ChR2-EYFP in Ai32 homozygous mice. Given the popularity of the Ai32 line, this finding suggests that researchers using this line should be vigilant of possible off-target ChR2-EYFP expression in their region of interest, especially in adult mice as ChR2-EFYP expression level accumulates with age.

Mouse strains
Mice were raised in a barrier facility in Hill Pavilion, the University of Pennsylvania. All procedures were conducted according to animal protocols approved by Institutional Animal Care and Use Committee (IACUC) (Protocol:804886) of the University of Pennsylvania and National Institutes of Health guidelines. Mice used in this paper were initially purchased from Jackson Labs or Charles River Laboratories and were subsequently propagated by our lab, and have been described previously: Ai32 Rosa ChR2 (

Biochemistry
Olfactory bulbs (~20-30 mg) of CO 2 euthanized adult (3-6 month old) Omp Cre ;Ai32/Ai32, Ai32/Ai32, or WT control mice were dissected out and snap frozen on dry ice. Samples were lysed with 1.4 mL of ice cold RIPA buffer (50 mM Tris pH = 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxychoate, 0.1% SDS) with added protease inhibitors (Sigma, P8340, St. Louis, MO) and were homogenized with a handheld rotor fixed with a sterile pestle. Sample were rocked for 2 hours at 4 degrees and centrifuged for 20 min at 12,000 rpm at 4 degrees. Supernatant was collected and moved to a fresh tube.

Results
While using Ai32 mice to drive various Cre-dependent expression of ChR2-EYFP, we have generated Cre + ; Rosa ChR2-EYFP/ChR2-EYFP (Ai32/Ai32 homozygous) mice to drive high level expression of ChR2-EYFP for optically controlled behavior studies. During these experiments, we noticed off-target expression of ChR2-EYFP. We sought to differentiate whether this was due to ectopic Cre expression or to 'leaky' expression from the Ai32 allele. Thus, we examined ChR2-EYFP expression in homozygous Ai32/Ai32 (Cre-negative) mice.
We first visualized ChR2-EYFP expression in Ai32/Ai32 mice using immunostaining against EYFP. We found leaky EYFP signals throughout the nervous system of Ai32 homozygous mice, and this expression was particularly high in a number of areas. Fig 1 shows representative immunostaining of Ai32/Ai32 adult olfactory bulb (OB). We found leaky expression of EFYP in the granular, mitral, and glomerular cell layers (Fig 1A-1C). EFYP+ cells express the neuronal marker NeuN in the mitral and granular layers (Fig 1C) but do not express the astrocyte marker GFAP in the OB (Fig 1B & 1C). Figs 2-4 show leaky expression of EYFP in  NeuN+ cells of the cingulate cortex (Fig 2A & 2B), hippocampus (Fig 2C & 2D), the islands of the Calleja in the olfactory tubercle (Fig 2E & 2F), in the cerebellar lobes (Fig 3A & 3B) and in the vermis (Fig 3C & 3D). In the brainstem, we saw EYFP expression in NeuN+ cells of the spinal trigeminal nucleus caudalis (Fig 4A & 4B), and in isolated regions of the ventral pallidum (Fig 4C & 4D).
We further characterized leaky expression of ChR2-EFYP in the dorsal root ganglion (DRG) and spinal cord dorsal horn (Fig 5). Leaky EYFP signals are found in NeuN+ interneurons of the cervical, thoracic, and lumbar dorsal horn of Ai32/Ai32 mice (Fig 5A-5E). Interestingly, in DRGs, EYFP is expressed in the GFAP+ satellite glia cells, but not in primary afferent neurons themselves (Fig 5F & 5G). In summary, this leaky expression occurs in both neurons and glial cells in many regions of the nervous system in homozygous Ai32/Ai32 (Cre-negative) mice.
Following immunostaining, we performed western blotting to confirm that EYFP fluorescence corresponds to the expression of full-length ChR2-EYFP protein. Fig 6 shows western blot detection of ChR2-EYFP expression in Ai32/Ai32 OB compared to the positive (Omp Cre ; Ai32/Ai32) and negative control mice. OB cell lysates were immunoprecipitated using rabbit anti-GFP antibody and blotted using chicken anti-GFP (OB lysates were chosen because of the high degree of fluorescence in immunostaining, Fig 1). Ai32/Ai32 OB lysates showed ã 60kDa positive band, which is as anticipated (ChR2-EYFP predicted weight = 62 kDa) and similar in size to the positive control ChR2-EYFP from Omp Cre ; Ai32 OB lysates (Fig 6 lanes  1-4). This band is absent from negative control CD1 (Ai32 negative) OB lysate (Fig 6 lane 5). This result confirms that the fluorescence visualized as leaky expression corresponded to full length ChR2-EYFP protein.
To test whether the leaky expression of ChR2-EYFP is functional, we examined light induced responses in ChR2-EYFP expressing dorsal horn cells in the spinal cord slices from 4-week old Ai32/Ai32 mice (Fig 7A). Out of 12 recorded cells, none showed detectable response upon light stimulation (0.1 ms-1 s duration). Based on our previous experience [3], 0.1ms blue laser pulse durations are sufficient to induce photocurrents in Cre-driven ChR2 expressing spinal cord DH neurons. In addition, these recorded cells are viable and responsive, as they show action potential firing upon current injection (Fig 7C). Thus, this result indicates that leaky ChR2-EYFP expression in dorsal spinal cord neurons is not high enough to generate detectable photocurrents (Fig 7B) at four weeks of age. However, our results do not exclude the possibility that this leaky expression of ChR2 could generate photocurrents with stronger light stimulation or with potentially higher expression levels in adult mice.

Discussion
We report Cre-independent, leaky ChR2-EYFP expression in Ai32/Ai32 homozygous mice as identified by EYFP immunostaining and IP/western blotting. While this expression occurred throughout the nervous system, we identified regions with particularly high ChR2-EYFP expression including the olfactory bulb, hippocampus, cerebellum, and other regions. While anti-EFYP immunostaining allows for clear identification of this off-target expression, the level of this expression is likely to be much lower compared to Cre-driven Ai32 ChR2-EYFP. Indeed, our western blotting results reveal much lower amounts of ChR2-EYFP purified from Ai32/Ai32 (Cre-negative) OB lysates in comparison with Omp Cre -driven positive control tissue (Fig 6). While previous work has reported no Cre-independent ChR2-EFYP mRNA expression in this line [4], it is possible that the off-target leaky expression we found is related to the use of Ai32/Ai32 homozygous tissue. We also found that, although the leaky expression of ChR2-E-FYP is obvious in some areas of nervous system, this expression is not sufficient to generate a detectable photo current at least in the spinal cord of 4-week old mice, likely due to the low level of ChR2 present in these cells.
The Ai32 line uses a CAG promoter cassette organization designed to drive high levels of transgene expression. The off-target expression we found is possibly a consequence of 'readthrough' transmission of the floxed stop element, especially given the strong promoter used to drive this cassette. Notably, other lines using this cassette organization have been reported to show off-target transgene mRNA expression [4]. While read-through transcription of similar floxed stop elements has been reported in other Cre-reporter transgene systems [10,11], such transcription has been shown to be considerably weaker than expression after Cre-mediated stop element excision, consistent with our results. It should be noted that, during propagation, the Ai32 line used in this study have been crossed to other lines, and this genetic background variability may contribute to this off-target leaky expression of ChR2-EFYP. The presence of this leaky ChR2-EYFP expression is an important consideration for labs using this line, specifically in the use of EYFP immunostaining to characterize ChR2 expression in Ai32 mice. Further, while we did not see functional photocurrents in our recordings with spinal cord dorsal horn neurons at 4 week old, we cannot rule out the possibility of functional effects of leaky ChR2 expression after light stimulation either through some modulatory effect or by direct activation in cells with higher expression levels in adult mice.