When using optogenetics to study circuit function or animal behavior, a critical prerequisite is that optogenetic protein expression does not, in itself, perturb the circuit being studied. While short-term expression is very commonly used without observable circuit disruption, whether this is also true for long-term expression is less clear. A recent paper by Miyashita et al. in Frontiers in Neural Circuits shows that long-term, high-level expression of ChR2 can induce abnormal axonal morphology and targeting in cerebral cortex. This underscores the importance of using the lowest expression possible, particularly for long-term studies.
Miyashita et al. expressed a common construct, CAG::hChR2 (H134R)-EYFP-WPRE, in L2/3 pyramidal neurons in rat somatosensory cortex via in utero electroporation (IUE). This same strategy was used in several prior studies of S1 circuit function, with one important difference: Miyashita et al. expressed hChR2 that was codon-optimized for mammalian expression, while prior studies expressed native ChR2 (discussed below). This strategy successfully conferred light-evoked spiking in vivo and in in vitro brain slices. However, long-term expression (> 40 d) also caused major abnormalities in axonal morphology, which included cylinders of axonal membrane that enveloped pyramidal cell proximal dendrites, and spherical, calyx-like axonal swellings that surrounded neuron cell bodies. These represent abnormal subcellular targets for L2/3 pyramidal cell axons, and many occurred in L4, which is an abnormal laminar target. These abnormalities increased with level and duration of ChR2 expression, and were observed in animals raised in normal room light (without specific blue light exposure). Fewer abnormalities were found using viral-mediated expression, which generally drives lower-level protein expression than IUE. However, some were still observed with >80 d expression using AAV-CaMKII-hChR2(H134R)-EYFP.
While it is not known whether these abnormalities represent functional synapses that may perturb circuit function, these results demonstrate that long-term, high-level expression of ChR2 can, under some conditions, generate structural abnormalities in cortical circuits. The axonal phenotype observed by Miyashita et al. may already be familiar to some expert laboratories. However, these results emphasize to others that it is critical to use the minimal expression level and duration possible for each experiment, and to verify the structural integrity of the transfected circuits.
An effect of ChR2 itself, or an interaction with GFP and DsRed?
In the Miyashita study, 3 separate plasmids were co-electroporated to improve fluorescent detection of transfected cells and brain regions: hChR2(H134R)-EYFP, cytosolic GFP and DsRed. To test whether expression of just ChR2-EYFP protein was sufficient to induce axonal abnormalities, the authors recently performed a follow-up experiment in which only the ChR2 construct was electroporated. Tissue was examined at 60 d of age. Dense axonal swellings were observed, including axonal cylinders and calyx-like structures, indicating that ChR2-EYFP protein itself is sufficient to induce axonal abnormalities (see Figure).
High protein expression with codon-optimized hChR2 contributed to abnormalities
It was not clear in the Miyashita paper why axonal abnormalities were not observed in an earlier study using long-term IUE of ChR2 under the same promoter (Huber et al., Nature 451: 61-64, 2008). In the text of their paper, Miyashita et al. hypothesized a possible species difference between rats (Miyashita) and mice (Huber). However, a methodological difference has been identified that likely explains this difference. Huber et al. expressed wildtype ChR2 (Chop2-315 from Nagel et al., PNAS 100: 13940-5, 2003), while Miyashita et al. expressed hChR2 that was codon-optimized for higher mammalian expression (http://www.stanford.edu/group/dlab/optogenetics/). This suggests that lower ChR2 protein levels in the Huber study enabled long-term expression without axonal malformations. This further supports Miyashita’s main conclusion that axonal abnormalities are associated with high-level, long-term expression of ChR2-EYFP protein.