When channelrhodopsin goes off-target: biophysical signatures of ectopic expression in neurons

The Cre-lox system allows targeted expression of optogenetic proteins such as channelrhodopsin-2 (ChR2), but can also produce unintended expression in non-target cells. In a VGLUT1-Cre::ChR2(H134R)-eYFP mouse line, we bred mice that correctly expressed ChR2 in VGLUT1 positive neurons. This was corroborated with whole cell recordings of dopaminergic (DA) neurons of the substantia nigra (SN), which elicited blue light evoked AMPA currents with a delay of a few milliseconds following the onset of light stimulation. However, at some point during breeding we discovered recurring ectopic expression of ChR2 that crept into our line. ChR2, which previously did not express in SN DA neurons, were now expressing in those neurons. These ectopically expressing DA neurons generated ChR2 currents that almost immediately followed (∼200 μs) the onset of blue light stimulation followed by a few milliseconds later with AMPA synaptic currents. While DA neurons from non-ectopic mice exhibited only synaptically mediated AMPA currents with onset latencies of a few milliseconds. In ectopic mice, the ChR2 currents in DA neurons had smaller amplitudes, more depolarized reversal potentials, and faster decay kinetics compared to canonical ChR2 responses recorded from cortical pyramidal neurons as control. Confocal imaging confirmed ChR2-YFP fluorescence within DA neurons from ectopically expressing mice, which were absent from DA neurons of non-ectopic mice. Together, these findings demonstrate that ectopically expressed ChR2 retains light sensitivity but may display altered biophysical properties that can mislead data interpretation. This work highlights the need to verify both expression fidelity and photocurrent behavior in Cre-lox based optogenetic models.