We have established 4 new transgenic mouse lines with robust Cre-dependent expression of ChR2, Arch-ER2 or eNpHR3.0. Compared to a previously published R26
ChR2-EGFP Cre-reporter mouse line 20
, our Ai32 ChR2-EYFP-expressing line exhibits significantly greater light sensitivity. In the R26
ChR2-EGFP mice, using very similar stimulation paradigms in cortical slices, very long and strong laser pulses (~20 ms, ~2 mW) were needed to induce single spiking in interneurons, and even so spiking could not be induced in pyramidal neurons. The necessity to use mice with homozygous floxed-ChR2 allele and to feed the mice with retinol was also reported. In contrast, the Ai32 mice possess excitability with 1-ms light pulses at intensities as low as 30 μW, much larger photocurrents, and much shorter latency to spiking, in pyramidal neurons. We further show that pyramidal neurons and interneurons are also readily excitable in vivo
with low light in heterozygous animals. These greatly improved properties are likely to be proven critical in future in vitro
and in vivo
studies. Using such an efficient transgenic expression strategy is particularly essential for expressing the silencing opsins (Arch and eNpHR) to functional levels.
The transgenic targeting strategy used in the R26
ChR2-EGFP line 20
is similar to our strategy described here. Both targeted the Rosa26
locus and used a strong and ubiquitous CAG promoter. But, two main differences may be responsible for the improved expression level in the new lines reported here. First, our expression cassettes included a WPRE sequence, which we previously showed to enhance protein expression of Rosa26
-targeted transgenes 9
. Second, while the R26
ChR2-EGFP line incorporated a commonly-utilized floxed-stop cassette that contains the bacterial neomycin-resistant (neo) gene in front of the stop, we intentionally excluded the neo gene from our own floxed-stop cassette and instead placed it separately downstream from the transgene expression cassette. Although it is unclear whether the relocation of the neo gene is helpful to the significantly improved expression in our mice, it is well-known that integration of bacterial sequences into mammalian genomes could cause epigenetic modifications that affect expression of nearby genes.
Each of our optogenetic lines exhibited distinct photo-response properties that could be useful for different applications. Neurons from Ai32 appeared significantly more photosensitive than those from Ai27, both in vitro
and in vivo
. Although the basis for this difference between Ai27 and Ai32 neurons is unresolved, and no apparent difference in expression level or membrane localization was seen, we speculate that the bulkier tdTomato fluorescent tag in the Ai27 transgene may interfere with some channel properties. Although Ai32’s photosensitivity seems desirable for most experiments, Ai27’s red label and/or preferential axonal excitation may be advantageous in some applications 28, 30
. Our in vitro
and in vivo
data on Ai35 and Ai39 neurons suggest that Arch-ER2 produces greater photocurrents and larger hyperpolarization than eNpHR3.0 under both green and yellow light. This could be due to the observed lower protein level for eNpHR3.0 even though the mice were generated using identical designs. However, due to its broad and red-shifted activation spectrum, eNpHR3.0 is equally effective as Arch-ER2 under red light, and may be preferable for dual-channel work together with blue light-responsive depolarizing opsins.
The kinetics of the in vivo
light response of ChR2-expressing neurons in Ai27 and Ai32 mice were identical to those observed with virally expressed opsins, with a rapid increase in spiking at light onset, followed by a stable, lower steady-state firing level during light stimulation and often a period of suppression immediately following light termination 32
. Photoinhibition of Arch-ER2 or eNpHR3.0-expressing neurons in Ai35 or Ai39 mice in vivo
showed a near instantaneous reduction in firing rate, consistent with that observed with viral infection method 6
. Many of the neurons recorded in Ai35 and Ai39 mice were nearly completely silenced. The homogenous and complete silencing in these mice presents a major advantage of the consistent expression achieved in these transgenic mice compared to viral infection method 6, 24
An important question is the specificity of neuronal activity manipulation in the targeted populations. While this can be addressed by exhaustive anatomical multiple labeling methods, physiological verification of specificity is also useful. We have chosen two brain areas, the CA1 pyramidal layer and the thalamus because identification of principal cells and interneurons in these regions is possible by physiological means 35-40
. While these methods cannot distinguish among the large family of interneurons 38
, light-activated neurons displayed well-known features of short-duration spikes and high firing rates, typical of perisomatic Pvalb
+ interneurons. Conversely, putative CA1 pyramidal neurons and thalamocortical cells with bursting properties and excitatory connections were never directly activated by light. Instead, the majority of them were suppressed by light stimulation. These excellent physiological-optogentic correlations in the intact brain support the cell type specificity of ChR2 activation. They also suggest that optogenetic activation of genetically labeled cell types, especially those difficult to distinguish by physiological or other means, will enable more refined in vivo
identification and characterization of their functional properties 43
Systematic expression characterization data from our previously generated fluorescent reporter lines (e.g. Ai14), crossed to dozens of different Cre-driver lines, have shown that Cre-dependent activation of transgene expression can be obtained in nearly all neuronal types 9
. Thus, although data presented here are confined to the light responses of cortical/hippocampal pyramidal neurons and hippocampal/thalamic Pvalb
+ interneurons, we believe it highly likely that these optogenetic tools will effectively modulate activity in a wide range of neurons. It should be noted that different types of neurons may have different excitability properties for both intrinsic and/or local circuitry reasons, and hence may require individual optimization of the conditions for activation and silencing.
Here we not only provide a novel set of transgenic tools with superior properties for both stimulating and silencing neuronal activity, we also demonstrate a transgenic expression strategy having several significant conceptual advantages. First, since the opsins are expressed as single copies in an identical manner from a consistent genomic environment, reliable comparisons of in vivo
performance can be made between different opsin genes and different transgenic lines. Second, the Cre-dependent on-off switch for transgene expression effectively prevents or minimizes leaky expression in non-targeted cells while enables strong expression from the well-characterized CAG promoter in targeted cells, a clear advantage over the specific promoter-driven single transgenic approach in which the promoters used could have variable (sometimes unknown or uncharacterized) expression in both targeted and non-targeted cells. Third, because of the efforts we and others have taken to systematically characterize expression in Cre-driver lines 8, 9
, especially when similar reporter lines are used, documented and publicly available information about Cre-recombination patterns (e.g. http://connectivity.brain-map.org/transgenic/search/basic
) can advise researchers about the cell-type specificity of expression expected when using a particular Cre driver with these reporter lines. This is of great importance also considering that unintended ectopic Cre expression may exist to varied degrees in some Cre lines, and informed choice about appropriate Cre (or inducible Cre) lines for specific cell types need to be made. Finally, this proven expression system will also facilitate the rapid incorporation of newly engineered optogenetic variants, and once validated, apply them to all the Cre lines. This presents a “one-for-all” opportunity than expressing one opsin in one cell type at a time, and will further increase the range of optogenetic capabilities for investigating neural circuits and brain function.