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Luminopsins are fusion proteins of luciferase and opsin that allow interrogation of neuronal circuits at different temporal and spatial resolutions by choosing either extrinsic physical or intrinsic biological light for its activation. Building on previous development of fusions of wild-type Gaussia luciferase with channelrhodopsin, here we expanded the utility of luminopsins by fusing bright Gaussia luciferase variants with either channelrhodopsin to excite neurons (luminescent opsin, LMO) or a proton pump to inhibit neurons (inhibitory LMO, iLMO). These improved LMOs could reliably activate or silence neurons in vitro and in vivo. Expression of the improved LMO in hippocampal circuits not only enabled mapping of synaptic activation of CA1 neurons with fine spatiotemporal resolution but also could drive rhythmic circuit excitation over a large spatiotemporal scale. Furthermore, virus-mediated expression of either LMO or iLMO in the substantia nigra in vivo produced not only the expected bidirectional control of single unit activity but also opposing effects on circling behavior in response to systemic injection of a luciferase substrate. Thus, although preserving the ability to be activated by external light sources, LMOs expand the use of optogenetics by making the same opsins accessible to noninvasive, chemogenetic control, thereby allowing the same probe to manipulate neuronal activity over a range of spatial and temporal scales.

Evolutionary transitions in individuality (ETIs) underlie the watershed events in the history of life on Earth, including the origins of cells, eukaryotes, plants, animals, and fungi. Each of these events constitutes an increase in the level of complexity, as groups of individuals become individuals in their own right. Among the best-studied ETIs is the origin of multicellularity in the green alga Volvox, a model system for the evolution of multicellularity and cellular differentiation. Since its divergence from unicellular ancestors, Volvox has evolved into a highly integrated multicellular organism with cellular specialization, a complex developmental program, and a high degree of coordination among cells. Remarkably, all of these changes were previously thought to have occurred in the last 50-75 million years. Here we estimate divergence times using a multigene data set with multiple fossil calibrations and use these estimates to infer the times of developmental changes relevant to the evolution of multicellularity. Our results show that Volvox diverged from unicellular ancestors at least 200 million years ago. Two key innovations resulting from an early cycle of cooperation, conflict and conflict mediation led to a rapid integration and radiation of multicellular forms in this group. This is the only ETI for which a detailed timeline has been established, but multilevel selection theory predicts that similar changes must have occurred during other ETIs.

Background The multicellular green alga Volvox carteri makes use of none less than 13 photoreceptors, which are mostly expressed in a cell-type specific manner. This gives reason to believe that trasncriptome pattern of each cell type could change differentially in response to environmental light. Here, the cell-type specific changes of various transcripts from different pathways in response to blue, red and far-red light were analyzed. Results In response to different light qualities, distinct changes in transcript accumulation of genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, circadian clock and cell cycle control were observed. Namely, blue light tends to be effective to accumulate transcripts in the somatic cells; while red light leads to accumulate transcripts predominantly in the reproductive cells. Blue light also induced marked accumulation of two components of circadian rhythms only in the somatic cells, indicating that these clock-relevant components are affected by blue light in a cell-type specific manner. Further, we show that photosynthetic associated genes are regulated distinctly among cell types by different light qualities. Conclusion Our results suggest that Volvox uses different sophisticated cell-type specific light signaling pathways to modulate expression of genes involved in various cellular and metabolic pathways including circadian rhythms and photosynthesis in response to environmental light.

The introduction of two microbial opsin–based tools, channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), to neuroscience has generated interest in fast, multimodal, cell type–specific neural circuit control. Here we describe a cation-conducting channelrhodopsin (VChR1) from Volvox carteri that can drive spiking at 589 nm, with excitation maximum red-shifted ∼70 nm compared with ChR2. These results demonstrate fast photostimulation with yellow light, thereby defining a functionally distinct third category of microbial rhodopsin proteins.

The C. elegans nervous system is particularly well suited for optogenetic analyses of circuit function: Essentially all connections have been mapped, and light can be directed at the neuron of interest in the freely moving, transparent animals, while behavior is observed. Thus, different nodes of a neuronal network can be probed for their role in controlling a particular behavior, using different optogenetic tools for photo-activation or -inhibition, which respond to different colors of light. As neurons may act in concert or in opposing ways to affect a behavior, one would further like to excite these neurons concomitantly, yet independent of each other. In addition to the blue-light activated Channelrhodopsin-2 (ChR2), spectrally red-shifted ChR variants have been explored recently. Here, we establish the green-light activated ChR chimera C1V1 (from Chlamydomonas and Volvox ChR1's) for use in C. elegans. We surveyed a number of red-shifted ChRs, and found that C1V1-ET/ET (E122T; E162T) works most reliable in C. elegans, with 540-580 nm excitation, which leaves ChR2 silent. However, as C1V1-ET/ET is very light sensitive, it still becomes activated when ChR2 is stimulated, even at 400 nm. Thus, we generated a highly efficient blue ChR2, the H134R; T159C double mutant (ChR2-HR/TC). Both proteins can be used in the same animal, in different neurons, to independently control each cell type with light, enabling a further level of complexity in circuit analyses.

Photoreceptor currents of the multicellular green alga Volvox carteri were analyzed using a dissolver mutant. The photocurrents are restricted to the eyespot region of somatic cells. Photocurrents are detectable from intact cells and excised eyes. The rhodopsin action spectrum suggests that the currents are induced by Volvox rhodopsin. Flash-induced photocurrents are a composition of a fast Ca 2+-carried current ( P F) and a slower current ( P S), which is carried by H +. P F is a high-intensity response that appears with a delay of less than 50 μs after flash. The stimulus-response curve of its initial rise is fit by a single exponential and parallels the rhodopsin bleaching. These two observations suggest that the responsible channel is closely connected to the rhodopsin, both forming a tight complex. At low flash energies P S is dominating. The current delay increases up to 10 ms, and the P S amplitude saturates when only a few percent of the rhodopsin is bleached. The data are in favor of a second signaling system, which includes a signal transducer mediating between rhodopsin and the channel. We present a model of how different modes of signal transduction are accomplished in this alga under different light conditions.