Defining parameters of specificity for bioluminescent optogenetic activation of neurons using in vitro multi electrode arrays (MEA).

In Bioluminescent Optogenetics (BL-OG) a biological, rather than a physical, light source is used to activate light-sensing opsins, such as channelrhodopsins or pumps. This is commonly achieved by utilizing a luminopsin (LMO), a fusion protein of a light-emitting luciferase tethered to a light-sensing opsin. Light of the wavelength matching the activation peak of the opsin is emitted by the luciferase upon application of its small molecule luciferin, resulting in activation of the fused opsin and subsequent effects on membrane potential. Using optimized protocols for culturing, transforming, and testing primary neurons in multi electrode arrays, we systematically defined parameters under which changes in neuronal activity are specific to bioluminescent activation of opsins, rather than due to off-target effects of either the luciferin or its solvent on neurons directly, or on opsins directly. We further tested if there is a direct effect of bioluminescence on neurons. Critical for assuring specific BL-OG effects are testing the concentration and formulation of the luciferin against proper controls, including testing effects of vehicle on LMO expressing and of luciferin on nonLMO expressing targets.

Bioluminescence-driven optogenetic activation of transplanted neural precursor cells improves motor deficits in a Parkinson's disease mouse model.

The need to develop efficient therapies for neurodegenerative diseases is urgent, especially given the increasing percentages of the population living longer, with increasing chances of being afflicted with conditions like Parkinson's disease (PD). A promising curative approach toward PD and other neurodegenerative diseases is the transplantation of stem cells to halt and potentially reverse neuronal degeneration. However, stem cell therapy does not consistently lead to improvement for patients. Using remote stimulation to optogenetically activate transplanted cells, we attempted to improve behavioral outcomes of stem cell transplantation. We generated a neuronal precursor cell line expressing luminopsin 3 (LMO3), a luciferase-channelrhodopsin fusion protein, which responds to the luciferase substrate coelenterazine (CTZ) with emission of blue light that in turn activates the opsin. Neuronal precursor cells were injected bilaterally into the striatum of homozygous aphakia mice, which carry a spontaneous mutation leading to lack of dopaminergic neurons and symptoms of PD. Following transplantation, the cells were stimulated over a period of 10 days by intraventricular injections of CTZ. Mice receiving CTZ demonstrated significantly improved motor skills in a rotarod test compared to mice receiving vehicle. Thus, bioluminescent optogenetic stimulation of transplanted neuronal precursor cells shows promising effects in improving locomotor behavior in the aphakia PD mouse model and encourages further studies to elucidate the mechanisms and long-term outcomes of these beneficial effects.

Motoneuron activity is required for enhancements in functional recovery after peripheral nerve injury in exercised female mice.

Inhibitory luminopsins (iLMO2) integrate opto- and chemo-genetic approaches and allow for cell-type specific inhibition of neuronal activity. When exposed to a Renilla luciferase substrate, Coelenterazine (CTZ), iLMO2 generates bioluminescence-mediated activation of its amino-terminal halorhodopsin, resulting in neuronal inhibition. Moderate daily exercise in the form of interval treadmill-training (IT) applied following a peripheral nerve injury results in enhanced motor axon regeneration and muscle fiber reinnervation in female mice. We hypothesized that iLMO2 mediated inhibition of motoneuron activity during IT would block this enhancement. Unilateral intramuscular injections of Cre-dependent AAV2/9-EF1a-DIO-iLMO2 (∼8.5 x 1013 vg/ml) were made into the gastrocnemius and tibialis anterior muscles of young female ChAT-IRES-Cre mice, thereby limiting iLMO2 expression specifically to their motoneurons. Four to six weeks were allowed for retrograde viral transduction after which a unilateral sciatic nerve transection (Tx) and repair was performed. Animals were randomized into four groups: IT only, IT + CTZ, CTZ only, and untreated (UT). Three weeks post Tx-repair, the maximal amplitude direct muscle responses (M-max) in both muscles in the IT only group were significantly greater than in UT mice, consistent with the enhancing effects of this exercise regimen. Inhibiting motoneuron activity during exercise by a single injection of CTZ, administered 30 minutes prior to exercise, completely blocked the enhancing effect of exercise. Similar treatments with CTZ in mice without iLMO2 had no effect on regeneration. Neuronal activity is required for successful enhancement of motor axon regeneration by exercise.

Chemically activated luminopsins allow optogenetic inhibition of distributed nodes in an epileptic network for non-invasive and multi-site suppression of seizure activity.

Although optogenetic techniques have proven to be invaluable for manipulating and understanding complex neural dynamics over the past decade, they still face practical and translational challenges in targeting networks involving multiple, large, or difficult-to-illuminate areas of the brain. We utilized inhibitory luminopsins to simultaneously inhibit the dentate gyrus and anterior nucleus of the thalamus of the rat brain in a hardware-independent and cell-type specific manner. This approach was more effective at suppressing behavioral seizures than inhibition of the individual structures in a rat model of epilepsy. In addition to elucidating mechanisms of seizure suppression never directly demonstrated before, this work also illustrates how precise multi-focal control of pathological circuits can be advantageous for the treatment and understanding of disorders involving broad neural circuits such as epilepsy.

Efficient opto- and chemogenetic control in a single molecule driven by FRET-modified bioluminescence.

Significance: Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). Aim: To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. Approach: We developed novel luciferases optimized for Förster resonance energy transfer when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). Results: A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard) and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. Conclusions: We report a robust new option for achieving multiple modes of control in a single actuator and a promising engineering strategy for continued improvement of BL-OG.

Bioluminescence-Driven Optogenetics.

Bioluminescence-based technologies are among the most commonly used methods to quantify and visualise physiology at the cellular and organismal levels. However, the potential of bioluminescence beyond reporter technologies remains largely unexplored. Here, we provide an overview of the emerging approaches employing bioluminescence as a biological light source that triggers physiological events and controls cell behaviour and discuss its possible future application in synthetic biology.

Combined Optogenetic and Chemogenetic Control of Neurons.

Optogenetics provides an array of elements for specific biophysical control, while designer chemogenetic receptors provide a minimally invasive method to control circuits in vivo by peripheral injection. We developed a strategy for selective regulation of activity in specific cells that integrates opto- and chemogenetic approaches, and thus allows manipulation of neuronal activity over a range of spatial and temporal scales in the same experimental animal. Light-sensing molecules (opsins) are activated by biologically produced light through luciferases upon peripheral injection of a small molecule substrate. Such luminescent opsins, luminopsins, allow conventional fiber optic use of optogenetic sensors, while at the same time providing chemogenetic access to the same sensors. We describe applications of this approach in cultured neurons in vitro, in brain slices ex vivo, and in awake and anesthetized animals in vivo.