Improved Locomotor Recovery in a Rat Model of Spinal Cord Injury by BioLuminescent-OptoGenetic (BL-OG) Stimulation with an Enhanced Luminopsin.

Irrespective of the many strategies focused on dealing with spinal cord injury (SCI), there is still no way to restore motor function efficiently or an adequate regenerative therapy. One promising method that could potentially prove highly beneficial for rehabilitation in patients is to re-engage specific neuronal populations of the spinal cord following SCI. Targeted activation may maintain and strengthen existing neuronal connections and/or facilitate the reorganization and development of new connections. BioLuminescent-OptoGenetics (BL-OG) presents an avenue to non-invasively and specifically stimulate neurons; genetically targeted neurons express luminopsins (LMOs), light-emitting luciferases tethered to light-sensitive channelrhodopsins that are activated by adding the luciferase substrate coelenterazine (CTZ). This approach employs ion channels for current conduction while activating the channels through treatment with the small molecule CTZ, thus allowing non-invasive stimulation of all targeted neurons. We previously showed the efficacy of this approach for improving locomotor recovery following severe spinal cord contusion injury in rats expressing the excitatory luminopsin 3 (LMO3) under control of a pan-neuronal and motor-neuron-specific promoter with CTZ applied through a lateral ventricle cannula. The goal of the present study was to test a new generation of LMOs based on opsins with higher light sensitivity which will allow for peripheral delivery of the CTZ. In this construct, the slow-burn Gaussia luciferase variant (sbGLuc) is fused to the opsin CheRiff, creating LMO3.2. Taking advantage of the high light sensitivity of this opsin, we stimulated transduced lumbar neurons after thoracic SCI by intraperitoneal application of CTZ, allowing for a less invasive treatment. The efficacy of this non-invasive BioLuminescent-OptoGenetic approach was confirmed by improved locomotor function. This study demonstrates that peripheral delivery of the luciferin CTZ can be used to activate LMOs expressed in spinal cord neurons that employ an opsin with increased light sensitivity.

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.

The BioLuminescent-OptoGenetic in vivo response to coelenterazine is proportional, sensitive, and specific in neocortex.

BioLuminescent (BL) light production can modulate neural activity and behavior through co-expressed OptoGenetic (OG) elements, an approach termed "BL-OG." Yet, the relationship between BL-OG effects and bioluminescent photon emission has not been characterized in vivo. Further, the degree to which BL-OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and nontoxic effects. We systematically tested the effects of four key components of the BL-OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin-3 (LMO3) BL-OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin-1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically nonsensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortex in vivo, there is a large CTZ range wherein BL-OG effects are specific to its active chemogenetic mechanism.

Step-function luminopsins for bimodal prolonged neuromodulation.

Although molecular tools for controlling neuronal activity by light have vastly expanded, there are still unmet needs which require development and refinement. For example, light delivery into the brain is still a major practical challenge that hinders potential translation of optogenetics in human patients. In addition, it would be advantageous to manipulate neuronal activity acutely and precisely as well as chronically and non-invasively, using the same genetic construct in animal models. We have previously addressed these challenges by employing bioluminescence and have created a new line of opto-chemogenetic probes termed luminopsins by fusing light-sensing opsins with light-emitting luciferases. In this report, we incorporated Chlamydomonas channelrhodopsin 2 with step-function mutations as the opsin moiety in the new luminopsin fusion protein termed step-function luminopsin (SFLMO). Bioluminescence-induced photocurrent lasted longer than the bioluminescence signal due to very slow deactivation of the mutated channel. In addition, bioluminescence was able to activate most of the channels on the cell surface due to the extremely high light sensitivity of the channel. This efficient channel activation was partly mediated by radiationless bioluminescence resonance energy transfer due to the proximity of luciferase and opsin. To test the utility of SFLMOs in vivo, we transduced the substantia nigra unilaterally via a viral vector in male rats. Injection of the luciferase substrate as well as conventional photostimulation via fiber optics elicited circling behaviors. Thus, SFLMOs expand the current approaches for manipulation of neuronal activity in the brain and add more versatility and practicality to optogenetics in freely behaving animals.

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.

Novel luciferase-opsin combinations for improved luminopsins.

Previous work has demonstrated that fusion of a luciferase to an opsin, to create a luminescent opsin or luminopsin, provides a genetically encoded means of manipulating neuronal activity via both chemogenetic and optogenetic approaches. Here we have expanded and refined the versatility of luminopsin tools by fusing an alternative luciferase variant with high light emission, Gaussia luciferase mutant GLucM23, to depolarizing and hyperpolarizing channelrhodopsins with increased light sensitivity. The combination of GLucM23 with Volvox channelrhodopsin-1 produced LMO4, while combining GLucM23 with the anion channelrhodopsin iChloC yielded iLMO4. We found efficient activation of these channelrhodopsins in the presence of the luciferase substrate, as indicated by responses measured in both single neurons and in neuronal populations of mice and rats, as well as by changes in male rat behavior during amphetamine-induced rotations. We conclude that these new luminopsins will be useful for bimodal opto- and chemogenetic analyses of brain function.

Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation.

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.

Novel E815K knock-in mouse model of alternating hemiplegia of childhood.

De novo mutations causing dysfunction of the ATP1A3 gene, which encodes the α3 subunit of Na+/K+-ATPase pump expressed in neurons, result in alternating hemiplegia of childhood (AHC). AHC manifests as paroxysmal episodes of hemiplegia, dystonia, behavioral abnormalities, and seizures. The first aim of this study was to characterize a novel knock-in mouse model (Atp1a3E815K+/-, Matoub, Matb+/-) containing the E815K mutation of the Atp1a3 gene recognized as causing the most severe and second most common phenotype of AHC with increased morbidity and mortality as compared to other mutations. The second aim was to investigate the effects of flunarizine, currently the most effective drug used in AHC, to further validate our model and to help address a question with significant clinical implications that has not been addressed in prior studies. Specifically, many E815K patients have clinical decompensation and catastrophic regression after discontinuing flunarizine therapy; however, it is not known whether this is congruent with the natural course of the disease and is a result of withdrawal from an acute beneficial effect, withdrawal from a long-term protective effect or from a detrimental effect of prior flunarizine exposure. Our behavioral and neurophysiological testing demonstrated that Matb+/- mice express a phenotype that bears a strong resemblance to the E815K phenotype in AHC. In addition, these mice developed spontaneous seizures with high incidence of mortality and required fewer electrical stimulations to reach the kindled state as compared to wild-type littermates. Matb+/- mice treated acutely with flunarizine had reduction in hemiplegic attacks as compared with vehicle-treated mice. After withdrawal of flunarizine, Matb+/- mice that had received flunarizine did neither better nor worse, on behavioral tests, than those who had received vehicle. We conclude that: 1) Our mouse model containing the E815K mutation manifests clinical and neurophysiological features of the most severe form of AHC, 2) Flunarizine demonstrated acute anti-hemiplegic effects but not long-term beneficial or detrimental behavioral effects after it was stopped, and 3) The Matb+/- mouse model can be used to investigate the underlying pathophysiology of ATP1A3 dysfunction and the efficacy of potential treatments for AHC.

Effects of Passage Number and Differentiation Protocol on the Generation of Dopaminergic Neurons from Rat Bone Marrow-Derived Mesenchymal Stem Cells.

Multiple studies have demonstrated the ability of mesenchymal stem cells (MSCs) to differentiate into dopamine-producing cells, in vitro and in vivo, indicating their potential to be used in the treatment of Parkinson's disease (PD). However, there are discrepancies among studies regarding the optimal time (i.e., passage number) and method for dopaminergic induction, in vitro. In the current study, we compared the ability of early (P4) and later (P40) passaged bone marrow-derived MSCs to differentiate into dopaminergic neurons using two growth-factor-based approaches. A direct dopaminergic induction (DDI) was used to directly convert MSCs into dopaminergic neurons, and an indirect dopaminergic induction (IDI) was used to direct MSCs toward a neuronal lineage prior to terminal dopaminergic differentiation. Results indicate that both early and later passaged MSCs exhibited positive expression of neuronal and dopaminergic markers following either the DDI or IDI protocols. Additionally, both early and later passaged MSCs released dopamine and exhibited spontaneous neuronal activity following either the DDI or IDI. Still, P4 MSCs exhibited significantly higher spiking and bursting frequencies as compared to P40 MSCs. Findings from this study provide evidence that early passaged MSCs, which have undergone the DDI, are more efficient at generating dopaminergic-like cells in vitro, as compared to later passaged MSCs or MSCs that have undergone the IDI.

Cre-dependent DREADD (Designer Receptors Exclusively Activated by Designer Drugs) mice.

DREADDs, designer receptors exclusively activated by designer drugs, are engineered G protein-coupled receptors (GPCR) which can precisely control GPCR signaling pathways (for example, Gq, Gs, and Gi). This chemogenetic technology for control of GPCR signaling has been successfully applied in a variety of in vivo studies, including in mice, to remotely control GPCR signaling, for example, in neurons, glia cells, pancreatic ß-cells, or cancer cells. In order to fully explore the in vivo applications of the DREADD technology, we generated hM3Dq and hM4Di strains of mice which allow for Cre recombinase-mediated restricted expression of these pathway-selective DREADDs. With the many Cre driver lines now available, these DREADD lines will be applicable to studying a wide array of research and preclinical questions. genesis 54:439-446, 2016. © 2016 Wiley Periodicals, Inc.

Mouse model of rare TOR1A variant found in sporadic focal dystonia impairs domains affected in DYT1 dystonia patients and animal models.

Rare de novo mutations in genes associated with inherited Mendelian disorders are potential contributors to sporadic disease. DYT1 dystonia is an autosomal dominant, early-onset, generalized dystonia associated with an in-frame, trinucleotide deletion (n. delGAG, p. ΔE 302/303) in the Tor1a gene. Here we examine the significance of a rare missense variant in the Tor1a gene (c. 613T>A, p. F205I), previously identified in a patient with sporadic late-onset focal dystonia, by modeling it in mice. Homozygous F205I mice have motor impairment, reduced steady-state levels of TorsinA, altered corticostriatal synaptic plasticity, and prominent brain imaging abnormalities in areas associated with motor function. Thus, the F205I variant causes abnormalities in domains affected in people and/or mouse models with the DYT1 Tor1a mutation (ΔE). Our findings establish the pathological significance of the F205I Tor1a variant and provide a model with both etiological and phenotypic relevance to further investigate dystonia mechanisms.

Knock-in mouse model of alternating hemiplegia of childhood: behavioral and electrophysiologic characterization.

OBJECTIVES: Mutations in the ATP1α3 subunit of the neuronal Na+/K+-ATPase are thought to be responsible for seizures, hemiplegias, and other symptoms of alternating hemiplegia of childhood (AHC). However, the mechanisms through which ATP1A3 mutations mediate their pathophysiologic consequences are not yet understood. The following hypotheses were investigated: (1) Our novel knock-in mouse carrying the most common heterozygous mutation causing AHC (D801N) will exhibit the manifestations of the human condition and display predisposition to seizures; and (2) the underlying pathophysiology in this mouse model involves increased excitability in response to electrical stimulation of Schaffer collaterals and abnormal predisposition to spreading depression (SD). METHODS: We generated the D801N mutant mouse (Mashlool, Mashl+/-) and compared mutant and wild-type (WT) littermates. Behavioral tests, amygdala kindling, flurothyl-induced seizure threshold, spontaneous recurrent seizures (SRS), and other paroxysmal activities were compared between groups. In vitro electrophysiologic slice experiments on hippocampus were performed to assess predisposition to hyperexcitability and SD. RESULTS: Mutant mice manifested a distinctive phenotype similar to that of humans with AHC. They had abnormal impulsivity, memory, gait, motor coordination, tremor, motor control, endogenous nociceptive response, paroxysmal hemiplegias, diplegias, dystonias, and SRS, as well as predisposition to kindling, to flurothyl-induced seizures, and to sudden unexpected death. Hippocampal slices of mutants, in contrast to WT animals, showed hyperexcitable responses to 1 Hz pulse-trains of electrical stimuli delivered to the Schaffer collaterals and had significantly longer duration of K+-induced SD responses. SIGNIFICANCE: Our model reproduces the major characteristics of human AHC, and indicates that ATP1α3 dysfunction results in abnormal short-term plasticity with increased excitability (potential mechanism for seizures) and a predisposition to more severe SD responses (potential mechanism for hemiplegias). This model of the human condition should help in understanding the molecular pathways underlying these phenotypes and may lead to identification of novel therapeutic strategies of ATP1α3 related disorders and seizures.

Special Section Guest Editorial: Molecular Neurophotonics.

The editorial introduces the Special Section on Molecular Neurophotonics.

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.

Engineering luminopsins with improved coupling efficiencies.

Significance: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. Aim: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. Approach: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array recordings in primary neurons. Results: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Conclusions: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer, the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

Behavioral context improves optogenetic stimulation of transplanted dopaminergic cells in unilateral 6-OHDA rats.

Stem cell therapy has long been a popular method of treatment for Parkinson's disease currently being researched in both preclinical and clinical settings. While early clinical results are based upon fetal tissue transplants rather than stem cell transplants, the lack of successful integration in some patients and gradual loss of effect in others suggests a more robust protocol is needed. We propose a two-front approach, one where transplants are directly stimulated in coordination with host activity elicited by behavioral tasks, which we refer to as behavioral context. After a pilot with unilateral 6-OHDA rats transplanted with dopaminergic cells differentiated from mesenchymal stem cells that were optogenetically stimulated during a swim task, we discovered that early stimulation predicted lasting reduction of motor deficits, even in the absence of later stimulation. This led to a follow-up with n = 21 rats split into three groups: one stimulated while performing a swim task (Stim-Swim; St-Sw), one not stimulated while swimming (NoStim-Swim; NSt-Sw), and one stimulated while stationary in a bowl (Stim-NoSwim; St-NSw). After initial stimulation (or lack thereof), all rats were retested two and seven days later with the swim task in the absence of stimulation. The St-Sw group gradually achieved and maintained symmetrical limb use, whereas the NSt-Sw group showed persistent asymmetry and the St-NSw group showed mixed results. This supports the notion that stem cell therapy should integrate targeted stimulation of the transplant with behavioral stimulation of the host tissue to encourage proper functional integration of the graft.

A New Highly Efficient Molecule for Both Optogenetic and Chemogenetic Control Driven by FRET Amplification of 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 Forster resonance energy transfer (FRET) 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.

A Bioluminescent Activity Dependent (BLADe) Platform for Converting Neuronal Activity to Photoreceptor Activation.

We developed a platform that utilizes a calcium-dependent luciferase to convert neuronal activity into activation of light sensing domains within the same cell. The platform is based on a Gaussia luciferase variant with high light emission split by calmodulin-M13 sequences that depends on influx of calcium ions (Ca2+) for functional reconstitution. In the presence of its luciferin, coelenterazine (CTZ), Ca2+ influx results in light emission that drives activation of photoreceptors, including optogenetic channels and LOV domains. Critical features of the converter luciferase are light emission low enough to not activate photoreceptors under baseline condition and high enough to activate photosensing elements in the presence of Ca2+ and luciferin. We demonstrate performance of this activity-dependent sensor and integrator for changing membrane potential and driving transcription in individual and populations of neurons in vitro and in vivo.

Bioluminescent Genetically Encoded Glutamate Indicators for Molecular Imaging of Neuronal Activity.

Genetically encoded optical sensors and advancements in microscopy instrumentation and techniques have revolutionized the scientific toolbox available for probing complex biological processes such as release of specific neurotransmitters. Most genetically encoded optical sensors currently used are based on fluorescence and have been highly successful tools for single-cell imaging in superficial brain regions. However, there remains a need to develop new tools for reporting neuronal activity in vivo within deeper structures without the need for hardware such as lenses or fibers to be implanted within the brain. Our approach to this problem is to replace the fluorescent elements of the existing biosensors with bioluminescent elements. This eliminates the need of external light sources to illuminate the sensor, thus allowing deeper brain regions to be imaged noninvasively. Here, we report the development of the first genetically encoded neurotransmitter indicators based on bioluminescent light emission. These probes were optimized by high-throughput screening of linker libraries. The selected probes exhibit robust changes in light output in response to the extracellular presence of the excitatory neurotransmitter glutamate. We expect this new approach to neurotransmitter indicator design to enable the engineering of specific bioluminescent probes for multiple additional neurotransmitters in the future, ultimately allowing neuroscientists to monitor activity associated with a specific neurotransmitter as it relates to behavior in a variety of neuronal and psychiatric disorders, among many other applications.

Engineering luminopsins with improved coupling efficiencies.

Significance: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. Aim: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. Approach: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array (MEAs) recordings in primary neurons. Results: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Conclusions: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer (FRET), the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.