Repeated alcohol experiences can produce long-lasting memories for sensory cues associated with intoxication. These memories can problematically trigger relapse in individuals recovering from alcohol use disorder (AUD). The molecular mechanisms by which ethanol changes memories to become long-lasting and inflexible remain unclear. New methods to analyze gene expression within precise neuronal cell types can provide further insight toward AUD prevention and treatment. Here, we used genetic tools in Drosophila melanogaster to investigate the lasting consequences of ethanol on transcription in memory-encoding neurons. Drosophila rely on mushroom body (MB) neurons to make associative memories, including memories of ethanol-associated sensory cues. Differential expression analyses revealed that distinct transcripts, but not genes, in the MB were associated with experiencing ethanol alone compared to forming a memory of an odor cue associated with ethanol. Adult MB-specific knockdown of spliceosome-associated proteins demonstrated the necessity of RNA-processing in ethanol memory formation. These findings highlight the dynamic, context-specific regulation of transcription in cue-encoding neurons, and the lasting effect of ethanol on transcript usage during memory formation.

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.

The ability to manipulate neuronal activity both opto- and chemogenetically with a single actuator molecule presents unique and flexible means to study neural circuit function. We previously developed methodology to enable such bimodal control using fusion molecules called luminopsins (LMOs), where a channelrhodopsin actuator can be activated using either physical (LED driven) or biological (bioluminescent) light. While activation of LMOs using bioluminescence has previously allowed manipulation of circuits and behavior in mice, further improvement would advance the utility of this technique. Thus, we here aimed to increase the efficiency of bioluminescent activation of channelrhodopsins by development of novel FRET-probes with bright and spectrally matched emission tailored to Volvox channelrhodopsin 1 (VChR1). We find that pairing of a molecularly evolved Oplophorus luciferase variant with mNeonGreen significantly improves the efficacy of bioluminescent activation when tethered to VChR1 (construct named LMO7) as compared to previous and other newly generated LMO variants. We proceed to extensively benchmark LMO7 against previous LMO standard (LMO3) and find that LMO7 outperforms LMO3 in the ability to drive bioluminescent activation of VChR1 both in vitro and in vivo, and efficiently modulates animal behavior following intraperitonial injection of fluorofurimazine. In conclusion, we demonstrate a rationale for improving bioluminescent activation of optogenetic actuators using a tailored molecular engineering approach and provide a new tool to bimodally manipulate neuronal activity with increased bioluminescence-driven efficacy.

We developed bioluminescent probes to image neural activity and harness biological light to serve as a multifunctional optogenetic actuator. This platform technology will enable noninvasive interrogation of activated circuits underlying behavior.