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.

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.

Objective: Transplantation of human embryonic dopaminergic progenitors within the striata of PD patients has provided encouraging results, but ethical concerns and tissue availability limit this approach. The use of mesenchymal stem cells (MSCs) provides a readily available source of cells, as they are derived from adult tissue. This in vitro study explored the use of MSCs as a cell source for DA neuronal induction utilizing a single adenovirus. Methods: Our lab developed a novel adenovirus expressing multiple viral 2A genes allowing for the polycistronic expression of multiple genes (Ascl1, Lmx1a, and Nurr1) for transcription factors that are involved in DA neuron differentiation and used the gene for green fluorescent protein (gfp) to track transfection. MSCs were cultured with the adenovirus, monitored morphological changes as well as expression of gfp as evidenced by fluorescence microscopy. The presence of the viral DNA within the transfected cells was confirmed with PCR, Immunocytochemistry and RT-PCR. Results: MSCs cultured with the adenovirus, resulted in morphological changes as well as expression of gfp as evidenced by fluorescence microscopy. The presence of the viral DNA within the transfected cells was confirmed with PCR. Immunocytochemistry and RT-PCR analyses revealed that, cells expressing gfp have nuclear co-labeling of translated transcription factors LMX1a, and NURR1, as well as an up-regulation of these genes, along with an up-regulation of downstream gene targets, such as tyrosine hydroxylase (TH), and the dopamine transporter (DAT).

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