RESUMEN
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.
Asunto(s)
Channelrhodopsins , Luciferasas , Neuronas/fisiología , Optogenética/métodos , Potenciales de Acción , Adenoviridae/fisiología , Animales , Channelrhodopsins/genética , Channelrhodopsins/fisiología , Femenino , Vectores Genéticos , Células HEK293 , Hipocampo/fisiología , Humanos , Luciferasas/genética , Luciferasas/fisiología , Masculino , Ratones , Cultivo Primario de Células , Ratas Sprague-Dawley , Volvox/genéticaRESUMEN
BACKGROUND: Putative treatments derived from in vivo stem cell transplant-derived dopamine (DA) in hemiparkinsonian rats have been assessed via DA-agonist-induced rotations involving imbalanced intra-hemispheric striatal DA receptor stimulation. However, such tests obscure the natural responses of grafts to sensory stimuli, and drug-induced plasticity can modify the circuit being tested. Thus, we propose an alternative testing strategy using a novel water tank swimming apparatus. NEW METHOD: Microdialysis was used to compare striatal DA levels when rats were: (1) in a rest-phase within a bowl-shaped apparatus, or (2) in an active forced-swim phase within a specially-equipped water tank. Resting-phase DA release levels were compared with active-phase levels obtained while rats were required to swim in the water-tank task. Behavioral variables such as asymmetric circling while swimming (rotations), front-limb strokes, and front-limb reaches were captured by a camera for analysis. RESULTS AND COMPARISON WITH EXISTING METHODS: Transplanted cells had a very modest effect on percentage of contralateral front-limb strokes, but did not reduce lesion-induced rotational asymmetry in the swim task. Neither striatal DA levels, nor their breakdown products, were significantly different between transplanted and sham-transplanted groups. Our new behavioral test eliminates the need for pharmacological stimulation, enabling simultaneous assessment of DA released in resting and active phases to explore graft control. CONCLUSIONS: Our new method allows for accurate assessments of stem cell therapy for PD as an alternative to "rotation" tests. Use of natural motivations to engage in sensory-driven motor tasks provides more accurate insights into ongoing graft-derived behavioral support.