Bioluminescent optogenetic (BL-OG) activation of neurons during mouse postnatal brain development.

Bioluminescent optogenetics (BL-OG) allows activation of photosensory proteins, such as opsins, by either fiberoptics or by administering a luciferin. BL-OG thus confers both optogenetic and chemogenetic access within the same genetically targeted neuron. This bimodality offers a powerful approach for non-invasive chemogenetic manipulation of neural activity during brain development and adult behaviors with standard optogenetic spatiotemporal precision. We detail protocols for bioluminescent stimulation of neurons in postnatally developing brain and its validation through bioluminescence imaging and electrophysiological recording in mice. For complete information on the use and execution of this protocol, please refer to Medendorp et al. (2021).

Selective postnatal excitation of neocortical pyramidal neurons results in distinctive behavioral and circuit deficits in adulthood.

In genetic and pharmacological models of neurodevelopmental disorders, and human data, neural activity is altered within the developing neocortical network. This commonality begs the question of whether early enhancement in excitation might be a common driver, across etiologies, of characteristic behaviors. We tested this concept by chemogenetically driving cortical pyramidal neurons during postnatal days 4-14. Hyperexcitation of Emx1-, but not dopamine transporter-, parvalbumin-, or Dlx5/6-expressing neurons, led to decreased social interaction and increased grooming activity in adult animals. In vivo optogenetic interrogation in adults revealed decreased baseline but increased stimulus-evoked firing rates of pyramidal neurons and impaired recruitment of inhibitory neurons. Slice recordings in adults from prefrontal cortex layer 5 pyramidal neurons revealed decreased intrinsic excitability and increased synaptic E/I ratio. Together these results support the prediction that enhanced pyramidal firing during development, in otherwise normal cortex, can selectively drive altered adult circuit function and maladaptive changes in behavior.

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.

Altered Behavior in Mice Socially Isolated During Adolescence Corresponds With Immature Dendritic Spine Morphology and Impaired Plasticity in the Prefrontal Cortex.

Mice socially isolated during adolescence exhibit behaviors of anxiety, depression and impaired social interaction. Although these behaviors are well documented, very little is known about the associated neurobiological changes that accompany these behaviors. It has been hypothesized that social isolation during adolescence alters the development of the prefrontal cortex, based on similar behavioral abnormalities observed in isolated mice and those with disruption of this structure. To establish relationships between behavior and underlying neurobiological changes in the prefrontal cortex, Thy-1-GFP mice were isolated from weaning until adulthood and compared to group-housed littermates regarding behavior, electrophysiological activity and dendritic morphology. Results indicate an immaturity of dendritic spines in single housed animals, with dendritic spines appearing smaller and thinner. Single housed mice additionally show impaired plasticity through measures of long-term potentiation. Together these findings suggest an altered development and impairment of the prefrontal cortex of these animals underlying their behavioral characteristics.