The ability to screen for agents that can promote the development and/or maintenance of neuronal networks creates opportunities for the discovery of novel agents for the treatment of central nervous system (CNS) disorders. Over the past 10 years, advances in robotics, artificial intelligence, and machine learning have paved the way for the improved implementation of live-cell imaging systems for drug discovery. These instruments have revolutionized our ability to quickly and accurately acquire large standardized datasets when studying complex cellular phenomena in real-time. This is particularly useful in the field of neuroscience because real-time analysis can allow efficient monitoring of the development, maturation, and conservation of neuronal networks by measuring neurite length. Unfortunately, due to the relative infancy of this type of analysis, standard practices for data acquisition and processing are lacking, and there is no standardized format for reporting the vast quantities of data generated by live-cell imaging systems. This paper reviews the current state of live-cell imaging instruments, with a focus on the most commonly used equipment (IncuCyte systems). We provide an in-depth analysis of the experimental conditions reported in publications utilizing these systems, particularly with regard to studying neurite outgrowth. This analysis sheds light on trends and patterns that will enhance the use of live-cell imaging instruments in CNS drug discovery.

Translation: The University of Toledo Journal of Medical Sciences is the online journal launched by the University of Toledo. Manuscripts will be considered on the understanding that they report original work and are not under consideration for publication by any other journal. The journal publishes original articles reporting experimental results of basic or clinical research, case reports, and reviews. The journal uses a single blind peer review system and each manuscript, based on the results presented in its original submission, will be evaluated by two student reviewers and one faculty reviewer. This process will provide an opportunity for medical students, graduate students, residents, fellows and faculty to publish research observation in a timely manner.

Nitric oxide (NO) plays a critical role in neuronal function and synaptic plasticity. Molecules capable of enhancing NO signaling have shown promise as potential therapeutics for neurodegenerative disorders. We are engaged in the development of a novel class of NO mimetics known as furoxans (1,2,5 oxadiazole-N-oxides). Furoxans are commonly described as thiol-dependent NO mimetics, which must react with biological thiols to exhibit NO bioactivity. We have created a series of attenuated furoxans, which do not readily react with generic cellular thiols, such as glutathione or L-cysteine. Nevertheless, our furoxans display neuroprotection in cell culture and the ability to reverse scopolamine induced cognition deficits in a model of contextual fear memory. We have demonstrated that furoxan neuroprotection is reversed upon blockade of NO/sGC (soluble guanylyl cyclase) signaling. We hypothesize our attenuated furoxans react with a subset of yet to be identified relatively reactive protein thiols. The work presented focuses on the synthesis and characterization of novel furoxan probes containing, 1) a photoaffinity labeling group, either a diazirine or perfluoroarylazide, for transient covalent modification of interacting proteins; and 2) a terminal alkyne for conjugation with a fluorophore and subsequent visualization of protein modification by SDS-PAGE, to elucidate this hypothesis. We have observed distinct advantages and disadvantages of each photoaffinity labeling moiety with regards to stability and rate of photolysis. Efforts are ongoing to identify furoxan target proteins in primary cell culture.

Translation: The University of Toledo Journal of Medical Sciences is the online journal launched by the University of Toledo. Manuscripts will be considered on the understanding that they report original work and are not under consideration for publication by any other journal. The journal publishes original articles reporting experimental results of basic or clinical research, case reports, and reviews. The journal uses a single blind peer review system and each manuscript, based on the results presented in its original submission, will be evaluated by two student reviewers and one faculty reviewer. This process will provide an opportunity for medical students, graduate students, residents, fellows and faculty to publish research observation in a timely manner.