Plate reader-based assays for measuring cell viability, neuroprotection and calcium in primary neuronal cultures.

Drug discovery and development efforts critically rely on cell-based assays for high-throughput screening. These assay systems mostly utilize immortalized cell lines, such as human embryonic kidney cells, and can provide information on cytotoxicity and cell viability, permeability and uptake of compounds as well as receptor pharmacology. While this approach has proven extremely useful for single-target pharmacology, there is an urgent need for neuropharmacological studies to screen novel drug candidates in a cellular environment resembles neurons in vivo more closely, in order to gain insight into the involvement of multiple signaling pathways. Primary cultured neuronal cells, such as cortical neurons, have long been used for basic research and low-throughput screening and assay development, and may thus be suitable candidates for the development of neuropharmacological high-throughput screening approaches. We here developed and optimized protocols for the use of primary cortical neuronal cells in high-throughput assays for neuropharmacology and neuroprotection, including calcium mobilization, cytotoxicity and viability as well as ion channel pharmacology. Our data show low inter-experimental variability and similar reproducibility as conventional cell line assays. We conclude that primary neuronal cultures provide a viable alternative to cell lines in high-throughput assay systems by providing a cellular environment more closely resembling physiological conditions in the central nervous system.

Quantification of deficits in spatial visual function of mouse models for glaucoma.

PURPOSE: DBA/2J mice are a standard preclinical glaucoma model, which spontaneously developed mutations resulting in chronic age-related pigmentary glaucoma. The goals of this study were to identify the degree of visual impairment in DBA/2J mice before and after disease onset by quantifying the optokinetic reflex responses and to compare them to the less-researched strain of DBA/2NHsd mice. METHODS: Visual performance was measured in healthy, nonglaucomatous, and glaucomatous male DBA/2NHsd or DBA/2J mice using a visuospatial testing box. The optokinetic reflex resulting in optomotor head tracking was manually detected. Measured threshold levels equate to the maximum contrast or spatial frequency the mouse responds to. Intraocular pressure (IOP) was measured by applanation tonometry. RESULTS: IOP increased with age in both DBA/2J and DBA/2NHsd mice and was not different between the two substrains. Both visual acuity and ability to detect contrast decreased significantly, and similarly with age in both substrains. However, DBA/2NHsd had poorer visual acuity even at a younger age compared to age-matched DBA/2J mice. CONCLUSIONS: Both DBA/2J and DBA/2NHsd mice show a progressive glaucomatous phenotype of age-related increases in IOP and loss of visual acuity and contrast sensitivity when compared to other inbred or outbred strains. Given the similar increases in IOP and contrast sensitivity threshold and loss of visual acuity between these two DBA/2 substrains, we also conclude that DBA/2NHsd mice are a suitable alternative model for pigmentary glaucoma.