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In certain embodiments, the present disclosure provides a method of identifying or determining a molecule having intolerable in vivo neurotoxicity comprising measuring calcium oscillations in vitro in neuronal cells after being in contact with the molecule in the neuronal cells. Examples of assays measuring the change in tubulin intensity in a cell are provided below. For example, long term toxicities can be determined by measuring the change in tubulin intensity in a cell by the molecule when the cell comes in contact with a molecule. In certain embodiments, the change in tubulin intensity is measured along with one or both of the change in calcium oscillation, sequence score, and/or in vivo tolerability assay. In some embodiments, the calcium oscillations for a molecule having tolerable in vivo acute neurotoxicity are compared with the calcium oscillations in a cell not exposed to the molecule. In another embodiment, the calcium oscillations measured in the present methods are the frequency of calcium oscillations. In an embodiment, the oscillation frequency can be determined by a time interval from commencement of a first oscillation in intracellular calcium flux to a commencement of a second oscillation in intracellular calcium flux.

In other embodiments, the calcium oscillations can be measured as shown in Murphy et al., J. Neurosci. The fluorescent measurements can be analyzed to identify oscillations in intracellular calcium flux and/or a «threshold» representing a point at which the intracellular calcium flux of a given oscillation is progressing either to a maximum or minimum. In other embodiments, an in vivo tolerability study can be used in combination with the calcium oscillation assay and/or the sequence score method. Fluorescent dyes useful for the calcium oscillation assay often provide for ratiometric detection on intracellular calcium flux by calibrating fluorescence intensities measured a wavelength. AMPA-dependent calcium oscillations. In some embodiments, the effective ion concentration allowing for detection of AMPA-dependent calcium oscillations is at least about 0.5 mM. In other embodiments, the calcium oscillations measured in the present methods are the combination of the oscillation frequency and magnitude. The term «oscillation frequency» refers to the time between oscillations. In certain embodiments, the behavioral test is a short term memory test, a spatial learning and memory test, a gait analysis test, or https://Watchonlineporns.com/ any combination thereof. In some embodiments, the behavioral score is measured by the following methods, including a novel object rejection test, a water maze test, a gait analysis test, and/or any combination thereof.

NOR testing is based on the spontaneous behavior of rodents to explore a novel object more than a familiar one (Dodart et. The laboratory animal can be a rodent, such as a mouse, rat, guinea pig or hamster, but can also be another animal typically used in laboratory testing. In some embodiments, fluorescence of the stained cells (including stained individual cells) can be measured by confocal, or standard, fluorescence microscopy, optionally at a number of time points or continuously (e.g., real-time) to provide, for example, time lapse measurements. In one embodiment, the calcium oscillations measured in the present methods are the cumulative increase in calcium oscillations within a culture of neuronal cells, whereby the time to reach the maximum fluorescence signal constitutes the magnitude of the calcium response. In some embodiments, the calcium oscillations were measured by using any methods known in the art. In other embodiments, the molecule comprises a small molecule, a polynucleotide, a protein, a peptide, or any combination thereof.

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