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. Calcium oscillations are important for the proper functions of neuronal cells. For example, Free Private Sexcam detection of intracellular calcium flux can be achieved by staining the cells with fluorescent dyes which bind to calcium ions (known as fluorescent calcium indicators) with a resultant, detectable change in fluorescence (e.g., Fluo-4 AM and Fura Red AM dyes available from Molecular Probes. 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 measured in the present methods are AMPA-dependent calcium oscillations. In some embodiments, calcium oscillations are measured in the present method through the use of fluorescent probes which detect the fluctuations of intracellular calcium levels. In one aspect, the disclosure sets forth a calcium oscillation assay as one way of measuring or predicting toxicity of a molecule. In some embodiments, the calcium oscillation assay can include a positive control cell (i.e., a cell exposed to a molecule that is known to reduce calcium oscillations to an untolerable level) or a negative control cell (i.e., a cell exposed to a molecule that is known not to affect calcium oscillations in the cell).



Calcium oscillations can also regulate interactions of neurons with associate glia, Free private sexcam in addition to other associated neurons in the network, to release other neurotransmitters in addition to glutamate. In other embodiments, the calcium oscillations can be measured as shown in Murphy et al., J. Neurosci. In some embodiments, the disclosure provides a method of selecting or identifying a molecule having tolerable in vivo acute neurotoxicity comprising a step of (i) adding a molecule to a culture of neuronal cells and (ii) measuring calcium oscillations in the neuronal cells in vitro, wherein the neuronal cells with the molecule exhibit calcium oscillations at a level comparable to or higher than that of control cells. In another embodiment, the disclosure provides a method of predicting in vivo acute neurotoxicity of a molecule comprising a step of (1) adding the molecule to a culture of neuronal cells and (2) measuring calcium oscillations in vitro in the neuronal cells.



In certain embodiments, the calcium oscillations measured in the present methods are AMPA-dependent calcium oscillations. 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. Therefore, identification of the correlations among the calcium oscillation assay, sequence score, and in vivo neurotoxicity allows one to predict the in vivo neurotoxicity based on the calcium oscillation in vitro assay and the sequence score. In other aspects, the disclosure provides a combined method of using a calcium oscillation assay and a sequence score method. In other embodiments, the disclosure includes a method of selecting or identifying a molecule having tolerable in vivo acute neurotoxicity comprising measuring calcium oscillations in vitro in neuronal cells which are in contact with the molecule, wherein the contacted neuronal cells exhibit calcium oscillations at a level comparable to or higher than that of control cells.



In one embodiment, the disclosure provides a method of testing, identifying, or determining in vivo acute neurotoxicity of a molecule comprising measuring calcium oscillations in vitro in neuronal cells which are in contact or have been in contact with the molecule. In some embodiments, the calcium oscillations were measured by using any methods known in the art. Most of the people search a lot of chat rooms where they can chat with anyone anytime by using this online software. 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. Fluorescent dyes useful for the calcium oscillation assay often provide for ratiometric detection on intracellular calcium flux by calibrating fluorescence intensities measured a wavelength. The disclosure also provides an in vivo tolerability assay that can be used separately or combined with the calcium oscillation assay and/or free private sexcam the sequence score method. In some embodiments, the calcium oscillations are gamma-aminobutyric acid (GABA)-dependent calcium oscillations.



In other embodiments, the calcium oscillations measured in the present methods are the combination of the oscillation frequency and magnitude. 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. Such methods are helpful to reduce unnecessary killing of animals during testing of the molecule's toxicity and/or enhance the possibilities that the molecules will be safe for in vivo administration. Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.