The World's Extremely Abnormal Thalidomide Saga

05. Our control experiments revealed a 10% change from baseline as the appropriate threshold value. For each cell, assess the reversible depolarization response based upon Non-specific serine/threonine protein kinase 2 criteria. First, the average percent change from baseline during minutes 18-23 must be greater than 10%, the threshold determined in the previous step. Second, the average percent change from baseline during minutes 35-40 must be less than half of the average percent change from baseline during minutes 18-23. The second criterion was chosen since it was found to be more rigorous than stimulation with glutamate or KCl. Unhealthy neurons often respond to stimulation with glutamate or KCl even though their responses to decreased glucose are not reversible. For each dish, calculate the % of depolarized neurons. This value Crizotinib is used to quantify the % of GI neurons among different treatment groups. Representative Results The precise dissection of the VMH away from other hypothalamic areas is important to obtain consistent results. The inclusion of other areas could dilute the VMH neuronal population, changing the % of depolarized neurons calculated. Furthermore, glucose sensing neurons have been identified in other hypothalamic regions, such as the lateral hypothalamus, which may differ functionally and mechanistically from VMH glucose sensing neurons. Figure 1 illustrates the correct anatomical locations for proper dissection. Following the protocol above, brain tissue containing the correct VMH region can be dissociated. Further dissection to precisely separate the VMN and ARC, while maintaining the entirety of ATM/ATR assay each subpopulation, may not be possible. Figure 2 shows an example of healthy dissociated VMH neurons. Using immunocytochemistry, we confirmed that our preparation is >90% neuronal. Only healthy neurons should be used for data analysis and dishes with too many unhealthy neurons should be discarded. Neurons that are unhealthy often have very dark edges, take up the MPD to a greater extent, and have irregular shapes. Additionally, neurons should be plated at a density such that most neurons are not touching each other during recording. Neurons chosen for analysis should not be touching other cells or debris. Data obtained from recordings of a GI neuron and a nonGI neuron are shown in Figure 3. After obtaining a baseline for 10 min, the extracellular glucose was decreased from 2.5-0.1 mM. The GI neuron shows a robust reversible response greater than the predetermined threshold of 10% change from baseline. The increase in fluorescence reflects depolarization. While GE neuron hyperpolarization responses are also detected, the frequency is too low (