Anti-Tyrosine Hydroxylase monoclonal antibody, clone MOD2 [Alexa Fluor® 555]

The functional state of the neurovascular unit (NVU), composed of the blood-brain barrier and the perivasculature that forms a dynamic interface between the blood and the central nervous system (CNS), plays a central role in the control of brain homeostasis and is strongly affected by CNS drugs. Human primary brain microvascular endothelium, astrocyte, pericyte, and neural cell cultures are often used to study NVU barrier functions as well as drug transport and efficacy; however, the proteomic and metabolomic responses of these different cell types are not well characterized. Culturing each cell type separately, using deep coverage proteomic analysis and characterization of the secreted metabolome, as well as measurements of mitochondrial activity, the responses of these cells under baseline conditions and when exposed to the NVU-impairing stimulant methamphetamine (Meth) are analyzed. These studies define the previously unknown metabolic and proteomic profiles of human brain pericytes and lead to improved characterization of the phenotype of each of the NVU cell types as well as cell-specific metabolic and proteomic responses to Meth.

Background Small conductance, calcium-activated (SK3) potassium channels control the intrinsic excitability of dopaminergic neurons (DN) in the midbrain and modulate their susceptibility to toxic insults during development. Methods We evaluated the age-dependency of the neuroprotective effect of an SK3 agonist, 1-Ethyl-1,3-dihydro-2H-benzimidazol-2-one (1-EBIO), on Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) excitotoxicity to DN in ventral mesencephalon (VM) organotypic cultures. Results Most tyrosine hydroxylase (TH)+ neurons were also SK3+; SK3+/TH- cells (DN+) were common at each developmental stage but more prominently at day in vitro (DIV) 8. Young DN+ neurons were small bipolar and fusiform, whereas mature ones were large and multipolar. Exposure of organotypic cultures to AMPA (100 mu m, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15 (n = 15, p = 0.005) and DIV 22 (n = 15, p<0.001). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures. Immature DN+ (small, bipolar) were increased after AMPA (100 mu m, 16 h) at DIV 8, at the expense of the number of differentiated (large, multipolar) DN+ (p = 0.039). This effect was larger at DIV 15 (p<<<0.0001) and at DIV 22 (p<<<0.0001). At DIV 8, 30 mu M 1-EBIO resulted in a large increase in DN+. At DIV 15, AMPA toxicity was prevented by exposure to 30 mu M, but not 100 mu M 1-EBIO. At DIV 22, excitotoxicity was unaffected by 30 mu M 1-EBIO, and partially reduced by 100 mu M 1-EBIO. Conclusion The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage.