Pharmacological and biochemical approaches were used to elucidate the involvement of growth factor signaling pathways mediating estrogen neuroprotection in primary cortical neurons after glutamate excitotoxicity. We addressed the activation of mitogen-activated protein kinase (MAPK) signaling pathways, which are activated by growth factors such as nerve growth factor (NGF). Inhibition of MAPK signaling with the MAPK kinase inhibitor PD98059 blocks both NGF and estrogen neuroprotection in these neurons. These results correlate with a rapid and sustained increase in MAPK activity within 30 min of estrogen exposure. The involvement of signaling molecules upstream from MAPK was also examined to determine whether activation of MAPK by estrogen is mediated by tyrosine kinase activity. Estrogen produces a rapid, transient activation of src-family tyrosine kinases and tyrosine phosphorylation of p21(ras)-guanine nucleotide activating protein. Effects of estrogen on neuroprotection, as well as rapid activation of tyrosine kinase and MAPK activity, are blocked by the anti-estrogen ICI 182,780. This provides evidence that activation of the MAPK pathway by estrogen participates in mediating neuroprotection via an estrogen receptor. These results describe a novel mechanism by which cytoplasmic actions of the estrogen receptor may activate the MAPK pathway, thus broadening the understanding of effects of estrogen in neurons.

Here we demonstrate a microfluidic perfusion system suitable for a long-term (>2 week) culture of muscle cells spanning the whole process of differentiation from myoblasts to myotubes. Cell-adhesive surface microdomains alternating with a robust cell-repellent coating mimic in vivo spatial cues for muscle cell assembly and allow for confining the fusion of myoblasts into aligned, isolated multinucleated myotubes. The microfluidic system provides accurate control of the perfusion rates and biochemical composition of the environment surrounding the cells. Comparing muscle cell-specific differentiation markers and the timing of fusion, we observed no differences in differentiation between microfluidic and traditional cultures. All differentiation assays were fully microfluidic, i.e. they were performed by sequentially changing the fluids in the micro-channels. By delivering fluorescent markers using heterogeneous laminar flows, it was possible to confine a membrane receptor labeling assay to a region smaller than a myotube. Our method can serve as an improved in vitro model for studying muscle cell differentiation and for characterizing extracellular molecules and mechanisms involved in neuromuscular differentiation.

Bcl-2, an antiapoptotic protein, protects cells against many but not all forms of apoptosis. For example, Bcl-2 does not protect non-neuronal cells against taxol, a microtubule-stabilizing agent. The underlying mechanism for the ineffectiveness of Bcl-2 against taxol has been the subject of intense interest. Data from non-neuronal cells indicate that taxol-induced apoptosis requires activation of N-terminal c-Jun protein kinase (JNK) that phosphorylates and inactivates Bcl-2. This suggests the interesting possibility that the apoptotic activity of JNK may be caused by phosphorylation of Bcl-2 and inhibition of the antiapoptotic activity of Bcl-2. Here we report that taxol induces apoptosis in cortical neurons but by a mechanism significantly different from that in non-neuronal cells. In contrast to human embryonic kidney 293 cells, expression of wild-type Bcl-2 in cortical neurons protected against taxol-induced apoptosis, and taxol did not induce Bcl-2 phosphorylation. Furthermore, cortical neurons express high basal JNK activity, and taxol did not stimulate total JNK activity. However, taxol activated a subpool of JNK in the nucleus and stimulated c-Jun phosphorylation. JNK inhibition or expression of a dominant-negative c-Jun abrogated taxol-induced apoptosis in cortical neurons, suggesting a role for JNK and JNK-mediated transcription in taxol-stimulated apoptosis. Furthermore, taxol-induced apoptosis in cortical neurons required inhibition of phosphatidylinositol 3-kinase signaling. These data suggest that taxol induces apoptosis in neurons by a mechanism quite distinct from that of non-neuronal cell lines and emphasize the importance of elucidating apoptotic mechanisms specific for neurons in the CNS.

We have developed a microfluidic cell culture method that allows for the formation of linear isolated myotubes organized in a parallel microarray. Attachment and spreading of cells are confined within microtracks of cell-adherent proteins separated by a protein-repellent coating. Signaling molecules or other molecules of interest can be focally delivered to the myotubes using heterogeneous microfluidic streams. We have used the method to focally deliver agrin (a molecule implicated as a postsynaptic organizer), which leads to localized acetylcholine receptor clustering. These techniques can be modified to accommodate other cell types and can be adapted to virtually any bioactive molecule such as signaling factors or drugs. This protocol features two major techniques that can be utilized simultaneously or independently to (i) micropattern cells using surface chemical modification and (ii) use a microfluidic platform for culturing and focal stimulation of cells with molecules of interest. Device design, fabrication and assembly can be completed in 3 days.