Spinal cord motor neuron cultures are an important tool for the study of mechanisms involved in motor neuron survival, degeneration and regeneration, volatile anesthetic-induced immobility, motor neuron disorders such as amyotrophic lateral sclerosis or spinal muscular atrophy as well as in spinal cord injury. Embryonic spinal cord motor neurons derived from rats have been successfully cultured; unfortunately, the culture of adult motor neurons has been problematic due to their short-term survival. Recently, by using a cocktail of target-derived factors, neurotrophins (brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor) and a permeable cyclic adenosine monophosphate analog, we have established a reproducible protocol for long-term cultures of healthy and functional adult motor neurons (Exp Neurol 220:303-315, 2009). Here, we now describe in detail the steps that we used for the optimization of the process of isolation and maintenance of adult rat ventral horn motor neurons in vitro.
In contrast to the adult brain, the adult spinal cord is a non-neurogenic environment. Understanding how to manipulate the spinal cord environment to promote the formation of new neurons is an attractive therapeutic strategy for spinal cord injury and disease. The cannabinoid 1 receptor (CB1R) has been implicated as a modulator of neural progenitor cell proliferation and fate specification in the brain; however, no evidence exists for modulation of adult spinal cord progenitor cells. Using adult rat spinal cord primary cultures, we demonstrated that CB1R antagonism with AM251 significantly decreased the number of Nestin(+) cells, and increased the number of βIII tubulin(+) and DCX(+) cells, indicative of neuronal differentiation. AM251’s effect was blocked by co-application of the CB1R agonists, WIN 55, 212-2, or ACEA. Consistent with our hypothesis, cultures, and spinal cord slices derived from CB1R knock-out (CB1−/−) mice had significantly higher levels of DCX(+) cells compared to those derived from wild type (CB1+/+) mice, indicative of enhanced neuronal differentiation in CB1−/− spinal cords. Moreover, AM251 promoted neuronal differentiation in CB1+/+, but not in CB1−/− cultures. Since CB1R modulates synaptic transmission, and synaptic transmission has been shown to influence progenitor cell fate, we evaluated whether AM251-induced neuronal differentiation was affected by chronic inactivity. Either the presence of the voltage-dependent sodium channel blocker tetrodotoxin (TTX), or the removal of mature neurons, inhibited the AM251-induced increase in DCX(+) cells. In summary, antagonism or absence of CB1R promotes neuronal differentiation in adult spinal cords, and this action appears to require TTX-sensitive neuronal activity. Our data suggest that the previously detected elevated levels of endocannabinoids in the injured adult spinal cord could contribute to the non-neurogenic environment and CB1R antagonists could potentially be used to enhance replacement of damaged neurons.
BACKGROUND Calmodulin activation by Ca2+, its translocation to the nucleus, and stimulation of phosphorylation of CREB (P-CREB) is necessary for new gene expression and has been linked to long term potentiation, a process important in memory formation. Since isoflurane is known to affect memory we tested whether isoflurane interfered with the translocation of calmodulin to the neuronal cell nucleus and attenuated the formation P-CREB. METHODS SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western Blotting, ELISA, and immunocytochemistry. The translocation of calmodulin from the cytosol to the nucleus was also examined following depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Prior to and during KCl depolarization cells were exposed to isoflurane, isoflurane plus Bay K8644, nitrendipine, and ω-conotoxin GVIa, respectively. RESULTS P-CREB increased following KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 seconds. The increase in P-CREB formation was inhibited by nitrendipine but not ω-conotoxin, and by isoflurane in a concentration dependent fashion. Pretreatment with the L-type Ca2+ channel agonist, Bay K8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred following KCl depolarization. CaM translocation was inhibited by nitrendipine, and attenuated by isoflurane. Bay K8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus. CONCLUSIONS Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca2+entry through L-type Ca2+ channels.
Background Volatile anesthetics decrease Ca2+ entry through voltage-dependent Ca2+ channels. Ca2+ influences neurotransmitter release and neuronal excitability. Because volatile anesthetics act specifically on the spinal cord to produce immobility, we examined the effect of isoflurane and the nonimmobilizers F6 (1, 2- dichlorohexafluorocyclobutane) and F8 (2, 3- dichlorooctafluorobutane) on CaV1 and CaV2 Ca2+ channels in spinal cord motor neurons and dorsal root ganglion neurons. Methods Using patch clamping, we compared the effects of isoflurane with those of F6 and F8 on CaV1 and CaV2 channels in isolated, cultured adult rat spinal cord motor neurons and on CaV1 and CaV2 channels in adult rat dorsal root ganglion sensory neurons. Results In spinal cord motor neurons, isoflurane, but not F6 or F8, inhibited currents through CaV1 channels. Isoflurane and at least one of the nonimmobilizers inhibited currents through CaV1 and CaV2 channels in dorsal root ganglion neurons and Cav2 in spinal cord motor neurons Conclusion The findings that isoflurane, but not nonimmobilizers, inhibited CaV1 Ca2+ channels in spinal cord motor neurons are consistent with the notion that spinal cord motor neurons might mediate isoflurane-induced immobility. Additional studies are required to examine whether inhibition of CaV1 calcium currents in spinal cord motor neurons are sufficient, or whether actions on other channels/proteins also contribute to isoflurane-induced immobility.
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