Ischemic stroke is a severe insult in the brain causing cell death, inflammation, and activation of microglia. Microglia are the immune cells of the brain and play a role in any inflammatory process during neurodegeneration. Microglia are round ameboid and migrate to the lesion site, where they differentiate into ramified forms and activated phagocytic microglia. On the other hand, microglia can also release growth factors to repair degeneration. The aim of the present study is to explore the migratory capacity of microglia after ischemic insults. Organotypic brain slices of the mouse cortex (300 μm) were prepared. In order to study migration, the slices were connected to collagen-loaded microcontact prints (with or without monocyte chemoattractant protein-1, MCP-1) on the membranes. Slices were stimulated with lipopolysaccharide (LPS) for maximal microglial activation. Ischemic insults were simulated with oxygen-glucose deprivation (OGD) and acidosis (pH 6.5) for 3 days. After 3 weeks in culture, slices were fixed and immunohistochemically stained for the microglial markers Iba1, CD11b and macrophage-like antigen. Our data show that Iba1+ microglia migrated along the microcontact prints, differentiate and phagocyte 1.0 μm fluorescent microbeads. LPS significantly enhanced the number of round ameboid migrating microglia, while OGD and acidosis enhanced the number of ramified activated microglia. The effect was not visible on slices without any μCP and was most potent in μCP with MCP-1. We conclude that OGD and acidosis activate ramification and exhibit a similar mechanism, while LPS only activates round ameboid microglia. Collagen-loaded microcontact prints connected to mouse brain slices are a potent method to study activation and migration of microglia ex vivo.

Voltage-gated Ca2+ channels are composed of a pore-forming α1 subunit and auxiliary β and α2δ subunits, which modulate Ca2+ current properties and channel trafficking. So far, the partial redundancy and specificity of α1 for α2δ subunits in the CNS have remained largely elusive. Mature spiral ganglion (SG) neurons express α2δ subunit isoforms 1, 2, and 3 and multiple Ca2+ channel subtypes. Differentiation and in vivo functions of their endbulb of Held synapses, which rely on presynaptic P/Q channels (Lin et al., 2011), require the α2δ3 subunit (Pirone et al., 2014). This led us to hypothesize that P/Q channels may preferentially co-assemble with α2δ3. Using a dissociated primary culture, we analyzed the effects of α2δ3 deletion on somatic Ca2+ currents (ICa ) of SG neurons isolated at postnatal day 20 (P20), when the cochlea is regarded to be mature. P/Q currents were the dominating steady-state Ca2+ currents (54% of total) followed by T-type, L-type, N-type, and R-type currents. Deletion of α2δ3 reduced P/Q- and R-type currents by 60 and 38%, respectively, whereas L-type, N-type, and T-type currents were not altered. A subset of ICa types was also analyzed in SG neurons isolated at P5, i.e., before the onset of hearing (P12). Both L-type and N-type current amplitudes of wildtype SG neurons were larger at P5 compared with P20. Deletion of α2δ3 reduced L-type and N-type currents by 23 and 44%, respectively. In contrast, small P/Q currents, which were just being up-regulated at P5, were unaffected by the lack of α2δ3. In summary, α2δ3 regulates amplitudes of L- and N-type currents of immature cultured SG neurons, whereas it regulates P/Q- and R-type currents at P20. Our data indicate a developmental switch from dominating somatic N- to P/Q-type currents in cultured SG neurons. A switch from N- to P/Q-type channels, which has been observed at several central synapses, may also occur at developing endbulbs of Held. In this case, reduction of both neonatal N- (P5) and more mature P/Q-type currents (around/after hearing onset) may contribute to the impaired morphology and function of endbulb synapses in α2δ3-deficient mice.