## 3 Results
### Findings
#### 3.2.2 Microglia  
Microglia are resident macrophage cells, acting as the first and main forms of active immune defense of the central nervous system. The application of 2 PI to microglia-focused epilepsy research demonstrates structural and functional changes of microglia in pathological conditions. Changes in cell morphology and activity that result from experimental seizure induction were predominantly assessed. 2 PI also enabled assessment of changes in microglial proliferation, translocation, as well as the frequency and nature of specific microglial-neuron interactions.  
[PERSON] et al. [42] and [PERSON] et al. [45] observed that seizure-activated microglia had significantly larger cell bodies, with fewer and shorter processes compared to inactivated microglia in control animals (see Figure 2). In terms of activity, seizures resulted in significant microglial proliferation in the hippocampus. Pharmacological inhibition of microglial proliferation could also be visualized with 2 PI, and this correlated with the attenuation of neuronal death during seizures, thereby suggesting a neurotoxic role of microglia during epileptic activity.  
Figure 2: Two-photon images of microglial cells obtained in control mice (**A**) and KA-induced status epilepticus mice (**B**) depicting significantly different cell morphologies. Cell body outlines were drawn manually to determine cell sizes. After treatment with KA, microglial cells are significantly larger and have significantly shorter processes (consider the cell depicted in the inset images). Scale bars: 10 \(\upmu\)m (main images) and 6 \(\upmu\)m (inset images). Adapted from “Altered morphological dynamics of activated microglia after induction of status epilepticus” ([PERSON] et al., _Journal of Neuroinflammation_, 2015 [45]). This figure has been modified. Creative Commons license: [[https://creativecommons.org/licenses/by/4.0/](https://creativecommons.org/licenses/by/4.0/)]([https://creativecommons.org/licenses/by/4.0/](https://creativecommons.org/licenses/by/4.0/)) (accessed on 17 November 2020).  
[PERSON] et al. [43] and [PERSON] et al. [45] visualized using 2 PI that microglial translocation and rearrangement significantly increase during epileptiform activity. In fact, [PERSON] et al. [45] described increased translocation activity of microglia in pathological conditions. In P2Y12-deficient mice, this microglial translocation was not observed, though these mice exhibited worsened seizure phenotypes, which is indicative of a neuroprotective role of microglia in epileptic conditions [43; 46].  
Furthermore, [PERSON] et al. [44] visualized a specific type of microglia-neuron interaction, namely microglial process convergence events (MPCs). They reported that the frequency of these MPCs dramatically increased upon extracellular calcium reduction (also visualized with 2 PI), leading to epileptiform activity. A decrease in the frequency of MPCs in fractalkine-deficient slices were seen, corresponding with increased seizure phenotypes. These findings identified fractalkine signaling as a regulator of neuroprotective microglia-neuron interactions during epileptiform activity. [PERSON] et al. [48] also described alterations to specific microglia-neuron interactions in synaptic integration. 2 PI was able to visualize that, under physiological conditions, microglia interacted more with distal dendrites of neurons, whereas following status epilepticus, microglia interacted more strongly with proximal dendrites.  
Finally, [PERSON] et al. [47] described differences in the activities of neural and glial networks during the transition from preictal to seizure states. Using a zebrafish model of epilepsy, it was demonstrated that glial networks are highly active and strongly synchronous preictally. However, in generalized seizure states, neural activity and connectivity abruptly increased, and this was associated with alterations in interactions between neurons and glial cells.