## 4 Discussion
### Two-Photon Imaging in Epilepsy Research  
The papers included in this review described several interesting and sometimes novel findings in the field of epilepsy research. The relevance of pathological hypererecitability, whereby individual neurons become increasingly capable of generating repeated action potentials, is a widely recognized phenomenon in epilepsy [75; 76]. It is thought this pathological hyperexcitability arises due to inhibitory control failure [77]. We found that several studies in this review applied 2 PI to shed more light on this process. For example, the described \"inhibitory model\" of seizure propagation, wherein the collapse of local inhibition leads to neuronal recruitment during ictal discharges, was suggested by the findings of multiple included studies [31; 32; 35; 39; 41]. In these studies, 2 PI allowed monitoring of [Ca2+] of neurons and interneurons, revealing their activity. In addition, the interactions between glial cells and neurons were revealed through 2 PI calcium imaging, underlining the importance of the glial environment in epileptic states. Additionally, we found that morphological changes to dendrites and axons can lead to dysfunction, possibly contributing to pathological hyperactivity [62]. Another characteristic feature of epilepsy is interictal spiking, which refers to abnormal neuronal discharges between seizures. Previous reports have addressed interictal spiking [78]; however, 2 PI was needed to demonstrate the widespread impact of interictal spiking on interconnected regions of the brain by visualizing [Ca2+]. This finding suggested a prominent role of interictalspiking in epilepsy [36; 37]. However, the exact mechanisms responsible for the emergence of spontaneous interictal activity remain unknown. Interestingly, 2 PI calcium imaging of neuronal activity in the in vitro human neocortex seemed viable and comparable to patterns in experimental animal models. Important morphological changes in microglia, astrocytes, and neurites due to epileptiform activity were revealed in studies that applied 2 PI. Using 2 PI, changes in the mean sizes of cell bodies and the quantification of microglial processes before and after seizure-induction were recorded [42; 45]. Such findings have not been reported previously. Alterations in the interactions of microglia and neurons in neuronal integration were described in epileptic states. Interestingly, microglia were identified to play a neurotoxic as well as a neuroprotective role in epileptic conditions, and the exact significance of this finding has yet to be explored. Furthermore, astrocytic [Ca2+] signaling and subsequent glutamate release was identified as a key process contributing to the development and progression of ictal discharges. These findings are highly relevant, as astrocytic [Ca2+] modulation provides a novel mechanism for seizure development and progression. This in turn indicates that astrocytes are capable of playing an active, dynamic role in modulating brain function [55; 79]. This led to the understanding that the role of astrocytes goes far beyond its previously delineated supportive role in maintaining local structures and metabolism. This finding has revolutionized our understanding of epilepsy and has resulted in the concept that \"astrocytic transformation\" plays a major role in its pathophysiology [80]. Given the recent change in epilepsy research from a neuronal-centered toward a vascular-centered concept of epileptogenesis [9; 10; 11; 12], neurovascular alterations in epilepsy are of major interest. By using 2 PI, changes in blood flow were visualized, with different states of vasoconstriction and vasodilation surrounding the epileptogenic focus [59; 60; 61]. Preictal vasoconstriction was found to be correlated with increased excitatory and reduced inhibitory neuronal activity levels [61].