Astrocytes & neural network synchronization: in vivo two-photon Calcium imaging and field recording of neocortical activity

“the brain’s ability to generate coherent thoughts derives from the spatiotemporal orchestration of neuronal activity” (Hebb, 1949). Oscillation in the degree of synchronization among neural cohorts, appears to be the epiphenomenon of a yet poorly understood process, through which information is computed in the central nervous system (Buzsàki G. ; 2013). Although considerable efforts have been made in order to shed light upon the basic mechanisms regulating neural network synchronization, several scenarios remain to be further explored. In the last 15 years the hypothesis that astrocytes, the “dark side of glia” (Miller G.; 2005), are involved in the fine modulation of neural activity, rapidly gained remarkable interest in the scientific community. These non classically excitable cells are organized in a circuit in parallel to that of neurons, operating on wider space and time scales, integrating and interacting with the excitable cell network (Navarrete M. and Araque A.; 2011). Nowadays a plethora of works demonstrate how their functions are deeply interwoven with the neural network physiology (Fellin T., et al.; 2004. Agulhon C. et al.; 2008) and how strongly astrocyte dysfunctions correlate with different neurologic disorders, such as epilepsy (Kimelberg and Nedergaard 2010). Epilepsy is an extremely complex and widespread neurological condition (Engel et al.; 2013). Epileptiform activity is characterized by peculiar patterns of neural activity, ranging from hyper-synchronous events to almost completely uncorrelated phases (Traub and Wong, 1982; Trevelyan et al., 2006; Menendez de la Prida and Trevelyan, 2011). If astrocytes are actually involved in the modulation of neural activity, then spontaneous, pathological or induced variations in the coordination of neurons shall be mirrored in correlated alterations in astrocytes behavior. Both neurons and astrocytes behavior can indeed be probed employing intra-vital fluorescent Calcium sensitive dyes while ensemble electrical activity is recorded with extracellular field electrodes. These joined approaches allow observations with tunable spatial and temporal resolution, ranging from large populations to single cells with millisecond precision. However, a mandatory requirement in order to investigate on such a topic is to keep both physiological and anatomical cues as preserved as possible. Minimally invasive in vivo two-photon imaging and local field recotrding techniques enable the endeavor. Target of the work presented here is to characterize how astrocytes Calcium dynamics change accordingly to a variety of different patterns of neural population activity. Employing the isolated guinea pig brain preparation it is possible, with the appropriate pharmacological treatment, to elicit recurrent epileptiform episodes in the entorhinal cortex (Uva L. et al.; 2005). Some of the data we collected from this model are published (Gómez-Gonzalo M. et al.; 2011. Gómez-Gonzalo et al.; 2010.) and summarized here as follows: 1) Epileptiform activity summons large coordinated Calcium transients in cortical astrocytes in the transition from ictal phase to synchronous activity. 2) Triggering Calcium elevation in astrocytes, induces phase transition in neural coordination. 3) During neural hyper-synchronous interictal phase, astrocytes remain silent. 4) Astrocytes endfeet, show Calcium transients during ictal phase. We then moved to the anesthetized mouse model in order to further investigate on the behavior of astrocyte in the context of spontaneous physiological activity and in visual processing. Moreover using transgenic mice, it is possible to discern between different neural cell types, here we used animals expressing green fluorescent protein in a subclass of inhibitory neurons considered involved in the synchronization of electrical activity and epilepsy (Fujiwara-Tsukamoto Y. et al.; 2010. Sohal et al.; 2009). Furthermore, pharmacological dishinibition induces a steady-state interictal phase that lasts for hours with highly synchronized electrographic events. The list that follows resumes some results obtained: 1) Spontaneous slow wave activity is associated with weak astrocyte activation. 2) Calcium activity of excitatory and inhibitory neurons, are phase locked in each interictal event. 3) Physiological processing of visual stimulation is abolished during interictal activity. 4) After each interictal event an “absolute refractory period” prevents neural firing. 5) Calcium transients in astrocytes are rare during interictal activity. 6) In long lasting interictal phase spontaneous de-synchronization is observed to be correlated with astrocytes Calcium elevation. 7) Astrocytes display Calcium oscillations preceding the manifestation of electrical coherence at the onset of hyper-synchronous phase. 8) Pharmacological tools reducing the synchronization of neural population events produce considerable increase in the astrocytes Calcium transients frequency.