Role of ATP and ATP-metabolizing enzymes in neuroreparative processes after hypoxic-ischemic injury in rodent organotypic brain slices

We characterized the developmental and regeneration features of a brain circuitry involved in working memory and reward processing, from the ventral tegmental area-substantia nigra [VTA-SN] fibers projecting to the prefrontal cortex [PFC] and the complementary glutamatergic pathways (Seamans and Yang, Prog. Neurobiol. 74:1-58). By utilizing a co-culture system (Heine et al., 2007, Neurosci., 149:165-181; Franke et al., 2003, Neurochem. Int., 42:431-439) adapted to multi electrode platforms (MEAs), we simultaneously recorded (and analyzed) from 60 electrodes, at brief (5-7 days in-vitro, div) and long-term (15-25 div) conditions, both spikes (bandwith 250-5000 Hz) and local field potentials (LFP, bandwith 1-200 Hz) activity (Gullo et al., 2009, J Neurosci. Methods, 181:186-198; Gullo et al., 2010, Front. Neural Circuits, 2010/04). After few days the co-cultures show a spontaneous activity in the form of bursts, trains of action potentials, and LFPs lasting ~0.3 and 1.5 s, respectively. Surprisingly, the activity increased in parallel with the growth of new projections from one slice to the other and there was no need of evoking activity in VTA to observe activity in PFC. We characterized three types of spontaneous activities as follows: 1) bursts only in VTA-SN, while PFC was silent (VTA events, 59±3%), 2) bursts originating in VTA and rapidly reaching PFC (with a delay of ~80 ms), that is a crosscorrelated activity (28±2%) and 3) bursts with synchronous activity in both regions (13±1%). This correlated activity is completely abolished if the new-born projections between the two areas are cut. Preliminary data suggest that the activity of the co-cultures is specifically modulated by dopaminergic, GABAergic and glutamatergic systems. We have also evaluated the damage induced by the application of 1h oxygen-glucose deprivation (OGD) to VTA-SN/PFC co-cultures, focusing on neurons and on the glial population (astroglia, microglia and oligodendroglia), in order to understand the damage extent and modification of the glial response after injury. Preliminary data suggest that, in the PFC, the number of astroglial cells is not affected by OGD, while an induction of microglial cells activation (approx. +50%) and VTA-SN is under evaluation. We will exploit the ability of endogenous stem/precursor cells of brain parenchyma to sustain the regeneration-remodeling of damaged circuitries and to possibly differentiate to new-born neurons and glia able to replace irreversibly damaged cells.