Proper neuronal functioning depends on a strictly regulated interstitial environment and tight coupling of neuronal and metabolic activity involving adequate vascular responses. These functions take place at the blood brain barrier (BBB) composed of endothelial cells, basal lamina covered with pericytes, and the endfeet of perivascular astrocytes. In conventional in vitro models of the BBB, some of these components are missing. Here we describe a new model system for studying BBB and neurovascular coupling by using confocal microscopy and fluorescence staining protocols in organotypic hippocampal slice cultures. An elaborated network of vessels is retained in culture in spite of the absence of blood flow. Application of calcein-AM either from the interstitial or from the luminal side resulted in different staining patterns indicating the maintenance of a barrier. By contrast, the ethidium derivative MitoSox penetrated perivascular basal lamina and revealed free radical formation in contractile cells embracing the vessels, likely pericytes. 1. Introduction Proper function of the central nervous system requires a meticulously controlled interstitial environment. Since its composition largely differs from that of blood plasma, its maintenance relies on selective filtering and active transport processes at the blood brain barrier (BBB). In order to keep pace with the energetic demand of neuronal activity, cerebral blood flow is tightly regulated by multiple and only partially understood mechanisms termed as neurovascular coupling. The structural substrate for BBB and neurovascular coupling is the neurovascular unit composed of tight junction coupled endothelial cells, capillary basal lamina covered with smooth muscle cells (SMCs)/pericytes, and the endfeet of perivascular astrocytes [1]. Studies on BBB and neurovascular coupling are frequently done in vivo although the exact control of systemic effects is difficult. Accordingly, the conclusions which can be drawn need careful interpretation. Studies on acute brain slices gave us new insights on the regulation of capillary microcirculation [2–5] as well as on consequences of BBB disruption [6, 7]. However, brain slices represent acutely injured tissue with severed BBB and ongoing cell damage that might negatively interfere with the mechanisms of neurovascular coupling [8, 9]. Widely used in vitro models of BBB are based on different cocultures of endothelial cells and astrocytes [10]. However, such models dismiss the intimate influence of the surrounding nervous tissue, pericytes, and perivascular microglia
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