Understanding how neural activity regulates local cerebral blood flow is essential for understanding the BOLD signals detected in functional magnetic resonance imaging. Glutamate has been shown to dilate microvessels in slices of neocortex and hippocampus, and this may be mediated by NO release from neurons or by activation of astrocytes to release prostaglandins (Fergus & Lee, 1997; Lovick, 1999; Zonta et al. 2003). Studying regulation of microvessel diameter in living brain slices offers advantages in determining the mechanisms of vascular regulation (though with the disadvantage that the vessels are not perfused), but many vessels are not easily visible in brain slices. We have used isolectin B4 (Laitinen, 1987) from Bandeiraea simplicifolia conjugated to FITC (Sigma), which mainly recognises terminal α-D-galactosyl residues, to label the endothelial cells of blood vessels in cerebellar slices or retina from P7 and P15 rats (killed humanely by cervical dislocation). Since the lectin recognises an extracellular site, it can be used to label vessels in both living and paraformaldehyde-fixed slices. Z-stacks (100 µm in depth) of FITC fluorescence images at different depths of the cerebellar cortex or retina were acquired, thresholded to segment the image into labelled and non-labelled areas, and rendered using Zeiss LSM 510 software to construct a 3D rotatable image of the microvessel network.

At both P7 and P15 the cerebellar vascular network defined by isolectin B4 labelling was similar to that seen with India ink injection (Yu et al. 1994), although isolectin B4 also revealed microglia in the white matter. Arterioles penetrate the cerebellar cortex from vessels outside the folia, forming a dense network of vessels in the Purkinje cell layer and sending branches into the granular layer and molecular layers. Application of noradrenaline (2-100 µM) to living cerebellar slices or retina caused constriction of vessels in the vasculature network, however further work is needed to investigate whether binding of the lectin to the endothelial cells alters the control of microvessel diameter by neurotransmitters.

This work was supported by the Wellcome Trust and a Wolfson-Royal Society Award.