Imaging Capillary Diameter Regulation

Slices were perfused with bicarbonate-buffered aCSF, as described above, but without the kynurenic acid, at 31-35°C. In experiments using 20% oxygen, the perfusion solution was bicarbonate-buffered aCSF, gassed with 20% O₂, 5% CO₂, and 75% N₂.
For bright-field recording of capillary diameter, sagittal cerebellar slices were prepared from postnatal day 10 (P10)-P21 Sprague-Dawley rats or coronal cortical slices were prepared from P12 rats. On average 1.3 capillary regions were imaged per slice. Capillaries were imaged^{1 (link)} at ~30 μm depth within the molecular layer of cerebellar slices or the grey matter of somatosensory/motor cortex slices, using a x40 water immersion objective, a Coolsnap HQ2 CCD camera, and ImagePro Plus or Metafluor acquisition software. Images were acquired every 1-5 sec, with an exposure time of 5 msec. Pixel size was 160 or 300 nm. Vessel internal diameters were measured by manually placing a measurement line (perpendicular to the vessel, Fig. 1d) on the image (at locations near visible pericytes which constricted when noradrenaline was applied), using ImagePro Analyzer, Metamorph or ImageJ software, with the measurer blinded as to the timing of drug applications. The end of the measurement line was placed at locations representing the measurer’s best estimate of where the rate of change of intensity was greatest across pixels under the vessel edge, and diameter was estimated to a precision of one pixel. Where necessary, images were aligned by manually tracking drift, or by using Image Pro “Align Global Images” macro. Experiments where changes in focus occurred were excluded from further analysis. Data in the presence of blockers of signalling pathways were compared with interleaved data obtained without the blockers.
For experiments in which the parallel fibres were stimulated in the molecular layer, coronal slices were used to preserve the parallel fibres, and stimuli of 60-100 μs duration, at 50–90 V and 12 Hz, were applied for 25 sec using a patch pipette electrode placed approximately 100 μm away from the imaged vessel. To check that parallel fibres were being successfully activated, the field potential was monitored in the molecular layer using a 4 MΩ patch pipette filled with aCSF. To ensure that pericytes were healthy we excluded capillaries that did not constrict to 1 μM noradrenaline. Stimulation evoked a dilation (Fig. 2e, f) except in 2 capillaries which constricted, presumably due to direct depolarization of a pericyte by the stimulus since when TTX was applied (to one of these vessels) a stimulation-evoked constriction was still seen in TTX: these 2 vessels were excluded from the analysis.