Oxygen tension within the arterial wall: relationship to altered bioenergetic metabolism and lipid accumulation.

Oxygen tension (pO2) was measured in upper thoracic arteries and in muscular foci at the celiac bifurcation from atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer pigeons at 6, 12, and 24 weeks of age. At each site the pO2 was measured within adventitial (outer), medial (middle), and subendothelial (inner) zones with polarographic oxygen microelectrodes while the animals were under dissociative anesthesia. The atherosclerosis-resistant Show Racer pigeons exhibited no significant age or site differences in pO2 levels. The medial pO2 was approximately 25 mm Hg, while the adventitial and subendothelial zones had pO2 levels of approximately 30 mm Hg. The pO2 profiles through the various zones of the arterial wall of 6-week-old atherosclerosis-susceptible White Carneau pigeons were similar to those of the Show Racers. However, significantly lower oxygen tension was found in the subendothelial zone from 12- and 24-week White Carneau celiac bifurcations (27 and 21 mm Hg) than in corresponding zones in Show Racer sites. Also, the medial zone in 24-week White Carneau celiac foci exhibited a significantly lower pO2 (20 mm Hg) than the medial zone of younger White Carneau. When correlated with previously described biochemical and morphological changes in White Carneau pigeons, the findings presented herein indicate that focal decreases in oxygen availability within arterial tissue occur after the onset of spontaneous atherogenesis, and probably result from decreased diffusion of oxygen into the arterial wall.

Identification of presynaptic neurons by laser photostimulation.

An optical method involving the use of a laser and a novel fluorescent dye as a photostimulation probe has been developed to identify presynaptic neurons in a large ensemble of cells. Illumination of an extracellularly stained neuron by the laser microbeam evokes action potentials. With this technique an interneuron connecting identified leech neurons was quickly located. The method speeds up the elucidation of neuronal networks, especially when small cells are involved.

Fluorescence monitoring of electrical responses from small neurons and their processes.

To improve the sensitivity of fluorescence measurements of electrical responses from small cells and their processes, we have optimized the optical measuring system. The fluorescence intensity from a stained cell was increased 40-fold relative to our previous apparatus. The increased fluorescence intensity permits the use of an inexpensive photodiode (or a photodiode array) that has a approximately 10-fold higher quantum efficiency relative to a photomultiplier. Utilizing the improved apparatus, we optically recorded an action potential of a 2 microns wide neuronal process with a signal-to-noise ratio of approximately 50 (root mean square noise) without averaging. We also report the design of an improved fluorescence voltage-sensitive probe; the fractional change of the fluorescence signal under optimal conditions was 21%/100 mV.

Improved fluorescent probes for the measurement of rapid changes in membrane potential.

To improve the quality of fluorescent voltage-sensitive probes twenty new styryl dyes were synthesized. Some of the new probes are significantly better than any used in the past. A signal-to-noise ratio of 90 root mean square (rms) noise was obtained for an optical recording of action potentials from neuroblastoma cells maintained in monolayer culture. The fluorescence fractional change of the optical signal is as large as 14%/100 mV. Photodynamic damage and bleaching are much less significant with the new probes. These fluorescent probes can be used to measure small and rapid changes in membrane potential from single cells maintained in monolayer cultures, from single cells in invertebrate ganglia, from their arborization, and from other preparations. The optical measurement can be made with a standard fluorescent microscope equipped with DC mercury illumination. Guidelines for the design of even better fluorescent probes and more efficient instruments are suggested.

Optical recording of calcium action potentials from growth cones of cultured neurons with a laser microbeam.

Simultaneous recordings of calcium action potentials directly from growth cones and from somata of neuroblastoma cells indicated that they could be generated in the neurites at or near growth cones. Growth cone responses were measured with a fluorescent voltage-sensitive dye and a 5-milliwatt helium-neon laser microbeam as a monitoring light source.