Distinct Cellular Patterns of Superoxide Production in the Murine Brainstem During Oxidative and Emotional Stress

Carolina Chavez1, Sergey Kasparov2, Julian Paton2, Dmitry Mayorov1. 1University of Melbourne, Melbourne, Victoria, Australia, 2University of Bristol, Bristol, United Kingdom.

Brain superoxide (O2¯) is implicated in the regulation of cardiovascular function. However, cellular sources of O2¯ in the brain remain elusive. In this study, we examined the role of neuronal, glial and perivascular cells in O2¯generation caused by oxidative and emotional stress in the rostral ventrolateral medulla (RVLM), an area critical in cardiovascular control.

Real-time O2¯ production within individual cells was detected in living rat brain slices containing the RVLM using a O2¯-sensitive fluorescent dye dihydroethidium (DHE) and confocal microscopy. After DHE (10 μM) superfusion, strong fluorescence was observed in most but not all neurons. By contrast, little fluorescence was detected in cells with astroglia-like morphology. Angiotensin II (AII, 1 μM) increased O2¯ levels by ∼40% in neurons and also initiated robust O2¯ production in astroglia. The O2¯ dismutase inhibitor (DETCA; 1-10 mM) dose-dependently increased DHE oxidation in the RVLM by up to 3-fold. This effect was mostly mediated by increases in O2¯ production in astroglia.

In another series of experiments, DHE was injected systemically in mice or rats, as this agent rapidly penetrates the brain and makes a stable “footprint” of O2¯ levels in individual cells at the time of reaction. Following DHE injection in mice, strong fluorescence was observed in neurons, but not astroglial cells in the brain. Emotional (restraint) stress increased DHE oxidation in the RVLM (+140%), caudal ventrolateral medulla (+60%), but not in the raphe pallidus (+9%). The stress-induced increase in fluorescence was mainly observed in neurons, but not astroglial cells. Likewise, isolation stress in rats was primarily associated with increased DHE oxidation in neurons, but not other cell types in the RVLM.

These results indicate that oxidative and emotional stress result in distinct cellular patterns of O2¯ production in the RVLM. Oxidative stress initiates a strong O2¯ generation in astroglia, which appears to become the principal source of O2¯. Conversely, emotional stress primarily increases O2¯ production in neurons, but not astroglial cells. The pattern of AII-induced O2¯ production in RVLM slices may reflect both pro-oxidative and normal neuromodulatory actions of this peptide.