Faculty Bibliography

Brains of hibernating mammals are protected against a variety of insults that are detrimental to humans and other nonhibernating species. Such protection is associated with a number of physiological adaptations including hypothermia, increased antioxidant defense, metabolic arrest, leukocytopenia, immunosuppression, and hypocoagulation. It is intriguing that similar manipulations provide considerable protection as experimental treatments for central nervous system injury. This review focuses on neuroprotective mechanisms employed during hibernation that may offer novel approaches in the treatment of stroke, traumatic brain injury, and neurodegenerative diseases in humans

During hibernation in Arctic ground squirrels (Spermophilus parryii), O(2) consumption and plasma leukocyte counts decrease by >90%, whereas plasma concentrations of the antioxidant ascorbate increase fourfold. During rewarming, O(2) consumption increases profoundly and plasma ascorbate and leukocyte counts return to normal. Here we investigated the dynamic interrelationships among these changes. Plasma ascorbate and uric acid (urate) concentrations were determined by HPLC from blood samples collected at approximately 15-min intervals via arterial catheter; leukocyte count and hematocrit were also determined. Body temperature, O(2) consumption, and electromyographic activity were recorded continuously. Ascorbate, urate, and glutathione contents in body and brain samples were determined during hibernation and after arousal. During rewarming, the maximum rate of plasma ascorbate decrease occurred at the time of peak O(2) consumption and peak plasma urate production. The ascorbate decrease did not correlate with mouth or abdominal temperature; uptake into leukocytes could account for only a small percentage. By contrast, liver and spleen ascorbate levels increased significantly after arousal, which could more than account for ascorbate clearance from plasma. Brain ascorbate levels remained constant. These data suggest that elevated concentrations of ascorbate [(Asc)] in plasma [(Asc)(p)] provide an antioxidant source that is redistributed to tissues during the metabolic stress that accompanies arousal

Recent evidence suggests that reactive oxygen species (ROS) might act as modulators of neuronal processes, including synaptic transmission. Here we report that synaptic dopamine (DA) release can be modulated by an endogenous ROS, H(2)O(2). Electrically stimulated DA release was monitored in guinea pig striatal slices using carbon-fiber microelectrodes with fast-scan cyclic voltammetry. Exogenously applied H(2)O(2) reversibly inhibited evoked release in the presence of 1.5 mM Ca(2+). The effectiveness of exogenous H(2)O(2), however, was abolished or decreased by conditions that enhance Ca(2+) entry, including increased extracellular Ca(2+) concentration ([Ca(2+)](o); to 2.4 mM), brief, high-frequency stimulation, and blockade of inhibitory D(2) autoreceptors. To test whether DA release could be modulated by endogenous H(2)O(2), release was evoked in the presence of the H(2)O(2)-scavenging enzyme, catalase. In the presence of catalase, evoked [DA](o) was 60% higher than after catalase washout, demonstrating that endogenously generated H(2)O(2) can also inhibit DA release. Importantly, the Ca(2+) dependence of the catalase-mediated effect was opposite to that of H(2)O(2): catalase had a greater enhancing effect in 2.4 mM Ca(2+) than in 1.5 mM, consistent with enhanced H(2)O(2) generation in higher [Ca(2+)](o). Together these data suggest that H(2)O(2) production is Ca(2+) dependent and that the inhibitory mechanism can be saturated, thus preventing further effects from exogenous H(2)O(2). These findings show for the first time that endogenous H(2)O(2) can modulate vesicular neurotransmitter release, thus revealing an important new signaling role for ROS in synaptic transmission

Brain slices gain water when maintained in bicarbonate-buffered artificial cerebro-spinal fluid (ACSF) at 35 degrees C. We previously showed that this edema is linked to glutamate receptor activation and oxidative stress. An additional factor that may contribute to swelling is acidosis, which arises from high CO(2) tension in brain slices. To examine the role of acidosis in slice edema, we added N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) to osmotically balanced ACSF (HEPES-ACSF), thereby increasing buffering capacity beyond that provided by bicarbonate/CO(2). Water gain was markedly inhibited in HEPES-ACSF. After 3 h incubation in HEPES-ACSF at 35 degrees C, water gain was limited to that of fresh slices after 1 h recovery in ACSF at room temperature. The effect of HEPES in decreasing slice water gain was concentration dependent from 0.3 to 20 mM. The inhibition of water gain by HEPES suggests that tissue acidosis is a contributing factor in brain slice edema

Somatodendritic release of dopamine (DA) in midbrain is, at least in part, nonsynaptic; moreover, midbrain DA receptors are predominantly extrasynaptic. Thus somatodendritic DA mediates volume transmission, with an efficacy regulated by the diffusion and uptake characteristics of the local extracellular microenvironment. Here, we quantitatively evaluated diffusion and uptake in substantia nigra pars compacta (SNc) and reticulata (SNr), ventral tegmental area (VTA), and cerebral cortex in guinea pig brain slices. The geometric parameters that govern diffusion, extracellular volume fraction (alpha) and tortuosity (lambda), together with linear uptake (k'), were determined for tetramethylammonium (TMA(+)), and for DA, using point-source diffusion combined with ion-selective and carbon-fiber microelectrodes. TMA(+)-diffusion measurements revealed a large alpha of 30% in SNc, SNr, and VTA, which was significantly higher than the 22% in cortex. Values for lambda and k' for TMA(+) were similar among regions. Point-source DA-diffusion curves fitted theory well with linear uptake, with significantly higher values of k' for DA in SNc and VTA (0.08--0.09 s(-1)) than in SNr (0.006 s(-1)), where DA processes are sparser. Inhibition of DA uptake by GBR-12909 caused a greater decrease in k' in SNc than in VTA. In addition, DA uptake was slightly decreased by the norepinephrine transport inhibitor, desipramine in both regions, although this was statistically significant only in VTA. We used these data to model the radius of influence of DA in midbrain. Simulated release from a 20-vesicle point source produced DA concentrations sufficient for receptor activation up to 20 microm away with a DA half-life at this distance of several hundred milliseconds. Most importantly, this model showed that diffusion rather than uptake was the most important determinant of DA time course in midbrain, which contrasts strikingly with the striatum where uptake dominates. The issues considered here, while specific for DA in midbrain, illustrate fundamental biophysical properties relevant for all extracellular communication

Ascorbic acid (vitamin C) occurs physiologically as the ascorbate anion: a water-soluble antioxidant that is found throughout the body. However, despite the high, homeostatically regulated levels of brain ascorbate, its specific functions in the CNS are only beginning to be elucidated. Certainly, it acts as part of the intracellular antioxidant network, and as such is normally neuroprotective. There is also evidence that it acts as a neuromodulator. A possibly unique role it might have is as an antioxidant in the brain extracellular microenvironment, where its concentration is modulated by glutamate-ascorbate heteroexchange at glutamate uptake sites. Ongoing studies of ascorbate and glutamate transporters should lead to rapid progress in understanding ascorbate regulation and function

Carbon-fiber microelectrodes and voltammetric methods have been used extensively for detection of neurochemical substances in brain tissue. We previously reported that calibration factors for dopamine obtained with these electrodes and fast-scan cyclic voltammetry are 2-3-fold higher in nonphysiological phosphate-buffered saline (PBS) than in artificial cerebrospinal fluid (ACSF) containing Ca2+ and Mg2+. In the present report, we describe the media-dependence of calibration factors for other neurochemicals. Strikingly, whereas electrode sensitivities to dopamine and the indoleamine serotonin were lower in ACSF than in PBS, those for the acid metabolites of these neurotransmitters were roughly 2-fold higher in ACSF than PBS. The data are consistent with adsorption or repulsion of these molecules by a negatively charged carbon-fiber electrode surface. One consequence of this pattern is that electrodes calibrated in PBS, rather than Ca2+-and Mg2+-containing media, will have a 2-6-fold lower amine-to-metabolite selectivity ratio in brain tissue than expected from in vitro calibration. More generally, the data suggest that the sensitivity of carbon-fiber-based microsensors to many organic molecules will be media-dependent. Thus, calibration in appropriate physiological media is essential for accurate tissue measurements

Carbon-fiber microelectrodes and voltammetric methods have been used extensively for the detection of dopamine in brain tissue in vivo and in vitro. Voltammetric microelectrodes are often calibrated in non-physiological media, like phosphate-buffered saline, rather than in oxygenated physiological media. Here, we determined dopamine calibration factors (nA microM-1) in several defined solutions for two types of carbon-fiber electrode used with fast-scan cyclic voltammetry. For both electrode types, dopamine calibration factors, and thus electrode sensitivities, were 2-3-fold higher in phosphate- or HEPES-buffered saline than in a bicarbonate-based artificial CSF (ACSF) that reflected that normal ionic composition of brain extracellular fluid. Removal of Ca2+ and Mg2+ from ACSF eliminated this difference. Because extracellular Ca2+ concentration ([Ca2+]o) can fall under stimulation conditions used to elicit dopamine release, we also evaluated the size of stimulated [Ca2+]o shifts in guinea pig midbrain slices using ion-selective microelectrodes. The [Ca2+]o decreases were less than 100 microM, which was well below the mM decreases observed to alter DA sensitivity. Consequently, calibration data obtained in normal physiological solutions should be valid under conditions of mild stimulation. Moreover, calibration in divalent cation-free media will cause calculated DA levels to be underestimated and should be avoided, unless appropriate for a given experimental paradigm

Compartmentalization of brain ascorbate and glutathione between neurons and glia has been a source of controversy. To address this question, we determined the ascorbate and glutathione contents of brain tissue with defined, but varying, densities of neurons and glia. In developing rat cortex and hippocampus, glutathione content rose during gliogenesis, while ascorbate fell. By contrast, ascorbate, but not glutathione, increased markedly during granule cell proliferation and maturation in the developing cerebellum. Similarly, in tissue from adult cerebral cortex of species with distinct neuron densities, ascorbate content increased linearly with increasing neuron density in the order: human<rabbit<guinea-pig<rat<mouse, whereas glutathione was relatively constant. These data suggest that ascorbate predominates in neurons, whereas glutathione is slightly predominant in glia. Quantitative analysis of ascorbate and glutathione contents in these studies combined with appropriate intra- and extracellular volume fraction data permitted calculation of concentrations of ascorbate in neurons (10 mM) and glia (0.9 mM), and glutathione in neurons (2.5 mM) and glia (3.8 mM). The relative accuracy of these values was confirmed by their use in a model that reliably predicted changes in ascorbate and glutathione levels in rat cortex during the first three postnatal weeks and into adulthood. These findings not only provide new information about the intracellular composition of neurons and glia, but also have implications for understanding the roles of ascorbate and glutathione in normal brain function, as well as neuron and glia involvement in disease states linked to oxidative stress