A well documented feature of brain hypoxia/ischemia is a change in pH, both intracellular pH )pHi) and extracellular pH (pHo). Recent evidence indicates that pH, especially pHo modulates hypoxic/ischemic injury in the mammalian central nervous system (CNS). It is clear that pHo disturbances accompanying hypoxicaanoxia are initiated by primary changes in neuron and/or astrocyte pHi. Disturbances in neuron and astrocyte pHi are critical in their own right because of the pH sensitivity of ion conductances and responses to neurotransmitters. Thus, it is critical to understand how pHi homeostasis is affected by hypoxica, as well as by the ancillary disturbances that accompany hypoxic/ischemia. The proposed research would investigate the mechanisms underlying pHi regulation in pyramidal neurons and astrocytes freshly isolated from CA1 region of the rat hippocampus, with the goal of understanding how the pHi physiologies of neurons and astrocytes interact with one another, via the extracellular space, during hypoxia/ischemia. We will load cells with a pH-sensitive dye, and compute pHi from fluorescence signals. We also will use electrophysiological approaches for monitoring membrane voltage (Vm) and assessing neuronal function. Our general approach will be to study pyramidal neurons and astrocytes freshly isolater from the CA1 region of the hippocampus, both from immature (3-10 day ole) and mature (22030 day old) rats. The proposal has three major aims. First, to understand how acute, graded hypoxia and chronic hypoxia affect steady- state pHi, as well as individual acid-base transporters responsible for pHi homeostasis in neurons and astrocytes. Of particular interest are the observations that the neurons can exist in both a low- and high-pHi state, that the distribution between low- and high pHi neurons is age dependent, and that the neurons sometimes spontaneously shift from the low-to the high-pHi state. Second, to understand how hypoxia/ischemia related disturbances such as [K+]o glutamate, GABA, (glu)o, and deltas in pHo affect pHi and individual transporters. Third, to determine how neuronal function is affected by pHi and pHo changes. We will use electrophysiological techniques to assess excitability both in freshly dissociated CA1 neurons, and in CA1 neurons examined in situ in hippocampal slices. We will corroborate the electrophysiological data in slices with confocal measurements of pHi. We will use conventional fluorescent microscopy and dyes to monitor pHi (and also [Ca++]i [Na+]i and voltage in single, freshly dissociated cells attached to cover slips. In addition, we will use the patch-clamp technique to monitor Vm. The proposed work would lead to the first comprehensive description of how hypoxia/ischemia and hypoxia/ischemia-related disturbances in extracellular parameters affect pHi regulation in either a neuron or an astrocyte from a mammalian brain. Its focus developmental changes could lead to a better understanding of how hypoxia/ischemia in the neonatal period affect brain function.
| Azad, Priti; Zhao, Huiwen W; Cabrales, Pedro J et al. (2016) Senp1 drives hypoxia-induced polycythemia via GATA1 and Bcl-xL in subjects with Monge's disease. J Exp Med 213:2729-2744 |
| Yao, Hang; Azad, Priti; Zhao, Huiwen W et al. (2016) The Na+/HCO3- co-transporter is protective during ischemia in astrocytes. Neuroscience 339:329-337 |
| Jha, Aashish R; Zhou, Dan; Brown, Christopher D et al. (2016) Shared Genetic Signals of Hypoxia Adaptation in Drosophila and in High-Altitude Human Populations. Mol Biol Evol 33:501-17 |
| Pamenter, Matthew E; Haddad, Gabriel G (2015) High-throughput cell death assays. Methods Mol Biol 1254:153-63 |
| Salameh, Ahlam Ibrahim; Ruffin, Vernon A; Boron, Walter F (2014) Effects of metabolic acidosis on intracellular pH responses in multiple cell types. Am J Physiol Regul Integr Comp Physiol 307:R1413-27 |
| Gu, Xiang Q; Pamenter, Matthew E; Siemen, Detlef et al. (2014) Mitochondrial but not plasmalemmal BK channels are hypoxia-sensitive in human glioma. Glia 62:504-13 |
| Gersten, Merril; Zhou, Dan; Azad, Priti et al. (2014) Wnt pathway activation increases hypoxia tolerance during development. PLoS One 9:e103292 |
| Udpa, Nitin; Ronen, Roy; Zhou, Dan et al. (2014) Whole genome sequencing of Ethiopian highlanders reveals conserved hypoxia tolerance genes. Genome Biol 15:R36 |
| Douglas, Robert M; Chen, Alice H; Iniguez, Alejandra et al. (2013) Chemokine receptor-like 2 is involved in ischemic brain injury. J Exp Stroke Transl Med 6:1-6 |
| Parker, Mark D; Boron, Walter F (2013) The divergence, actions, roles, and relatives of sodium-coupled bicarbonate transporters. Physiol Rev 93:803-959 |
Showing the most recent 10 out of 173 publications
