A quantitative analysis of l‐glutamate‐regulated Na⁺ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics

Abstract

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.

Astrocytes generate Na + -mediated metabolic waves

“…Glutamate-evoked Na ϩ uptake in astrocytes and the resulting activation of the Na ϩ ͞K ϩ -ATPase have been identified as a signal coupling excitatory neuronal activity to increased glucose utilization (5,7). In addition, astrocytes have the ability of transmitting Ca i 2ϩ elevations from cell to cell (17).…”

“…extracellular concentration of glutamate, reaching maximal values of 30-40 mM (5). In addition, an extracellular glutamate concentration of 10 M, inducing an Na i ϩ elevation of Ϸ8 mM, already markedly increases the energy demands of the astrocytes by activating the Na ϩ ͞K ϩ ATPase (5, 6).…”

“…Cortical astrocytes in primary culture were obtained from 1-to 3-day-old OF1 mice as described (5). Cells were grown for 2-5 weeks on glass coverslips in DME medium supplemented with 10% FCS.…”

“…Glutamate is cotransported with three Na ϩ ions by excitatory amino acid transporters expressed in astrocytes (3,4), inducing an intracellular Na ϩ (Na i ϩ ) elevation. This Na i ϩ elevation results in an activation of the Na ϩ ͞K ϩ -ATPase, causing an increased energy demand in astrocytes (5,6), which in turn enhances cellular glucose utilization and glycolysis (7). Lactate, the end-product of glycolysis, is released by astrocytes and could serve as metabolic substrate for neurons in conjunction with glucose (2).…”

Differential Effects of NMDA and AMPA Glutamate Receptors on Functional Magnetic Resonance Imaging Signals and Evoked Neuronal Activity during Forepaw Stimulation of the Rat

“…The metabolic response, on the one hand, is probably mediated by glial transport of glutamate and its recycling through mechanisms involving changes in intracellular Na ϩ concentration (Pellerin and Magistretti, 1994;Chatton et al, 2000;Bernardinelli et al, 2004). This hypothesis has received in vivo experimental support from both magnetic resonance spectroscopy studies (for review, see Rothman et al, 2003) and recent findings that increases in cerebral glucose utilization after somatosensory or visual stimulation are markedly reduced in knock-out mice deficient for each of the two glial glutamate transporters GLAST and GLT-1 (Voutsinos-Porche et al, 2003a,b;Herard et al, 2005).…”