Channel-mediated lactate release by K⁺-stimulated astrocytes

Abstract

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

Keywords: fluorescence microscopy; genetically encoded nanosensor; gliotransmission; membrane depolarization.

Figure 1.

Astrocytes maintain a cytosolic lactate reservoir that is depleted in the short term by high [K⁺]_o. A, The FRET lactate sensor Laconic expressed in the cytosol of cultured astrocytes, showing mTFP (blue), Venus (green), and the ratio between mTFP and Venus. Scale bar, 20 μm. B, Laconic was first depleted of lactate by superfusion with 10 mm pyruvate (pyr), and then saturated with 5 mm lactate and 2 mm glucose (San Martín et al., 2013). C, Resting lactate level in 183 cells (17 experiments), estimated with the protocol in B. The top x-axis indicates lactate concentration, according to the kinetic parameters estimated in vitro. Equilibrium concentration (MCT_eq) of the monocarboxylate transporters. D, Response of intracellular lactate to 1 μm AR-C155858. The closed symbol represents the average change after 5 min of MCT blockage. E, Effect of 0.5, 1, and 9 mm [K⁺]_o additions on the lactate level of an astrocyte. Resting [K⁺]_o = 3 mm. F, Initial rates of K⁺-induced lactate depletion. The open symbol represents the initial rate of lactate depletion after exposure to 3 mm Ba²⁺. G–I, Effect of a 9 mm [K⁺]_o rise on glucose consumption (G), cytosolic NADH/NAD⁺ ratio (H), and cytosolic pyruvate level (I). J, Protoplasmic astrocytes expressing Laconic observed in an acute cortical slice at low (top) and high (bottom) magnification. Scale bar, 20 μm. K, L, The effects of increasing [K⁺]_o by 9 mm (K) or by adding 3 mm Ba²⁺ (L) on the lactate level of protoplasmic astrocytes are shown.

Figure 2.

Intracellular lactate accumulation in response to OXPHOS inhibition. Astrocytes were exposed to 5 mm azide while measuring glucose, NADH/NAD⁺ ratio, pyruvate or lactate. The effect of 12 mm [K⁺]_o on pyruvate levels (from Fig. 1I) is shown for comparison.

Figure 3.

Early depletion of astrocytic lactate during local electrical stimulation in vivo. The strength of electrical stimulation was modulated by varying the distance between cells and the tip of the stimulation pipette, giving a weaker stimulation at 300–500 μm and a stronger stimulation at 20–200 μm. A, Imaging of Laconic expressed in somatosensory cortex astrocytes. The location of the stimulation pipette is indicated. Scale bar, 20 μm. B, Data from a single experiment. C, Early response to weaker (n = 3 experiments) and stronger stimulation (n = 7 experiments). D, Extended time course for stronger stimulation (n = 7 experiments) E, Left, Intracellular lactate level in cultured astrocytes exposed first to a 9 mm increase in [K⁺]_o, and 30 s later, to a rise in extracellular lactate level from 1 to 5 mm. Right, Data from three cells from the same field (black) are compared with the average depletion elicited by a 9 mm [K⁺]_o increase at constant extracellular lactate concentration of 1 mm (white).

Figure 4.

Fast astrocytic lactate release detected with a lactate sniffer. A, HEK293 cells expressing Laconic (sniffers) were seeded on top of an astrocytic culture and imaged by 3D confocal microscopy (green). A second 3D reconstruction was performed after ester loading the culture with calcein (gray). B, Response of a sniffer positioned on top of an astrocytic culture to increasing concentrations of [K⁺]_o (4, 6, and 12 mm), 3 mm Ba²⁺, 1 mm lactate, and 10 mm pyruvate. The experiment was performed in 2 mm glucose and 0 mm lactate. C, Typical response of sniffers to astrocytic culture exposure to 12 mm K⁺. D, Correlation between the amplitude and the initial rate of the response of the sniffer to 12 mm K⁺. E, Astrocytic lactate depletion by 12 mm K⁺ in the absence (gray symbols and bars) and presence (white symbols and bars) of 1 μm AR-C155858.

Figure 5.

A lactate-permeable channel modulated by membrane depolarization and lactate. A, Traces obtained under lactate-rich (145 mm) and chloride-low (10 mm) pipette conditions in the cell-attached mode at 100 and 40 mV applied potential. B, Left, Recording from an astrocyte patch held at 0 mV (80 mV applied potential) before and during exposure of the cell to 3 mm Ba²⁺. Right, The channel activity in eight similar experiments is illustrated as NP_o, the product of the number of channels in the patch and the open probability of each channel. C, Left, Effect of increasing bath lactate concentration from 1 to 10 mm on channel activity. Right, Summary of four similar experiments. D, A cell was sequentially bathed with 3 mm Ba²⁺ and/or 10 mm lactate for periods of 5 min as shown.

Figure 6.

Further characterization of the lactate-permeable channel. A, Traces obtained with a lactate-rich (145 mm), chloride-low (10 mm) medium, with pipette in the cell-attached mode at 120, 100, 60, and 0 applied potential. B, Single-channel current obtained at increasing nominal patch potentials (calculated assuming an astrocytic membrane potential of −80 mV). The equilibrium potentials estimated for lactate and chloride are indicated. C, A cell was sequentially bathed with 10 mm lactate and 3 mm Ba²⁺ for periods of 5 min as shown.

Figure 7.

Stimulated astrocytes can extrude lactate against a lactate gradient. A, Astrocytes were sequentially exposed to 10 mm pyruvate and then to 5 and 10 mm lactate in the presence of 3 or 12 mm [K⁺]_o. B, Summary of three similar experiments showing the initial rates of 5 mm lactate accumulation in 3 or 12 mm [K⁺]_o. C, Left, The effect of a 9 mm increase in [K⁺]_o on intracellular lactate was monitored in a single astrocyte before and during exposure to 200 μm DIDS. Right, Summary of similar experiments, with 200 μm DIDS, 500 μm NFA, 500 μm NPPB, 200 μm Cd²⁺, 1 mm probenecid, or 10 μm CBX. Data are the rates of lactate depletion measured over 2 min (n = 3 experiments and 16–32 cells were used for each inhibitor). D, Top, The effects of a 9 mm increase in [K⁺]_o or the addition of 3 mm Ba²⁺ on intracellular lactate level were monitored in single astrocytes before and during exposure to 200 μm Cd²⁺. Bottom, Initial rates measured during the first minute of exposure to [K⁺]_o or 3 mm Ba²⁺ in the absence or presence of 200 μm Cd²⁺.

Figure 8.

Activity-dependent channel-mediated lactate release by astrocytes. Resting astrocytes maintain a standing reservoir of cytosolic lactate, the result of a dynamic balance between glycolytic production and MCT-mediated lactate export. Active neurons release K⁺, which depolarizes the astrocytic plasma membrane (ΔV_m) and activates the lactate-permeable channel, resulting in lactate release, leading to higher [lactate]_o and further lactate release through a positive feedback. Neurons may sense lactate through HCA1 (Bozzo et al., 2013; Lauritzen et al., 2013) and other surface lactate receptors (Tang et al., 2014), or after internalization of lactate via MCTs.