Author response: The Na+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cellular ATP that is consumed to perform energetically expensive tasks must be replenished by new ATP through the activation of metabolism. Neuronal stimulation, an energetically demanding process, transiently activates aerobic glycolysis, but the precise mechanism underlying this glycolysis activation has not been determined. We previously showed that neuronal glycolysis is correlated with Ca2+ influx, but is not activated by feedforward Ca2+ signaling (Díaz-García et al., 2021a). Since ATP-powered Na+ and Ca2+ pumping activities are increased following stimulation to restore ion gradients and are estimated to consume most neuronal ATP, we aimed to determine if they are coupled to neuronal glycolysis activation. By using two-photon imaging of fluorescent biosensors and dyes in dentate granule cell somas of acute mouse hippocampal slices, we observed that production of cytoplasmic NADH, a byproduct of glycolysis, is strongly coupled to changes in intracellular Na+, while intracellular Ca2+ could only increase NADH production if both forward Na+/Ca2+ exchange and Na+/K+ pump activity were intact. Additionally, antidromic stimulation-induced intracellular [Na+] increases were reduced >50% by blocking Ca2+ entry. These results indicate that neuronal glycolysis activation is predominantly a response to an increase in activity of the Na+/K+ pump, which is strongly potentiated by Na+ influx through the Na+/Ca2+ exchanger during extrusion of Ca2+ following stimulation. Editor's evaluation The authors investigate mechanisms that regulate glycolytic ATP production in neurons. They conclude that the cytosolic Na+, not Ca2+, and the activity of the Na+/K+ pump drive glycolysis. The study is conceptually significant as it seeks to determine how neuronal glycolysis is coupled to electrical activity. The study is thoughtful, uses sophisticated fluorescence lifetime imaging technology and clever experimental designs, and as such provides new insights into how electrical activity regulates glycolysis. https://doi.org/10.7554/eLife.81645.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Cellular energy consumption must be balanced by new energy production through the activation of metabolism. In neurons, energy consumption is strongest following excitation (Attwell and Iadecola, 2002; Attwell and Laughlin, 2001; Engl and Attwell, 2015; Howarth et al., 2012; Lennie, 2003; Yu et al., 2018). Accordingly, neuronal excitation triggers a transient activation of glycolysis, which is measured as an increase to neuronal cytoplasmic NADH:NAD+ (NADHCYT) that can last for several minutes after the excitatory event (Díaz-García et al., 2017; Díaz-García et al., 2021a). The specific mechanism that drives neuronal glycolysis activation has not been identified. It seems highly plausible that glycolysis activation in neurons could be driven primarily by an increase in the activity of ion-ATPases, the ATP-powered ion transporters that move ions against their electrochemical gradients by (through a series of reaction steps; Post et al., 1965; Sen and Post, 1964) converting ATP into ADP and Pi (Biondo et al., 2021; Calì et al., 2018; Chemaly et al., 2018; Kaplan, 2002). Ion-ATPases are estimated to be responsible for most of the energy consumption associated with neuronal signaling and are closely coupled to glycolytic enzymes across many cell types (Ames, 2000; Dhar-Chowdhury et al., 2007; Dzeja and Terzic, 2003; Xu et al., 1995). Moreover, activation of glycolysis by increased ion-ATPase activity could be multimodal: a decrease to cellular ATP would stimulate glycolysis by mass action at the ATP-generating steps, while an increase to cellular ADP and Pi would stimulate glycolysis by allosterically activating phosphofructokinase and by mass action at both the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase enzyme complex and pyruvate kinase steps (Fothergill-Gilmore and Michels, 1993; Kemp and Foe, 1983; Nelson et al., 2008; Schöneberg et al., 2013; Tomokuni et al., 2010). There are several ion-ATPases (also called ion pumps) that regulate neuronal Na+ and Ca2+ changes (Figure 1A). Na+ is handled exclusively by the Na+/K+-ATPase (or Na+/K+ pump), which uses 1 ATP molecule to export 3 Na+ and import 2 K+ across the plasma membrane. Ca2+ is handled by a diverse set of active transporters, including the plasma membrane Ca2+-ATPase (PMCA), which exports 1 Ca2+ across the plasma membrane per ATP (Niggli et al., 1982; Thomas, 2009), and the sarco-/endo-plasmic reticulum Ca2+-ATPase (SERCA), which pumps 2 Ca2+ into the endoplasmic reticulum lumen per ATP (Tran et al., 2009). Aside from ATPases, Ca2+ is also regulated by the Na+/Ca2+-exchanger (NCX) (Lee et al., 2009), a secondary active transporter that uses the electrochemical energy stored in the plasma membrane Na+ gradient (built by the Na+/K+ pump) to actively export 1 Ca2+ by importing 3 Na+. Any increase to neuronal Na+ or Ca2+ will increase the activity of their respective ion transporters. Figure 1 with 3 supplements see all Download asset Open asset NADHCYT production is strongly influenced by Na+, but not by Ca2+CYT. (A) Cartoon showing the NCX transport modes activated by different external [Na+] and [Ca2+] conditions and their expected effects on the activities of ion pumps and the production of NADHCYT from glycolysis activation. Reverse NCX transport (left schematic) increases intracellular [Ca2+], which increases the activities Ca2+ pumps and Ca2+ transport into mitochondria (magenta arrows). Forward NCX transport (right schematic) increases intracellular [Na+], which increases the activity of the Na+/K+ pump (orange arrows). The bracket below each schematic indicates the NCX transport mode activated by the external solution changes in (C). Transport stoichiometries are not indicated. Abbreviations: Na+/Ca2+-exchanger (NCX), Na+/K+-ATPase (NKA), plasma membrane Ca2+-ATPase (PMCA), sarco-/endo-plasmic reticulum Ca2+-ATPase (SERCA), mitochondrial Ca2+ uniporter (MCU), mitochondrial Na+/Ca2+-exchanger (mNCX), endoplasmic reticulum (ER). (B) Representative fluorescence lifetime (LT) traces of Peredox (top trace) and RCaMP (bottom trace) from a DGC bathed in ACSF. Antidromic stimulation was delivered at the time point indicated by the arrow along the RCaMP trace, which transiently increases both NADHCYT and Ca2+CYT. (C) Fluorescence LT traces of Peredox (top) and RCaMP (bottom) from a DGC showing how external Na+ and Ca2+ changes affect NADHCYT and Ca2+CYT. The bars above the Peredox trace indicate the external [Na+] and [Ca2+]. NADHCYT was decreased by switching the bath solution from ACSF (147 mM Na+ and 2 mM Ca2+) to a solution with nominally 0 Na+ and 0 Ca2+ (cyan shading). Ca2+CYT was elevated by applying 0.5 mM Ca2+ with 0 Na+ to activate reverse NCX transport (magenta shading), and NADHCYT decreased further. NADHCYT was strongly increased after activating forward NCX transport by the subsequent removal of external Ca2+ and application of 147 mM Na+ (orange shading). (D) Box plots of the fluorescence LTs of Peredox (top) and RCaMP (bottom) showing the effects of the external Na+ and Ca2+ changes performed in panel C across many DGCs (n=53). The external bath conditions for each box plot are listed at the bottom of the RCaMP plot in chronological order from left to right. The colors of each box plot correspond to the colors indicated in (C). The mean Peredox LT values in each condition were: 1.63±0.06 ns in ACSF, 1.52±0.05 ns in 0 Na+ and 0 Ca2+, 1.46±0.09 ns in 0 Na and 0.5 mM Ca2+, and 1.72±0.06 ns in 147 mM Na+ and 0 Ca2+. The mean RCaMP LT values in each condition were: 0.88±0.05 ns in ACSF, 0.88±0.05 ns in 0 Na+ and 0 Ca2+, 1.52±0.15 ns in 0 Na+ and 0.5 mM Ca2+, and 0.93±0.07 ns in 147 mM Na+ and 0 Ca2+. (E) Changes to the Peredox LT relative to the 0 Na+ and 0 Ca2+ condition, after either a Ca2+CYT elevation from reverse NCX transport (Ca, black box, magenta filled diamonds), an influx of Na+ due to forward NCX transport (Na after Ca, black box, orange filled diamonds), or application of Na+ without forward NCX (Na no Ca, gray box, orange open diamonds). The mean Peredox LT changes were: –0.07±0.08 ns (n=53) for Ca2+CYT elevation, 0.20±0.05 ns (n=53) for Na+ influx via forward NCX, and 0.07364±0.04565 ns (n=19) for Na+ application without forward NCX. Statistical significance between ‘Ca’ and ‘Na after Ca’ is indicated by a paired Wilcoxon test and between ‘Na after Ca’ and ‘Na no Ca’ by a Mann-Whitney test. (F) Effect of external Na+ on the return of Ca2+CYT to baseline following a reverse NCX transport-mediated Ca2+ influx. The mean decay of the RCaMP LT following the Ca2+CYT increase (normalized to the peak RCaMP LT value) is shown when the external solution contained either 147 mM Na+ (solid line, orange SD shading, n=53) or 0 Na+ (dashed line, green SD shading, n=49). Decay data in 147 mM Na+ were from the same DGCs as in (D) and (E), while data in 0 Na+ were from the same DGCs as Figure 1—figure supplement 1B; a representative trace of this experiment is shown in Figure 1—figure supplement 1A. Transient increases to neuronal cytoplasmic Ca2+ (Ca2+CYT) induced by excitation are positively correlated with transient increases in subsequent NADHCYT production (Díaz-García et al., 2017; Díaz-García et al., 2021a); in other words, stronger stimulations evoke larger increases to both Ca2+CYT and NADHCYT. At first glance, this positive correlation could be (mis)interpreted as glycolysis being driven primarily by an increase in the activities of Ca2+ pumps (PMCA and SERCA) as Ca2+ is pumped from the cytoplasm. However, this readout of neuronal NADHCYT and Ca2+CYT lacks information about stimulation-induced increases to intracellular Na+, which also likely covary with stimulation strength, so a contribution of increased Na+/K+ pump activity to glycolysis activation cannot be excluded. The Na+/K+ pump is primed to be the predominant driving force underlying neuronal glycolysis activation. Many reports estimate that the Na+/K+ pump consumes ~50% of total brain energy (Ames, 2000; Astrup et al., 1981; Engl and Attwell, 2015; Milligan and McBride, 1985; Whittam, 1962) and uses substantially more ATP than Ca2+ pumps both during neurotransmission and at rest (Attwell and Laughlin, 2001; Harris et al., 2012; Rolfe and Brown, 1997). Furthermore, since Ca2+CYT regulation in some neurons depends strongly on the NCX (Lee et al., 2009), a transient Ca2+CYT increase could indirectly elevate intracellular [Na+], which would potentiate any increase in Na+/K+ pump activity due to channel-mediated Na+ entry. To shed light on the mechanism of neuronal glycolysis activation, we investigated the coupling of Na+/K+ pump and Ca2+ pump activities to NADHCYT production in the somas of hippocampal dentate granule cells (DGCs) within acute brain slices by using two-photon fluorescence imaging of genetically encoded fluorescent biosensors and fluorescent dyes. The data provide compelling evidence indicating that increased activity of the Na+/K+ pump is the predominant driver of neuronal glycolysis activation, whereas increased activities of Ca2+ pumps appear to be negligibly coupled to glycolysis. Results Cytoplasmic NADH production is strongly influenced by Na+, but not Ca2+ We simultaneously monitored changes in neuronal metabolism and ion fluxes in DGC somas by expressing Peredox and RCaMP, two genetically encoded fluorescent biosensors that report NADH:NAD+ (NADHCYT) and free [Ca2+], respectively (Akerboom et al., 2013; Hung et al., 2011; Mongeon et al., 2016). Analyte binding to Peredox (NADH in competition with NAD+) or RCaMP (Ca2+) alters each sensor’s fluorescence lifetime (LT), the average time that the fluorophore spends in the excited state prior to its return to the ground state (Lakowicz, 2006). The fluorescence LTs of Peredox and RCaMP are increased (i.e., extended in average duration) by NADHCYT or Ca2+CYT increases, respectively. Our previous observations demonstrated that NADHCYT increases are strongly tied to glycolysis (Díaz-García et al., 2017; Díaz-García et al., 2021a) and occur regardless of changes in mitochondrial metabolism: electrical-stimulation-induced increases to NADHCYT were substantially diminished by iodoacetic acid (an inhibitor of GAPDH) but not by AOA (an inhibitor of MAS) and were not affected by impaired NADH production in mitochondria (i.e., in the presence of inhibitors of both the mitochondrial pyruvate carrier and lactate dehydrogenase; Figure 2D in Díaz-García et al., 2021a). Thus, acute NADHCYT changes arise directly from changes in glycolytic flux and are not strongly influenced by changes in NADH consumption by the mitochondria. We began our investigations on the coupling of the Na+/K+ pump or Ca2+ pumps to glycolysis activation by recapitulating our previous findings: that electrical stimulation of DGCs transiently increases their NADHCYT production (Díaz-García et al., 2017; Díaz-García et al., 2021a). Representative LT traces of Peredox and RCaMP, when both sensors were expressed in the cytoplasm of a DGC within a hippocampal slice that was bathed in ACSF (Figure 1B), show that an antidromic stimulation event delivered from an electrode placed in the hippocampal hilus causes both a fast transient increase of the RCaMP LT, reflecting a transient increase to Ca2+CYT, and a slower, longer-lasting transient increase of the Peredox LT, reflecting transient overproduction of NADHCYT by glycolysis. But since neuronal stimulation triggers influxes of both Na+ and Ca2+, the glycolysis activation could result from increases to both Na+/K+ pump and Ca2+ pump activities. To differentiate how changes in the activities of the Na+/K+ pump or Ca2+ pumps affect glycolysis, we measured how NADHCYT production is affected by separate elevations of either intracellular Na+ or Ca2+CYT (Figure 1C). If glycolysis is preferentially coupled to the activity of either the Na+/K+ pump or Ca2+ pumps, then NADHCYT production should be coupled to changes in the levels of the transported ion (Na+ or Ca2+). Simultaneous removal of both Na+ and Ca2+ from the bath solution (by external ion substitution) decreased the Peredox LT from its baseline in ACSF (Figure 1C), which indicates a decrease to NADHCYT; we can attribute this NADHCYT decrease entirely to the removal of Na+, since removing only Ca2+ slightly increases NADHCYT (cf. Figure 6—figure supplement 1b in Díaz-García et al., 2021a). This NADHCYT decrease suggests that the rate of glycolysis can be slowed by depleting the intracellular [Na+], which would decrease the activity of the Na+/K+ pump. The RCaMP LT was not affected by the removal of Na+ and Ca2+ (Figure 1C), indicating that Ca2+CYT was maintained at or below resting levels, near the floor of RCaMP’s dynamic range (Akerboom et al., 2013). Following the removal of external Na+ and Ca2+, we added each ion back one at a time to separately increase either intracellular Na+ or Ca2+. We increased Ca2+ by applying 0.5 mM external Ca2+ in the nominal absence of external Na+ to facilitate Ca2+ entry via reverse NCX transport, and the expected strong increase in Ca2+CYT was confirmed by an increase in the RCaMP LT (Figure 1C, during the magenta shaded interval). However, this increase to Ca2+CYT was not associated with an increase to NADHCYT. In fact, the opposite occurred: Ca2+CYT elevation decreased the Peredox LT even further from the 0 Na+ 0 Ca2+ bath condition (possibly due to the extrusion of Na+ during reverse NCX; see Discussion). At this point, with Ca2+CYT elevated, we removed 0.5 mM external Ca2+ and re-applied external Na+ to facilitate strong Na+ influx via forward NCX transport (in exchange for Ca2+CYT), which promptly increased NADHCYT. Following the external Na+ application, Ca2+CYT returned to baseline levels (Figure 1C, orange shaded interval). The decrease to Peredox LT by removing external Na+ and the increase to Peredox LT from re-applying external Na+, across many DGCs (Figure 1D), show that NADHCYT is strongly influenced by Na+. Relative to the Peredox LT in the 0 Na+ 0 Ca2+ bath condition, the Peredox LT was decreased when Ca2+CYT was elevated but increased following external Na+ re-application (Figure 1E), which suggests that glycolysis activation is strongly tied to the activity of the Na+/K+ pump, but not strongly tied to the activity of Ca2+ pumps (nor to the increase in Ca2+CYT per se, e.g., via a signaling mechanism). Our ion substitution experiments also hinted that both glycolysis activation and Ca2+CYT regulation in DGCs are dependent on forward NCX transport. A Ca2+CYT increase (from reverse NCX transport) returned faster to baseline levels if external Na+ was present (i.e., if forward NCX was activated, Figure 1F; and compare Figure 1C with Figure 1—figure supplement 1A), and NADHCYT did not increase following a Ca2+CYT increase if external Ca2+ was removed without re-applying external Na+ (Figure 1—figure supplement 1A and B). NADHCYT production increased when external Na+ was removed and re-applied without previously elevating Ca2+CYT (i.e., without activating forward NCX, Figure 1—figure supplement 1C and D), but this NADHCYT increase was weaker than when external Na+ was re-applied while Ca2+CYT was elevated (i.e., when forward NCX was activated, Figure 1D and E). The inability of a Ca2+CYT elevation to measurably stimulate NADHCYT production in the absence of external Na+ suggests that any increase in activity of PMCA and SERCA to pump Ca2+ from the cytoplasm does not substantially activate glycolysis, even during the prolonged Ca2+CYT increases in our ion substitution experiments. But maybe inhibiting one of these Ca2+ pumps, to divert more Ca2+CYT to be extruded by the non-inhibited pump, could boost the activity of the other and consequently amplify its associated effect on glycolysis. We tested this possibility by inhibiting SERCA, which compared to PMCA has more specific pharmacology and uses less ATP per Ca2+ transported. Thapsigargin (1 μM, a specific SERCA inhibitor) increased both the NADHCYT and Ca2+CYT transients evoked by antidromic electrical stimulations in ACSF (Figure 1—figure supplement 2A and B) as well as the ∆Peredox/∆RCaMP ratio (Figure 1—figure supplement 2C). These effects are consistent with SERCA inhibition and a diversion of Ca2+CYT to other clearance pathways that are either more closely coupled to glycolysis or that transport fewer ions per ATP utilized. However, reverse-NCX-mediated Ca2+CYT increases in the presence of 1 μM thapsigargin (and absence of external Na+) still failed to increase NADHCYT (Figure 1—figure supplement 1B), meaning that any putative amplification of PMCA activity after SERCA inhibition was still not enough to drive measurable glycolysis activation. We have previously shown that electrical stimulation increases DGC mitochondrial Ca2+ (Ca2+MITO) from Ca2+ entry through the mitochondrial Ca2+ uniporter (MCU) (Díaz-García et al., 2021a). Could Ca2+CYT’s failure to activate NADHCYT production during our ion substitution experiments be due to a lack of activation of some unknown Ca2+MITO-dependent process? This seems unlikely, as bath-applied 0.5 mM external Ca2+ in the absence of external Na+ increased the fluorescence LT of mitochondrially targeted RCaMP (Figure 1—figure supplement 3A and B), indicating that Ca2+MITO is increased by reverse-NCX-mediated Ca2+ entry. Ca2+MITO slightly decreased when external Ca2+ was removed, but complete return of Ca2+MITO to baseline levels was not achieved until external Na+ was applied, demonstrating that Ca2+MITO regulation is dependent on Na+, like Ca2+CYT. In summary, these ion substitution experiments show that neuronal NADHCYT production is closely coupled to the flux of Na+, and that a Ca2+CYT elevation is not sufficient to elevate NADHCYT regardless of whether it is pumped by SERCA, pumped by PMCA, or taken up into mitochondria via MCU. They also show that NADHCYT production is activated more strongly by conditions that favor forward NCX transport that exchanges Ca2+CYT for Na+, and that regulation of both DGC Ca2+CYT and Ca2+MITO depends on Na+. These observations suggest that neuronal glycolysis is both strongly coupled to the activity of the Na+/K+ pump and sensitive to Na+ import via forward NCX transport, which would become activated following an excitatory event where Ca2+CYT must be removed. Malate-aspartate shuttle activity does not mask a Ca2+-induced increase to NADHCYT The malate-aspartate shuttle (MAS) is a major pathway that recycles NADHCYT by transferring reducing equivalents from NADHCYT to mitochondria (McKenna et al., 2006; Satrústegui and Bak, 2015). MAS activity is activated by Ca2+CYT elevations but is reduced by Ca2+MITO elevations and Satrústegui and Bak, which likely that neuronal excitation would a MAS activation by a the Ca2+CYT and Ca2+MITO transient time and 2009). But the state of MAS activity in our ion substitution experiments was since reverse NCX transport prolonged increases to both Ca2+CYT and Ca2+MITO (Figure 1 and Figure 1—figure supplement so it a possibility that Ca2+CYT activation of MAS could increase NADHCYT and any increase to NADHCYT production due to increased activity of Ca2+ we tested if a Ca2+CYT elevation could increase NADHCYT after MAS inhibition (Figure Figure 2 Download asset Open asset Malate-aspartate shuttle activity does not a NADHCYT increase by elevated Ca2+CYT. (A) Representative fluorescence lifetime (LT) traces of Peredox (top) and RCaMP (bottom) from a solution changes are indicated by the bars above the Peredox The in ACSF (147 mM Na+ and 2 mM where the of mM AOA strongly increases the Peredox of mM pyruvate the Peredox LT back to Following external Na+ and Ca2+ were removed (cyan shading), which slightly decreased the Peredox The of 0.5 mM Ca2+ in 0 Na+ to stimulate reverse NCX transport increased the RCaMP LT and further decreased the Peredox LT (magenta shading). the removal of external Ca2+ and of external Na+ to stimulate forward NCX transport (orange increased the Peredox LT and the RCaMP LT back to (B) Box plots of the Peredox (top) and RCaMP (bottom) fluorescence LTs in the different external solution conditions performed in across many DGCs The external [Na+] and [Ca2+] condition after applying AOA and pyruvate is listed across the bottom in chronological order from left to right. The of the data to the in The mean Peredox and RCaMP LTs in each external condition were (in in 0 Na+ and 0 Ca2+, in 0 Na+ and 0.5 mM Ca2+, and in 147 Na+ and 0 Ca2+. The mean RCaMP LTs were (in in 0 Na+ and 0 Ca2+, in 0 Na+ and 0.5 mM Ca2+, and in 147 Na+ and 0 Ca2+. (C) Relative to the Peredox fluorescence LT induced by the of 0.5 mM Ca2+ in the absence of external Na+ to stimulate reverse NCX transport or 147 mM Na+ in the absence of external Ca2+ to stimulate forward NCX transport The data are from the same DGCs as in The mean values were: ns for ‘Ca’ and ns for Statistical significance from a paired Wilcoxon test is indicated. Representative fluorescence LT traces from a DGC show that Peredox LT was substantially increased by the application of AOA to the enzyme (Figure as previously Díaz-García et al., which is consistent with an inhibition of NADHCYT and elevation of the cytoplasmic This strong increase to Peredox LT, which the of its dynamic the to further NADHCYT increases, so we pyruvate to the of NADH to via lactate and restore the Peredox LT back to its baseline LT in ACSF. We then removed external Na+ and Ca2+, which decreased the Peredox LT from the AOA and pyruvate condition (Figure and then increased Ca2+CYT by applying 0.5 mM external Ca2+ in external Na+ to drive reverse NCX transport. We confirmed the Ca2+CYT increase from the increase to RCaMP LT, but the Ca2+CYT elevation still did not with an increase to NADHCYT. NADHCYT decreased relative to the 0 Na+ 0 Ca2+ baseline (Figure and This that an increase to Ca2+CYT still to stimulate NADHCYT production even when NADHCYT by the MAS is Following the Ca2+CYT we removed 0.5 mM external Ca2+ and re-applied external Na+ to activate forward NCX transport (Figure which increased the Peredox LT (Figure and This that NADHCYT production is coupled to the flux of Na+. these experiments against a of NADHCYT production by a Ca2+CYT activation of NADHCYT via the MAS and that an increase to Na+/K+ pump activity is the major driver of glycolysis. of glycolysis by Ca2+ is by coupling between the Na+/Ca2+-exchanger and the Na+/K+ pump The increase to NADHCYT production by an influx of Na+ (Figure 1 and Figure suggests that glycolysis activation in DGCs is strongly tied to an increase in the activity of the Na+/K+ pump. the NADHCYT increase induced by external Na+ application was stronger if Ca2+CYT was elevated compared to when Ca2+CYT was not elevated (Figure 1E), meaning that forward NCX transport, which Ca2+CYT for Na+, can potentiate glycolysis activation. By these observations with the of glycolysis to increases in Ca2+ pump it that a transient Ca2+CYT elevation would activate glycolysis predominantly by Na+ influx through forward NCX transport, which would then increase the activity of the Na+/K+ pump. We for a to directly test how NADHCYT is affected by inhibition of the Na+/K+ pump or inhibition of forward NCX transport (Figure Na+/K+ pumps are by the Na+/K+ pump have and inhibitor for complete inhibition conditions and and of 2 mM to DGCs bathed in ACSF to Ca2+ and cell (Figure supplement which it to how electrical stimulation-induced NADHCYT transients are affected by complete Na+/K+ pump the activity of the Na+/K+ pump must be even at but complete Na+/K+ pump inhibition neuronal conditions is DGCs bathed in ACSF were more to application of which only the Na+/K+ pump and that have inhibitor and but the stimulation-induced NADHCYT transient was not (Figure supplement This result that in these the is to the activation of glycolysis. Figure 3 with 2 supplements see all Download asset Open asset Ca2+CYT transients induced by external Ca2+ only increase NADHCYT production when both forward NCX transport and Na+/K+ pump activity are intact. (A) Cartoon the expected effect of a external Ca2+ on ion transport by the NCX and subsequent activation of the Na+/K+ pump. A Ca2+ is placed near the of a DGC within a slice that is bathed in solution mM Na+ and 0 Ca2+ A of positive is to the to transiently increase the external [Ca2+] and stimulate reverse NCX transport (magenta to Ca2+ import and an increase to Ca2+CYT. In the of the the external [Ca2+] as the of Ca2+ with of the mM Na+ 0 Ca2+ bath which to forward NCX transport that Na+ extrusion and an increase to Na+/K+ pump activity (orange arrows). Abbreviations: Na+/Ca2+-exchanger (NCX), Na+/K+-ATPase Representative fluorescence lifetime (LT) traces of Peredox (top and RCaMP (bottom from a DGC bathed in either mM Na+ and 0 Ca2+ solution 0 Na+ and 0 Ca2+ to forward NCX transport 0 or in mM Na+ and 0 Ca2+ with mM to the Na+/K+ pump of Ca2+ were delivered at the indicated along the bottom of the RCaMP LT traces (magenta arrows). (E) of the transient changes to the Peredox and RCaMP LTs of DGCs in mM Na+ and 0 Ca2+ black in 0 Na+ and 0 Ca2+ Na+, or in mM Na+ and 0 Ca2+ with mM gray The indicates where was to with transient RCaMP LT