Figures and data in The Na+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells

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Version of Record published: October 24, 2022 (This version)
Accepted Manuscript published: October 12, 2022 (Go to version)
Accepted: October 11, 2022
Preprint posted: July 9, 2022 (Go to version)
Received: July 7, 2022

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4 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements

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NADH_CYT 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 NADH_CYT 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 NADH_CYT and Ca²⁺_CYT. (C) Fluorescence LT traces of Peredox (*top*) and RCaMP (*bottom*) from a DGC showing how external Na⁺ and Ca²⁺ changes affect NADH_CYT and Ca²⁺_CYT. The bars above the Peredox trace indicate the external [Na⁺] and [Ca²⁺]. NADH_CYT 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 NADH_CYT decreased further. NADH_CYT 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.

Figure 1—figure supplement 1

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NADH_CYT production does not increase following external Ca²⁺ removal if external Na⁺ is absent.

(A) Representative traces showing the effects of external [Na⁺] and [Ca²⁺] changes on Peredox (*top*) and RCaMP (*bottom*) lifetimes (LTs). The bars above the Peredox trace indicate the external [Na⁺] and [Ca²⁺]. After removing external Na⁺ and Ca²⁺ (*cyan shading*), Ca²⁺_CYT was increased by applying 0.5 mM Ca²⁺ in 0 Na⁺ to stimulate reverse NCX transport (*magenta shading*). Following the Ca²⁺_CYT increase, external Ca²⁺ was removed without re-addition of external Na⁺ (*green shading*) to follow the NADH_CYT and Ca²⁺_CYT levels without stimulating forward NCX transport. (B) Box plots of the fluorescence LTs of Peredox (*top*) and RCaMP (*bottom*) summarizing the effects of the external [Na⁺] and [Ca²⁺] changes performed in (A) across many DGCs. These experiments were performed either in the absence (*black boxes, filled symbols*, n=49) or presence of thapsigargin (*blue boxes, open symbols*, n=24). The external solution conditions for each box plot are listed at the bottom of the RCaMP plot in chronological order from left to right. The coloring of the data corresponds to the shading in (A). The mean Peredox and RCaMP LTs in each external condition are shown in Figure 1—figure supplement 1—source data 1. (C) Representative fluorescence LT traces of Peredox (*top*) and RCaMP (*bottom*) from a DGC showing the effect of external Na⁺ removal for 20 min (*cyan shading*) and re-addition (*orange shading*) in the absence of a Ca²⁺_CYT increase on NADH_CYT. The external [Na⁺] and [Ca²⁺] are indicated by bars above the Peredox trace.

Figure 1—figure supplement 1—source data 1 The mean Peredox and RCaMP lifetimes in each external condition for Figure 1—figure supplement 1B.: https://cdn.elifesciences.org/articles/81645/elife-81645-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 2

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Effect of thapsigargin on antidromic stimulation-induced NADH_CYT and Ca²⁺_CYT transients.

(A) Representative fluorescence lifetime (LT) traces of Peredox (*top*) and RCaMP (*bottom*) from a DGC bathed in ACSF showing transient increases to NADH_CYT and Ca²⁺_CYT induced by antidromic stimulation before and after application of 1 μM thapsigargin. Antidromic stimulation events are indicated by arrows below the RCaMP trace. (B) Scatterplot of the antidromic stimulation-induced transient changes in Peredox LT (∆Peredox) and RCaMP LT (∆RCaMP) before (ACSF, *gray circles*) or in the presence of 1 μM thapsigargin (TG, *magenta squares*) across many DGCs (n=52). (C) Box plots showing the effect of thapsigargin (TG) on the ratio of ∆Peredox to ∆RCaMP from the same DGCs as (B). The mean ∆Peredox/∆RCaMP was: 0.22±0.05 in ACSF and 0.25±0.06 with 1 μM TG. Statistical significance of a paired sample t-test is indicated.

Figure 1—figure supplement 3

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Ca²⁺ entry from reverse NCX transport increases Ca²⁺_MITO, which depends on Na⁺ for removal.

(A) Representative fluorescence lifetime (LT) trace of mito-RCaMP from a DGC showing how Ca²⁺_MITO is affected by changes to the external [Na⁺] or [Ca²⁺] (indicated by the bars above the trace). Following the removal of external Na⁺ and Ca²⁺ (*cyan shading*), addition of 0.5 mM Ca²⁺ in the absence of Na⁺ (*magenta shading*) to stimulate reverse NCX transport increased Ca²⁺_MITO. The mito-RCaMP LT partially returned to baseline from the subsequent removal of external Ca²⁺ to turn off reverse NCX transport (*green shading*) but did not return completely to baseline until external Na⁺ was applied (orange shading). (B) Box plots showing the changes in mito-RCaMP LT induced by the external [Na⁺] or [Ca²⁺] changes performed in (A), across many DGCs (n=37). The external [Na⁺] and [Ca²⁺] conditions for each box plot are listed along the bottom in chronological order from left to right. The coloring of the data corresponds to the shading in (A). The LT values for the ‘0 Na, 0 Ca (20 min)’ condition (*green symbols*) were taken 20 min after removing 0.5 mM external Ca²⁺ in the absence of external Na⁺ (i.e., at the end of the *green shading* in A). The mean mito-RCaMP LT values in each condition are listed in Figure 1—figure supplement 3—source data 1.

Figure 1—figure supplement 3—source data 1 The mean Peredox and RCaMP lifetimes in each external condition for Figure 1—figure supplement 3B.: https://cdn.elifesciences.org/articles/81645/elife-81645-fig1-figsupp3-data1-v2.zip
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Figure 2

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Malate-aspartate shuttle activity does not conceal a possible NADH_CYT increase produced by elevated Ca²⁺_CYT.

(A) Representative fluorescence lifetime (LT) traces of Peredox (*top*) and RCaMP (*bottom*) from a DGC. External solution changes are indicated by the bars above the Peredox trace. The recording begins in ACSF (147 mM Na⁺ and 2 mM Ca²⁺), where the addition of 10 mM AOA strongly increases the Peredox LT. Addition of 1.5 mM pyruvate reverted the Peredox LT approximately back to baseline. Following this, external Na⁺ and Ca²⁺ were removed (*cyan shading*), which slightly decreased the Peredox LT. The addition 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*). Subsequently, the removal of external Ca²⁺ and re-addition of external Na⁺ to stimulate forward NCX transport (*orange shading*) increased the Peredox LT and brought the RCaMP LT back to baseline. (B) Box plots of the Peredox (*top*) and RCaMP (*bottom*) fluorescence LTs in the different external solution conditions performed in (A), across many DGCs (n=29). The external [Na⁺] and [Ca²⁺] condition after applying AOA and pyruvate is listed across the bottom in chronological order from left to right. The coloring of the data corresponds to the shading in A. The mean Peredox and RCaMP LTs in each external condition were (in ns): 1.50±0.04 in 0 Na⁺ and 0 Ca²⁺, 1.45±0.05 in 0 Na⁺ and 0.5 mM Ca²⁺, and 1.61±0.09 in 147 Na⁺ and 0 Ca²⁺. The mean RCaMP LTs were (in ns): 0.90±0.06 in 0 Na⁺ and 0 Ca²⁺, 1.49±0.20 in 0 Na⁺ and 0.5 mM Ca²⁺, and 0.91±0.07 in 147 Na⁺ and 0 Ca²⁺. (C) Relative change to the Peredox fluorescence LT induced by the addition of 0.5 mM Ca²⁺ in the absence of external Na⁺ to stimulate reverse NCX transport (Ca) or 147 mM Na⁺ in the absence of external Ca²⁺ to stimulate forward NCX transport (Na). The data are from the same DGCs as in (B). The mean ∆Peredox values were: –0.05±0.04 ns for ‘Ca’ and 0.11±0.07 ns for ‘Na’. Statistical significance from a paired Wilcoxon test is indicated.

Figure 3 with 2 supplements

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Ca²⁺_CYT transients induced by external Ca²⁺ puffs only increase NADH_CYT production when both forward NCX transport and Na⁺/K⁺ pump activity are intact.

(A) Cartoon depicting the expected effect of a local external Ca²⁺ puff on ion transport by the NCX and subsequent activation of the Na⁺/K⁺ pump. (*Left*) A pipette containing Ca²⁺ is placed near the soma of a DGC within a slice that is bathed in solution containing 40 mM Na⁺ and 0 Ca²⁺ solution. A brief pulse of positive pressure is applied to the pipette to transiently increase the local external [Ca²⁺] and stimulate reverse NCX transport (*magenta arrows*), leading to Ca²⁺ import and an increase to Ca²⁺_CYT. (*Right*) In the aftermath of the puff, the local external [Ca²⁺] decreases as the small volume of puffed Ca²⁺ mixes with large volume of the 40 mM Na⁺ 0 Ca²⁺ bath solution, which leads to forward NCX transport that stimulates Na⁺ extrusion and an increase to Na⁺/K⁺ pump activity (*orange arrows*). Abbreviations: Na⁺/Ca²⁺-exchanger (NCX), Na⁺/K⁺-ATPase (NKA). (**B–D**) Representative fluorescence lifetime (LT) traces of Peredox (*top traces*) and RCaMP (*bottom traces*) from a DGC bathed in either 40 mM Na⁺ and 0 Ca²⁺ solution (B, *Control*), 0 Na⁺ and 0 Ca²⁺ to block forward NCX transport (C, *0 Na⁺*), or in 40 mM Na⁺ and 0 Ca²⁺ with 5 mM ouabain to block the Na⁺/K⁺ pump (D, *Ouabain*). Puffs of Ca²⁺ were delivered at the timepoint indicated along the bottom of the RCaMP LT traces (*magenta arrows*). (E) Scatterplot of the Ca²⁺-puff-induced transient changes to the Peredox and RCaMP LTs of DGCs in 40 mM Na⁺ and 0 Ca²⁺ (*Control, black circles,* n=172), in 0 Na⁺ and 0 Ca²⁺ (*0 Na⁺, white triangles*, n=102), or in 40 mM Na⁺ and 0 Ca²⁺ with 5 mM ouabain (*Ouabain, gray squares*, n=114). The dashed line indicates where ∆Peredox=0. Analysis was restricted to recordings with transient RCaMP LT half-decay times <35 s. (F) Ratio of ∆Peredox to ∆RCaMP for the same Ca²⁺-puff-induced LT transients as in panel E, except excluding those recordings where the ∆RCaMP LT was <0.2 ns to avoid noisy ratio calculations due to small ∆RCaMP values. The dashed line indicates where ∆Peredox/∆RCaMP=0. The mean ∆Peredox/∆RCaMP in each condition was: 0.12±0.07 in 40 mM Na⁺ and 0 Ca²⁺ (*Control*, n=147), 0.01±0.02 in 0 Na⁺ and 0 Ca²⁺ (*0 Na⁺*, n=96), and 0.00±0.02 in 40 mM Na⁺ and 0 Ca²⁺ with 5 mM ouabain (*Ouabain*, n=99). Statistical significance from Mann-Whitney tests is indicated.

Figure 3—figure supplement 1

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Complete Na⁺/K⁺ pump inhibition in ACSF leads to cell rupture.

Representative fluorescence lifetime traces of Peredox (*top*) and RCaMP (*bottom*) from a DGC bathed in ACSF. Synaptic stimulation of the afferent fibers in the absence of synaptic blockers (*stim*, indicated by the arrow at the bottom of the RCaMP trace) transiently increased Ca²⁺_CYT and NADH_CYT. Application of 2 mM ouabain irreversibly increased Ca²⁺_CYT and ruptured the DGCs (*cell rupture,* arrow in the RCaMP trace).

Figure 3—figure supplement 2

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Effect of low dose (5 μM) ouabain on transient Peredox and RCaMP lifetime (LT) increases evoked by synaptic or antidromic stimulation.

(**A–C**) Effect of low dose ouabain on synaptic stimulation-induced transients in the absence of synaptic blockers. (A) Representative fluorescence LT traces of Peredox (*top*) and RCaMP (*bottom*) from a DGC bathed in ACSF. The DGC was stimulated synaptically with an electrode placed in the afferent fibers at the timepoints indicated by the arrows along the bottom of the RCaMP LT trace. Application of 5 μM ouabain is indicated by the bar above the Peredox trace. Repetitive synaptic stimulations in ouabain often ruptured the DGCs (*cell rupture,* arrow in the RCaMP trace). (B) Scatterplot of the transient Peredox and RCaMP LT increases (n=34) induced by synaptic stimulation in ACSF (*ACSF, gray circles*) and in 5 μM ouabain (*Ouab, orange squares*). (C) The ∆Peredox/∆RCaMP ratio from synaptic stimulations in ACSF and with 5 μM ouabain, calculated from the same DGCs as in (B). The mean ∆Peredox/∆RCaMP in each condition was: 0.20±0.07 in ACSF and 0.19±0.04 in ouabain. Statistical significance from a paired Wilcoxon test is indicated. (**D–F**) Effect of low dose ouabain on antidromic stimulation-induced transients. (D) Representative fluorescence LT traces of Peredox (*top*) and RCaMP (*bottom*) from a DGC bathed in ACSF with synaptic blockers. The DGC was stimulated antidromically at the timepoints indicated by the arrows along the bottom of the RCaMP trace. Application of 5 μM ouabain is indicated by the bar above the Peredox trace. (E) Scatterplot of the transient Peredox and RCaMP LT increases (n=16) induced by antidromic stimulation in ACSF with synaptic blockers (*ACSF, gray circles*) and with 5 μM ouabain (*Ouab, orange squares*). (F) The ∆Peredox/∆RCaMP ratio from antidromic stimulations in ACSF and in 5 μM ouabain, calculated from the same DGCs as in (E). The mean ∆Peredox/∆RCaMP in each condition was: 0.18±0.07 in ACSF with synaptic blockers and 0.16±0.04 in ouabain. Statistical significance from a paired sample t-test is indicated.

Figure 4 with 1 supplement

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Antidromic stimulation-induced [Na⁺] transients depend strongly on Ca²⁺ entry.

(A) Cartoon depicting antidromic stimulation-induced Na⁺ and Ca²⁺ fluxes. *Left, Control*: Stimulation in ACSF triggers Na⁺ and Ca²⁺ influx through their respective voltage-gated Na⁺ and Ca²⁺ channels. Following the Ca²⁺_CYT elevation, the NCX will import Na⁺ to drive Ca²⁺ extrusion. Both the Na⁺ influx through Na_V and through the NCX contribute to the total intracellular [Na⁺] increase (*blue arrows*). *Right, After Ca_V Block*: Inhibiting Ca²⁺ influx through Ca_V (indicated by the *red X*) prevents Ca²⁺_CYT elevation, which strongly reduces Na⁺ influx through the NCX. Abbreviations: voltage-gated Na⁺ channel (Na_V), voltage-gated Ca²⁺ channel (Ca_V), Na⁺/Ca²⁺-exchanger (NCX). (B) Representative fluorescence traces showing antidromic stimulation-induced transient changes to SBFI ∆F/F (*top*) and RCaMP lifetime (LT) (*bottom*) from a DGC bathed in ACSF with synaptic blockers. The stimulation was delivered at the timepoint indicated by the arrow at the bottom of the RCaMP trace. The SBFI ∆F/F axis is inverted to illustrate an increase to [Na⁺] as an upward deflection. (C) Antidromic stimulation-induced transient changes to SBFI ∆F/F and RCaMP LT in the presence of 3 μM isradipine and 20 μM CdCl₂ for the same DGC as in (B). The stimulation was delivered at the timepoint indicated by the arrow below the RCaMP trace. (D) Average, interpolated, antidromic stimulation-induced SBFI ∆F/F and RCaMP LT transients (n=13) before (*ACSF, black trace and shading*) and after Ca_V block (*Israd+Cd²⁺, magenta trace and shading*). The means are indicated by the solid line, SEM is indicated by the darker shading, and SD is indicated by the lighter shading. (E) Paired box plots showing the antidromic stimulation-induced peak transient SBFI ∆F/F amplitudes before (*ACSF, black box*) and after Ca_V block (*Israd+Cd²⁺, magenta box*) for the same DGCs as in (D). The mean peak SBFI ∆F/F values were: –0.29±0.07 in ACSF and –0.15±0.04 with Israd+Cd²⁺. Statistical significance from a paired sample t-test is indicated. (F) Paired box plots showing the ∆RCaMP LT before (*ACSF, black box*) after Ca_V block (*Israd+Cd²⁺, magenta box*) for the same DGCs as in (D) and (E). The mean ∆RCaMP LTs were: 0.30±0.13 ns in ACSF and 0.04±0.03 ns with Israd+Cd²⁺. Statistical significance from a paired sample t-test is indicated. (G) Scatterplot of antidromic stimulation-induced transient changes to SBFI ∆F/F against ∆RCaMP LT before (*ACSF, gray circles*) and after Ca_V block (*Israd +Cd²⁺, magenta squares*). Data are from the same DGCs as in (**D–F**).

Figure 4—figure supplement 1

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Somatic intracellular [Na⁺] and Ca²⁺_CYT transients induced by synaptic or antidromic stimulation.

(A) Representative fluorescence traces of SBFI ∆F/F (*top*) and RCaMP lifetime (LT; *bottom*) from a DGC when stimulated synaptically in the absence of synaptic blockers (indicated by the arrow along the bottom of the RCaMP trace). The SBFI ∆F/F axis is inverted to illustrate an increase to intracellular [Na⁺] as an upward deflection. (**B–C**) Average, interpolated, transient changes to SBFI ∆F/F (*top*) and RCaMP LT (*bottom*) triggered by synaptic ((B), n=24) or antidromic ((C), n=48) stimulation. The stimulation was delivered at the time indicated by the arrow at the bottom of the RCaMP trace. The mean is shown as the solid line, SEM is the dark gray shading, and SD is the light gray shading. (D) Scatterplot of the peak transient SBFI ∆F/F against the transient ∆RCaMP LT in response to either synaptic stimulation (*Synaptic, open squares*) or antidromic stimulation (*Antidromic, filled circles*) from the same set of DGCs as in (B) and (C).

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Mus musculus, M and F)	C57BL/6NCrl	Charles River	RRID:IMSR_CRL:27
Recombinant DNA reagent	AAV.CAG.Peredox.WPRE.SV40	Mongeon et al., 2016		Addgene #73807
Recombinant DNA reagent	AAV.hSyn.RCaMP1h.WPRE.SV40	Akerboom et al., 2013
Recombinant DNA reagent	AAV.hSyn.mito-RCaMP1h.WPRE.SV40	Díaz-García et al., 2021a
Chemical compound, drug	Isradipine	Abcam	Cat: ab120142, CAS: 75695-93-1
Chemical compound, drug	NBQX (6-Nitro-7- sulfamoylbenzo [f]quinoxaline-2,3-dione, Disodium Salt)	Toronto Research Chemicals	Cat: N550005, CAS: 479347-86-9
Chemical compound, drug	D-AP5 (D-(-)–2-Amino- 5-phosphonopentanoic acid)	Abcam	Cat: ab120003, CAS: 79055-68-8
Chemical compound, drug	Picrotoxin	Sigma-Aldrich	Cat: P1675 CAS: 124-87-8
Chemical compound, drug	CdCl₂	Sigma-Aldrich	Cat: 202908, CAS: 10108-64-2
Chemical compound, drug	Poly-L-lysine	Sigma-Aldrich	Cat: P4832
Chemical compound, drug	Aminooxyacetate (O-(carboxymethyl)hydroxylamine hemihydrate)	Sigma-Aldrich	Cat: C13408 CAS: 2921-14-4
Chemical compound, drug	Pyruvic acid	Sigma-Aldrich	Cat: 107360, CAS: 127-17-3
Chemical compound, drug	Thapsigargin	Santa Cruz Biotechnology	Cat: sc-24017A, CAS: 67526-95-8
Chemical compound, drug	Ouabain	Sigma-Aldrich	Cat: O3125, CAS: 11018-89-6
Chemical compound, drug	SBFI K⁺ salt (fluorescent dye)	Ion Biosciences	Cat: 2022B
Other	Glass capillaries, Borosilicate, standard wall, no filament, 4 in., O.D. 1.5 mm	WPI	Cat: 1B150-4	For microelectrodes and pipettes
Other	Glass coverslips, 12 mm circle No.1	VWR	Cat: 48366–251	For brain slice handling

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MDAR checklist: https://cdn.elifesciences.org/articles/81645/elife-81645-mdarchecklist1-v2.pdf
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Dylan J Meyer
Carlos Manlio Díaz-García
Nidhi Nathwani
Mahia Rahman
Gary Yellen

(2022)

The Na⁺/K⁺ pump dominates control of glycolysis in hippocampal dentate granule cells

eLife 11:e81645.

https://doi.org/10.7554/eLife.81645

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NADHCYT production is strongly influenced by Na+, but not by Ca2+CYT.

NADHCYT production does not increase following external Ca2+ removal if external Na+ is absent.

Figure 1—figure supplement 1—source data 1

Effect of thapsigargin on antidromic stimulation-induced NADHCYT and Ca2+CYT transients.

Ca2+ entry from reverse NCX transport increases Ca2+MITO, which depends on Na+ for removal.

Figure 1—figure supplement 3—source data 1

Malate-aspartate shuttle activity does not conceal a possible NADHCYT increase produced by elevated Ca2+CYT.

Ca2+CYT transients induced by external Ca2+ puffs only increase NADHCYT production when both forward NCX transport and Na+/K+ pump activity are intact.

Complete Na+/K+ pump inhibition in ACSF leads to cell rupture.

Effect of low dose (5 μM) ouabain on transient Peredox and RCaMP lifetime (LT) increases evoked by synaptic or antidromic stimulation.

Antidromic stimulation-induced [Na+] transients depend strongly on Ca2+ entry.

Somatic intracellular [Na+] and Ca2+CYT transients induced by synaptic or antidromic stimulation.

MDAR checklist

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Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

NADH_CYT production is strongly influenced by Na⁺, but not by Ca²⁺_CYT.

NADH_CYT production does not increase following external Ca²⁺ removal if external Na⁺ is absent.

Effect of thapsigargin on antidromic stimulation-induced NADH_CYT and Ca²⁺_CYT transients.

Ca²⁺ entry from reverse NCX transport increases Ca²⁺_MITO, which depends on Na⁺ for removal.

Malate-aspartate shuttle activity does not conceal a possible NADH_CYT increase produced by elevated Ca²⁺_CYT.

Ca²⁺_CYT transients induced by external Ca²⁺ puffs only increase NADH_CYT production when both forward NCX transport and Na⁺/K⁺ pump activity are intact.

Complete Na⁺/K⁺ pump inhibition in ACSF leads to cell rupture.

Antidromic stimulation-induced [Na⁺] transients depend strongly on Ca²⁺ entry.

Somatic intracellular [Na⁺] and Ca²⁺_CYT transients induced by synaptic or antidromic stimulation.