Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity
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Evaluation Summary:
This is a rigorous study that confirms the existence of functional KATP and dominant oxidative metabolism in several types of juvenile somatosensory cortical neurons. The authors present multiple lines of experimental results examining the effects of lactate on neocortical neuron types. They also report a mechanism by which lactate is likely to enhance neuronal firing. The data is convincing in supporting the conclusions in the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (K ATP ) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through K ATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.
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Author Response:
Reviewer #1:
This work demonstrates that functional KATP channels exist in most neuronal cell types in the mouse somatosensory cortex. While the transcriptomic profiling of electrophysiologically characterized neurons is only indicative of the existence of the Kir6.2/SUR1 KATP channel, the acute slice pharmacological/electrophysiological experiments convincingly supports this notion.
The uncertainty of single-cell RT-PCR is likely due to a small amount of starting material inherent to the sample collection method. As the authors discuss, low copy numbers of target transcripts may also have contributed to the negative/uncertain results.
We fully agree that scRT-PCR analysis underdetected Kir6.2 (kcnj11) and SUR1 (abcc8) mRNAs. This is likely due to their low abundance at the single-cell level, the sample collection …
Author Response:
Reviewer #1:
This work demonstrates that functional KATP channels exist in most neuronal cell types in the mouse somatosensory cortex. While the transcriptomic profiling of electrophysiologically characterized neurons is only indicative of the existence of the Kir6.2/SUR1 KATP channel, the acute slice pharmacological/electrophysiological experiments convincingly supports this notion.
The uncertainty of single-cell RT-PCR is likely due to a small amount of starting material inherent to the sample collection method. As the authors discuss, low copy numbers of target transcripts may also have contributed to the negative/uncertain results.
We fully agree that scRT-PCR analysis underdetected Kir6.2 (kcnj11) and SUR1 (abcc8) mRNAs. This is likely due to their low abundance at the single-cell level, the sample collection method and the low efficiency of the reverse transcription (RT).
As requested by reviewer 2 we now report the low detection rate of these subunits in neurons responsive to diazoxide and tolbutamide and acknowledge the limitation of scRT- PCR (pages 7,8, lines, 34,1-6).
We have also improved the discussion by providing the copy number of these mRNAs detected by single cell RNAseq (Zeisel et al. 2015, DOI: 10.1126/science.aaa1934, data available online https://linnarssonlab.org/cortex/) and the estimated sensitivity limit of the scRT-PCR (page 13, lines 29-33).
Next, the authors demonstrate that lactate is taken up by neurons and elevates the discharge rate via an increased ATP production due to the oxidative metabolism downstream of lactate, which is in line with earlier studies including Ivanov et al. (2011, doi: 10.3389/fnene.2011.00002).
We thank the reviewer for pointing out this reference that we have added in the discussion (page 17, line 16).
The authors showed this by introducing 15 mM lactate, and discuss a possibility that extracellular lactate can be elevated by a systemic increase of lactate. However, such an increase is likely more modest in the brain (Carrard et al., 2018, doi: 10.1038/mp.2016.179). So, the lactate-enhanced firing might occur in extreme conditions such as during anoxia or ischemia; however, intracellular ATP would most probably decrease and hence KATP channels would open in this case. A discussion on extracellular lactate levels in physiological conditions would be helpful.
We have improved the discussion on the physiological extracellular level of lactate which can be as high as 5 mM at rest. Since during neuronal activity lactate levels are almost doubled (i.e. up to 10 mM), lactate-enhanced firing might occur under physiological conditions (page 18, lines 9-13). We agree that a systemic lactate increase modestly elevates its extracellular concentration to a level with little or no effect on firing rate. Accordingly we now also quote references reporting this observation. Nonetheless, peripheral lactate could represent an additional source facilitating lactate-sensing when both the brain and the body are active, as during physical exercise (page 18, lines 13-19).
Overall, this is a rigorous study that confirms the existence of functional KATP and dominant oxidative metabolism in most types of juvenile somatosensory cortical neurons.
Thank you.
Reviewer #2:
The authors present an impressive array of experiments testing the effect of lactate on a number of neocortical cell types. They uncover a mechanism by which lactate might enhance neuronal firing although direct physiological relevance needs further support for CSF lactate concentrations. Most of the experiments are sound and interesting and the remaining experiments have limitations inherent to the methodology and presented accordingly in the discussion. The results are convincing, however a number of specific points need to be addressed.
We thank the reviewer for the specific points raised that helped us to improve and clarify the manuscript.
Specific points:
• Page 6 line 21 onwards. The authors state consistent expression of Kir6.2 and SUR1 in various cortical cell types. Data presented in Fig1 challenge this statement showing that Kir6.2 and/or SUR1 was expressed in the minority of cells tested regardless of cell type. For example, out of the 10 intrinsically bursting cells shown in the Ward cluster plot on Fig1A-B, only two was positive for Kir6.2 according to Fig1D. Surprisingly, Fig1F shows that 10% of intrinsically bursting cells express Kir6.2 which is clearly not the case (it is 20%).
We thank the reviewer for pointing out this apparent incoherence. Indeed, Fig. 1D showed two intrinsically bursting cells that appeared positive for Kir6.2. However, one of them was also positive for genomic control and was discarded from the calculation of detection rate, as already discussed (pages 13,14 lines 34,1-5). For the sake of clarity Fig. 1D now depicts potential Kir6.2 false positive as shaded colored rectangles.
Amplification was used for the detection of mRNAs by the authors, thus it is unlikely that detection threshold plays a role in having Kir6.2 or SUR1 negative cells.
We agree that PCR amplification can detect a single DNA molecule (e.g. Li et al 1988, DOI: 10.1038/335414a0). However, the low reverse transcription (RT) efficiency is an important limiting factor for the mRNA detection by scRT-PCR. In addition, dendritic mRNAs are almost inaccessible to the harvesting from a somatic patch pipette, thereby decreasing the detection rate. Similar issues of mRNA detection by scRT-PCR have been reported for neuropeptide receptors despite a functional expression in a majority of recorded pyramidal cells (Gallopin et al. 2006, DOI: 10.1093/cercor/bhj081). scRT-PCR detection limit was estimated to be around 25 molecules of mRNA in a previous study quantifying at the single-cell level AMPA receptor mRNAs harvested in the patch pipette (Tsuzuki et al. 2001, DOI: 10.1046/j.1471-4159.2001.00388.x).
We have now improved the discussion by providing the copy number of Kir6.2 (kcnj11) and SUR1 (abcc8) mRNAs detected by RNAseq from single isolated cells (Zeisel et al. 2015, data available online https://linnarssonlab.org/cortex/). The estimated sensitivity limit of the scRT-PCR is also now provided (page 13, lines 29-33).
Along the same vein, amplification makes it difficult to understand what the authors mean by "low copy number at single cell level". Specifically, the sentence (p6l22-25) is self-conflicting suggesting reliable detection of KATP subunits yet downplaying the significance of moderate single cell detection rates.
Since the point on the "low copy number" is now discussed in more detail the sentence has been removed from the results section. To avoid confusion between detection and expression we now use only "detection" for scRT-PCR data and "expression" for functional data. Accordingly, in Figures 1F, 3B, 6A and S5, "Occurrence" was changed to "Detection rate".
I think a moderate statement with percentages of expression would adequately describe the findings with an emphasis on potential variability between individual cells regardless of cell type. Throughout the text, the authors should avoid the use of uniform expression of KATP channels in neurons.
• Page 6 line 30. The authors conclude co-expression of Kir6.2 and SUR1 subunits. Fig1D shows that out of approximately n=71 Kir6.2 positive cells and n=28 SUR1 positive cells only n=16 expresses Kir6.2 and SUR1 together and the evidence presented shows that n=83 cells do not co-express Kir6.2 and SUR1. Again, the conclusion in the manuscript seems biased towards the minority of cases and does not reflect the overall dataset. Accordingly, the suggestion that neurons and beta cells use the same KATP channel is not supported (p6l32).
The statement has been mitigated as follows (page 6, lines 21-27): "Apart from a single Adapting NPY neuron (Figure 1D), where Kir6.1 mRNA was observed, only the Kir6.2 and SUR1 subunits were detected in cortical neurons (in 25%, n=63 of 248 neurons; and in 10%, n=28 of 277 of neurons; respectively). The single-cell detection rate was similar between the different neuronal subtypes (Figure 1F). We also codetected Kir6.2 and SUR1 in cortical neurons (n=14 of 248, Figure 1D) suggesting the expression of functional KATP channels."
We have also avoided the use of uniform expression throughout the text and do not refer anymore to pancreatic beta-cell like KATP channels in the results section.
• KATP channel presence in neurons. With respect to the points above, it would be helpful to see in the results section and possibly on Fig2 whether there is an electrophysiological indication of pharmacologically unresponsive cells. This would help in assessing the relative sensitivity of the two approaches. Fig.2G is helpful here, however signal to noise is hard to assess in the current version in individual experiments. Please state if single cell PCR was performed on any pharmacologically examined cells.
We now clearly report that all neurons pharmacologically analyzed in voltage clamp were responsive to diazoxide and tolbutamide. We also mention the range of the effects of these KATP channel modulators on membrane resistance and whole-cell current (page 7, lines 12-15).
We thank the reviewer for suggesting to state if scRT-PCR was performed on pharmacologically examined cells, which helps to evaluate the relative sensitivity of scRT- PCR and pharmacological/electrophysiological experiments. We now report the number of neurons pharmacologically characterized and successfully analyzed by scRT-PCR (pages 7,8, lines 34,1-6). All these neurons were found to express functional KATP channels, but Kir6.2 and SUR1 subunits were detected in only a minority of them. We thus conclude that scRT-PCR underdetects these mRNAs.
Fig3B recapitulates the results of Fig1 that only a small fraction of RS cells express Kir6.2 and SUR1.
Since scRT-PCR is less sensitive than electrophysiological investigations, as just discussed above, the absence of detection of mRNAs does not mean an absence of functional expression of KATP channels. The absence of outward ATP-washout current in Kir6.2 KO neurons, in marked contrast with neurons from wild-type mice, supports the notion of a widespread functional expression of Kir6.2-containing KATP channels in cortical neurons. To avoid the confusion between detection and expression, we have reformulated the sentence (page 8, lines 11-12) as follows: "We first verified that Kir6.2 and SUR1 subunits can be detected in pyramidal cells from wild type mice by scRT-PCR".
In spite having a clever pharmacological design, due to limitations inherent to spatially nonspecific drug application methods, one cannot exclude that the results measured on individual cells could also reflect network interactions with astrocytes and/or neurons and should be discussed.
We agree with the reviewer that bath applications of drugs can induce network effects leading to potential confounding results. However, the kinetics and biophysical properties of the whole-cell currents recorded during pharmacological manipulations do not support such a network effect. This possibility, nonetheless, is now discussed page 13, lines 18- 23.
We have also discussed the possibility that the blockade of lactate transport by 4-CIN could reflect an impairment of lactate uptake by neurons but also of lactate release by astrocytes. However, under our conditions the contribution of astrocyte-derived lactate is expected to be negligible (page 16, lines 10-18).
• Lactate concentration in blood vs CSF. As the authors point out, there is a discrepancy in glucose concentration between the blood and CSF, yet they use lactate concentrations measured in the blood (and not in the CSF) during exercise in their experiments. The physiological relevance of these experiments is unclear unless there is evidence that lactate concentration in the CSF is indeed in the range found effective here.
We thank the reviewer for pointing out the discrepancies between plasma and extracellular levels of glucose vs. lactate. Although surprising at first, and in contrast to glucose, extracellular lactate level is higher than its plasma level. Such a difference, most likely reflects the ability of the brain produce lactate and not glucose.
As also requested by reviewer 1 we have improved the discussion on the physiological extracellular level which can be as high as 5 mM at rest. Since during neuronal activity lactate levels are almost doubled (i.e. up to 10 mM), we believe that lactate-enhanced firing might occur under physiological conditions (page 18, lines 9-13).
We have improved the rationale of the lactate concentration used which is an isoenergetic condition to 10 mM glucose for having the same number of carbon atoms (page 10, lines 4-5).
We also discuss the possibility, that peripheral lactate could represent an additional source facilitating lactate-sensing when both the brain and the body are active, as during physical exercise (page 18, lines 13-19).
• MCT1 and MCT2 expression and widespread lactate effects. Here, the authors admit that relatively low single cell detection rates were observed for MCT1 (19%) and MCT2 (28%). It seems consistent (and a bit worrisome) throughout the manuscript that expression of mRNAs additionally tested functionally have a limited range of PCR detection yet (again) ubiquitous presence was found when tested pharmacologically.
Similar to KATP channels subunits and as reported by single cell RNAseq data (Zeisel et al. 2015, DOI: 10.1126/science.aaa1934, data available online https://linnarssonlab.org/cortex/), MCT1 (slc16a1) and MCT2 (slc16a7) are expressed in cortical neurons at a copy number below the detection limit of scRT-PCR.
We have now discussed the discrepancy between MCT1 and MCT2 detection and the widespread lactate effects which are most likely due to their low abundance at the single cell level (pages 15,16, lines 32-34, 1-6). We also provide a counter example with LDH subunits which are expressed at higher single-cell levels, and for which a higher scRT- PCR detection rate was found to match the functional data (page 16, lines 6-9).
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Evaluation Summary:
This is a rigorous study that confirms the existence of functional KATP and dominant oxidative metabolism in several types of juvenile somatosensory cortical neurons. The authors present multiple lines of experimental results examining the effects of lactate on neocortical neuron types. They also report a mechanism by which lactate is likely to enhance neuronal firing. The data is convincing in supporting the conclusions in the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This work demonstrates that functional KATP channels exist in most neuronal cell types in the mouse somatosensory cortex. While the transcriptomic profiling of electrophysiologically characterized neurons is only indicative of the existence of the Kir6.2/SUR1 KATP channel, the acute slice pharmacological/electrophysiological experiments convincingly supports this notion. The uncertainty of single-cell RT-PCR is likely due to a small amount of starting material inherent to the sample collection method. As the authors discuss, low copy numbers of target transcripts may also have contributed to the negative/uncertain results. Next, the authors demonstrate that lactate is taken up by neurons and elevates the discharge rate via an increased ATP production due to the oxidative metabolism downstream of lactate, …
Reviewer #1 (Public Review):
This work demonstrates that functional KATP channels exist in most neuronal cell types in the mouse somatosensory cortex. While the transcriptomic profiling of electrophysiologically characterized neurons is only indicative of the existence of the Kir6.2/SUR1 KATP channel, the acute slice pharmacological/electrophysiological experiments convincingly supports this notion. The uncertainty of single-cell RT-PCR is likely due to a small amount of starting material inherent to the sample collection method. As the authors discuss, low copy numbers of target transcripts may also have contributed to the negative/uncertain results. Next, the authors demonstrate that lactate is taken up by neurons and elevates the discharge rate via an increased ATP production due to the oxidative metabolism downstream of lactate, which is in line with earlier studies including Ivanov et al. (2011, doi: 10.3389/fnene.2011.00002). The authors showed this by introducing 15 mM lactate, and discuss a possibility that extracellular lactate can be elevated by a systemic increase of lactate. However, such an increase is likely more modest in the brain (Carrard et al., 2018, doi: 10.1038/mp.2016.179). So, the lactate-enhanced firing might occur in extreme conditions such as during anoxia or ischemia; however, intracellular ATP would most probably decrease and hence KATP channels would open in this case. A discussion on extracellular lactate levels in physiological conditions would be helpful. Overall, this is a rigorous study that confirms the existence of functional KATP and dominant oxidative metabolism in most types of juvenile somatosensory cortical neurons.
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Reviewer #2 (Public Review):
The authors present an impressive array of experiments testing the effect of lactate on a number of neocortical cell types. They uncover a mechanism by which lactate might enhance neuronal firing although direct physiological relevance needs further support for CSF lactate concentrations. Most of the experiments are sound and interesting and the remaining experiments have limitations inherent to the methodology and presented accordingly in the discussion. The results are convincing, however a number of specific points need to be addressed.
Specific points:
• Page 6 line 21 onwards. The authors state consistent expression of Kir6.2 and SUR1 in various cortical cell types. Data presented in Fig1 challenge this statement showing that Kir6.2 and/or SUR1 was expressed in the minority of cells tested regardless of …
Reviewer #2 (Public Review):
The authors present an impressive array of experiments testing the effect of lactate on a number of neocortical cell types. They uncover a mechanism by which lactate might enhance neuronal firing although direct physiological relevance needs further support for CSF lactate concentrations. Most of the experiments are sound and interesting and the remaining experiments have limitations inherent to the methodology and presented accordingly in the discussion. The results are convincing, however a number of specific points need to be addressed.
Specific points:
• Page 6 line 21 onwards. The authors state consistent expression of Kir6.2 and SUR1 in various cortical cell types. Data presented in Fig1 challenge this statement showing that Kir6.2 and/or SUR1 was expressed in the minority of cells tested regardless of cell type. For example, out of the 10 intrinsically bursting cells shown in the Ward cluster plot on Fig1A-B, only two was positive for Kir6.2 according to Fig1D. Surprisingly, Fig1F shows that 10% of intrinsically bursting cells express Kir6.2 which is clearly not the case (it is 20%). Amplification was used for the detection of mRNAs by the authors, thus it is unlikely that detection threshold plays a role in having Kir6.2 or SUR1 negative cells. Along the same vein, amplification makes it difficult to understand what the authors mean by "low copy number at single cell level". Specifically, the sentence (p6l22-25) is self-conflicting suggesting reliable detection of KATP subunits yet downplaying the significance of moderate single cell detection rates. I think a moderate statement with percentages of expression would adequately describe the findings with an emphasis on potential variability between individual cells regardless of cell type. Throughout the text, the authors should avoid the use of uniform expression of KATP channels in neurons.
• Page 6 line 30. The authors conclude co-expression of Kir6.2 and SUR1 subunits. Fig1D shows that out of approximately n=71 Kir6.2 positive cells and n=28 SUR1 positive cells only n=16 expresses Kir6.2 and SUR1 together and the evidence presented shows that n=83 cells do not co-express Kir6.2 and SUR1. Again, the conclusion in the manuscript seems biased towards the minority of cases and does not reflect the overall dataset. Accordingly, the suggestion that neurons and beta cells use the same KATP channel is not supported (p6l32).
• KATP channel presence in neurons. With respect to the points above, it would be helpful to see in the results section and possibly on Fig2 whether there is an electrophysiological indication of pharmacologically unresponsive cells. This would help in assessing the relative sensitivity of the two approaches. Fig.2G is helpful here, however signal to noise is hard to assess in the current version in individual experiments. Please state if single cell PCR was performed on any pharmacologically examined cells. Fig3B recapitulates the results of Fig1 that only a small fraction of RS cells express Kir6.2 and SUR1. In spite having a clever pharmacological design, due to limitations inherent to spatially nonspecific drug application methods, one cannot exclude that the results measured on individual cells could also reflect network interactions with astrocytes and/or neurons and should be discussed.
• Lactate concentration in blood vs CSF. As the authors point out, there is a discrepancy in glucose concentration between the blood and CSF, yet they use lactate concentrations measured in the blood (and not in the CSF) during exercise in their experiments. The physiological relevance of these experiments is unclear unless there is evidence that lactate concentration in the CSF is indeed in the range found effective here.
• MCT1 and MCT2 expression and widespread lactate effects. Here, the authors admit that relatively low single cell detection rates were observed for MCT1 (19%) and MCT2 (28%). It seems consistent (and a bit worrisome) throughout the manuscript that expression of mRNAs additionally tested functionally have a limited range of PCR detection yet (again) ubiquitous presence was found when tested pharmacologically.
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