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A neuronal MCT2 knockdown in the rat somatosensory cortex reduces both the NMR lactate signal and the BOLD response during whisker stimulation

View Article: PubMed Central - PubMed

ABSTRACT

Although several in vitro and ex vivo evidence support the existence of lactate exchange between astrocytes and neurons, a direct demonstration in vivo is still lacking. In the present study, a lentiviral vector carrying a short hairpin RNA (shRNA) was used to downregulate the expression of the monocarboxylate transporter type 2 (MCT2) in neurons of the rat somatosensory cortex (called S1BF) by ~ 25%. After one hour of whisker stimulation, HRMAS 1H-NMR spectroscopy analysis of S1BF perchloric acid extracts showed that while an increase in lactate content is observed in both uninjected and shRNA-control injected extracts, such an effect was abrogated in shMCT2 injected rats. A 13C-incorporation analysis following [1-13C]glucose infusion during the stimulation confirmed that the elevated lactate observed during activation originates from newly synthesized [3-13C]lactate, with blood-derived [1-13C]glucose being the precursor. Moreover, the analysis of the 13C-labeling of glutamate in position C3 and C4 indicates that upon activation, there is an increase in TCA cycle velocity for control rats while a decrease is observed for MCT2 knockdown animals. Using in vivo localized 1H-NMR spectroscopy, an increase in lactate levels is observed in the S1BF area upon whisker stimulation for shRNA-control injected rats but not for MCT2 knockdown animals. Finally, while a robust BOLD fMRI response was evidenced in control rats, it was absent in MCT2 knockdown rats. These data not only demonstrate that glucose-derived lactate is locally produced following neuronal activation but also suggest that its use by neurons via MCT2 is probably essential to maintain synaptic activity within the barrel cortex.

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A: 13C-Specific enrichments of lactate C3 in the S1BF areas at rest or activated of control, MCT2 and UNIV rats (n = 12 10 and 7, respectively). Values represent the % of carbone-13 that was incorporated into the carbon position 3 of lactate from [1-13C]glucose infused in the tail vein during the one-hour right whisker stimulation. 13C-Specific enrichments of lactate C3 was quantified from the doublet observed on the 1H-NMR spectra. **: p = 0.003, *: p = 0.016. B: Linear regression between increase in lactate content during brain activation (ratio of lactate content between activated and rest states) and evolution in 13C-SE Lact C3 (ratio of 13C-SE Lact between activated and rest states, %). Each plot represent on individual rat (blue dot, control rats, r2 = 0.795; red squares, MCT2 rats, r2 = 0.001 and green triangle, UNIV rats, r2 = 0.892).
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pone.0174990.g003: A: 13C-Specific enrichments of lactate C3 in the S1BF areas at rest or activated of control, MCT2 and UNIV rats (n = 12 10 and 7, respectively). Values represent the % of carbone-13 that was incorporated into the carbon position 3 of lactate from [1-13C]glucose infused in the tail vein during the one-hour right whisker stimulation. 13C-Specific enrichments of lactate C3 was quantified from the doublet observed on the 1H-NMR spectra. **: p = 0.003, *: p = 0.016. B: Linear regression between increase in lactate content during brain activation (ratio of lactate content between activated and rest states) and evolution in 13C-SE Lact C3 (ratio of 13C-SE Lact between activated and rest states, %). Each plot represent on individual rat (blue dot, control rats, r2 = 0.795; red squares, MCT2 rats, r2 = 0.001 and green triangle, UNIV rats, r2 = 0.892).

Mentions: During whisker stimulation, rats were infused with 13C-labeled glucose. The incorporation of 13C from glucose into lactate was quantified in the S1BF area of control rats, MCT2 rats and UNIV rats, and compared between resting and activated states. Results are presented in Fig 3A and show a 27% and 24% increase in the lactate C3 specific enrichment (13C-SE lactate C3) during brain activation in control and UNIV rats, respectively. No difference in 13C-SE lactate C3 between resting and activated states was detected in MCT2 rats. A linear regression was performed between the increase in lactate content and the increase in 13C-SE lactate C3 during brain activation using each individual rat value. Fig 3B shows a correlation between these two parameters for control and UNIV rats (r2 = 0.80 and 0.89, respectively) but not for MCT2 rats.


A neuronal MCT2 knockdown in the rat somatosensory cortex reduces both the NMR lactate signal and the BOLD response during whisker stimulation
A: 13C-Specific enrichments of lactate C3 in the S1BF areas at rest or activated of control, MCT2 and UNIV rats (n = 12 10 and 7, respectively). Values represent the % of carbone-13 that was incorporated into the carbon position 3 of lactate from [1-13C]glucose infused in the tail vein during the one-hour right whisker stimulation. 13C-Specific enrichments of lactate C3 was quantified from the doublet observed on the 1H-NMR spectra. **: p = 0.003, *: p = 0.016. B: Linear regression between increase in lactate content during brain activation (ratio of lactate content between activated and rest states) and evolution in 13C-SE Lact C3 (ratio of 13C-SE Lact between activated and rest states, %). Each plot represent on individual rat (blue dot, control rats, r2 = 0.795; red squares, MCT2 rats, r2 = 0.001 and green triangle, UNIV rats, r2 = 0.892).
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Related In: Results  -  Collection

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pone.0174990.g003: A: 13C-Specific enrichments of lactate C3 in the S1BF areas at rest or activated of control, MCT2 and UNIV rats (n = 12 10 and 7, respectively). Values represent the % of carbone-13 that was incorporated into the carbon position 3 of lactate from [1-13C]glucose infused in the tail vein during the one-hour right whisker stimulation. 13C-Specific enrichments of lactate C3 was quantified from the doublet observed on the 1H-NMR spectra. **: p = 0.003, *: p = 0.016. B: Linear regression between increase in lactate content during brain activation (ratio of lactate content between activated and rest states) and evolution in 13C-SE Lact C3 (ratio of 13C-SE Lact between activated and rest states, %). Each plot represent on individual rat (blue dot, control rats, r2 = 0.795; red squares, MCT2 rats, r2 = 0.001 and green triangle, UNIV rats, r2 = 0.892).
Mentions: During whisker stimulation, rats were infused with 13C-labeled glucose. The incorporation of 13C from glucose into lactate was quantified in the S1BF area of control rats, MCT2 rats and UNIV rats, and compared between resting and activated states. Results are presented in Fig 3A and show a 27% and 24% increase in the lactate C3 specific enrichment (13C-SE lactate C3) during brain activation in control and UNIV rats, respectively. No difference in 13C-SE lactate C3 between resting and activated states was detected in MCT2 rats. A linear regression was performed between the increase in lactate content and the increase in 13C-SE lactate C3 during brain activation using each individual rat value. Fig 3B shows a correlation between these two parameters for control and UNIV rats (r2 = 0.80 and 0.89, respectively) but not for MCT2 rats.

View Article: PubMed Central - PubMed

ABSTRACT

Although several in vitro and ex vivo evidence support the existence of lactate exchange between astrocytes and neurons, a direct demonstration in vivo is still lacking. In the present study, a lentiviral vector carrying a short hairpin RNA (shRNA) was used to downregulate the expression of the monocarboxylate transporter type 2 (MCT2) in neurons of the rat somatosensory cortex (called S1BF) by ~ 25%. After one hour of whisker stimulation, HRMAS 1H-NMR spectroscopy analysis of S1BF perchloric acid extracts showed that while an increase in lactate content is observed in both uninjected and shRNA-control injected extracts, such an effect was abrogated in shMCT2 injected rats. A 13C-incorporation analysis following [1-13C]glucose infusion during the stimulation confirmed that the elevated lactate observed during activation originates from newly synthesized [3-13C]lactate, with blood-derived [1-13C]glucose being the precursor. Moreover, the analysis of the 13C-labeling of glutamate in position C3 and C4 indicates that upon activation, there is an increase in TCA cycle velocity for control rats while a decrease is observed for MCT2 knockdown animals. Using in vivo localized 1H-NMR spectroscopy, an increase in lactate levels is observed in the S1BF area upon whisker stimulation for shRNA-control injected rats but not for MCT2 knockdown animals. Finally, while a robust BOLD fMRI response was evidenced in control rats, it was absent in MCT2 knockdown rats. These data not only demonstrate that glucose-derived lactate is locally produced following neuronal activation but also suggest that its use by neurons via MCT2 is probably essential to maintain synaptic activity within the barrel cortex.

No MeSH data available.


Related in: MedlinePlus