Astrocyte and L-lactate in the anterior cingulate cortex modulate schema memory and neuronal mitochondrial biogenesis

  1. Mastura Akter
  2. Mahadi Hasan
  3. Aruna Surendran Ramkrishnan
  4. Zafar Iqbal
  5. Xianlin Zheng
  6. Zhongqi Fu
  7. Zhuogui Lei
  8. Anwarul Karim
  9. Ying Li  Is a corresponding author
  1. Department of Neuroscience, City University of Hong Kong, China
  2. Department of Biomedical Sciences, City University of Hong Kong, China
  3. Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, China
  4. Centre for Biosystems, Neuroscience, and Nanotechnology, City University of Hong Kong, China
6 figures, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement
Expression of hM4Di in anterior cingulate cortex (ACC) astrocytes.

Injection of AAV8-GFAP-hM4Di-mCherry into the ACC resulted in expression of hM4Di (A) in 91.1 ± 2.4% of GFAP-positive cells (B) with 96.3 ± 0.9% specificity (C), whereas 3.6 ± 0.8% of NeuN-positive …

Figure 1—source data 1

Zip file containing data for Figure 1B–D in GraphPad Prism file format.

https://cdn.elifesciences.org/articles/85751/elife-85751-fig1-data1-v1.zip
Figure 1—figure supplement 1
Microinjection and cannulation in the anterior cingulate cortex (ACC).

(A) Representative sections 3.24 mm and 2.52 mm anterior to bregma showing hM4Di-mCherry expression (arrowheads) in the ACC. The arrow shows the site of microinjection and cannula placement into the …

Figure 2 with 3 supplements
Astrocytic Gi pathway activation in the anterior cingulate cortex (ACC) impairs paired associate (PA) learning, schema consolidation, memory retrieval, and assimilation of new PAs (NPAs) into existing schema whereas L-lactate can rescue these impairments.

(A) Performance index (PI) (mean ± SD) during the acquisition of the six original PAs (OPAs) (S1–2, 4–8, 10–17) and NPAs (S19) of the control (n=8), hM4Di-CNO (n=15), and rescue …

Figure 2—figure supplement 1
Schema experimental design.

(A) Event arena for multiple flavor-place paired associate training. (B) Timeline for schema experiments.

Figure 2—figure supplement 2
Clozapine-N-oxide (CNO) application itself has no effect on paired associate (PA) learning and memory retrieval.

(A) Performance index (PI) (mean ± SD) during the acquisition of the six original PAs (OPAs) (S1–2, 4–8, 10–17) and new PAs (NPAs) (S19) of the control (n=6) and control-CNO (n=4) groups. The …

Figure 2—figure supplement 3
Results of the open field test (OFT).

(A) Timeline for the experiments. OFT was conducted for all groups (hM4Di-saline, hM4Di-CNO, and hM4Di-CNO+L-lactate; n=8 in each group) after two paired associate (PA) training sessions. …

Effect of anterior cingulate cortex (ACC) astrocytic Gi activation on cyclic adenosine monophosphate (cAMP) and L-lactate levels in the ACC.

(A) Experimental design to investigate the effect of Gi activation of ACC astrocytes on cAMP and L-lactate levels. (B and C) CNO decreases cAMP in the hM4Di-expressed cells. (B) Confocal micrograph …

Figure 3—source data 1

Zip file containing data for Figure 3C–E in GraphPad Prism file format.

https://cdn.elifesciences.org/articles/85751/elife-85751-fig3-data1-v1.zip
Gi activation in anterior cingulate cortex (ACC) astrocytes reduces neuronal mitochondrial biogenesis whereas concurrent exogenous L-lactate administration rescues the impairment.

(A–C) Representative confocal micrograph of PGC-1α (A)/SIRT3 (B)/ATPB (C) co-labeled with NeuN, glial fibrillary acidic protein (GFAP), and DAPI (4′,6-diamidino-2-phenylindole) in the ACC of the …

Figure 4—source data 1

Zip file containing data for Figure 4D–G in GraphPad Prism file format.

https://cdn.elifesciences.org/articles/85751/elife-85751-fig4-data1-v1.zip
Figure 5 with 1 supplement
Mitochondrial biogenesis by L-lactate is dependent on monocarboxylate transporter 2 (MCT2) and NMDA receptor (NMDAR).

(A) Experimental design to investigate whether MCT2 and NMDAR activity are required for L-lactate-induced mitochondrial biogenesis. (B and C) mtDNA copy number abundance in the anterior cingulate …

Figure 5—figure supplement 1
Western blot of monocarboxylate transporter 2 (MCT2).

(A) Western blot showing decreased expression of MCT2 in the anterior cingulate cortex (ACC) 12 hr after injection of MCT2 antisense oligodeoxynucleotide. (B) Intensity of MCT2 was quantified and …

Schematic diagram showing astrocytic Gi signaling and L-lactate modulating schema memory and mitochondrial biogenesis.

L-lactate in the anterior cingulate cortex (ACC) is required for schema memory formation and neuronal mitochondrial biogenesis. Astrocytic Gi activation results in decreased L-lactate in the ACC …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Rattus norvegicus, male)Adult Sprague- Dawley rats (250–300 g)Laboratory Animal Services Centre, Chinese University of Hong Kong, SAR, China
Transfected construct (Rattus norvegicus)AAV8-GFAP-hM4Di-mCherryShanghai Taitool Bioscience Co. Ltd
Transfected construct (Rattus norvegicus)AAV8-GFAP-mCherryShanghai Taitool Bioscience Co. Ltd
Sequence-based reagentRat MCT2 antisense oligodeoxynucleotide (ODN), 200 nmol (HPLC purified)Integrated DNA Technologies (IDT)Cat #: 107968967
Sequence-based reagentRat Relative scrambled ODN, 200 nmol (HPLC purified)Integrated DNA Technologies (IDT)Cat #: 107969138
Sequence-based reagentRat D-loop Forward and Reverse PrimerIntegrated DNA Technologies (IDT)Cat #: 107056074 and Cat #: 107056075Used to measure mtDNA by real-time PCR
Sequence-based reagentRat β-actin Forward and Reverse PrimerIntegrated DNA Technologies (IDT)Cat #: 107056076 and Cat #: 107056077Used to measure nDNA by real-time PCR
AntibodyAnti-GFAP (Mouse Monoclonal)AbcamCat #: ab46481:500 (IHC)
AntibodyAnti-NeuN (Rabbit Polyclonal)Merck MilliporeCat #: AB9781:500 (IHC)
AntibodyAnti-mCherry (Chicken Polyclonal)AbcamCat #: ab2054021:1000 (IHC)
AntibodyAnti-cAMP (Rabbit Monoclonal)AbcamCat #: ab1349011:500 (IHC)
AntibodyAnti-SIRT3 (Rabbit Polyclonal)Sigma-AldrichCat #: SAB57002221: 250 (IHC)
AntibodyAnti-PGC-1α (Rabbit Polyclonal)AbcamCat #: ab1918381: 500 (IHC)
AntibodyAnti-ATPB (Mouse Monoclonal)AbcamCat #: ab147301: 500 (IHC)
AntibodyAnti-MCT2 (Rabbit Polyclonal)Merck MilliporeCat #: AB35421: 500 (WB)
AntibodyAnti-β-actin (Mouse Monoclonal)ImmunowayCat #: YM30281: 5000 (WB)
AntibodyAlexa Flour 488 (Goat Anti-Mouse Polyclonal)Thermo Fisher ScientificCat #: A110011:300 (IHC)
AntibodyAlexa Flour 594 (Goat Anti-Mouse Polyclonal)Thermo Fisher ScientificCat #: A110321:300 (IHC)
AntibodyAlexa Flour 488 (Goat Anti-Rabbit Polyclonal)Thermo Fisher ScientificCat #: A110341:300 (IHC)
AntibodyAlexa Flour 594 (Goat Anti-Mouse Polyclonal)Thermo Fisher ScientificCat #: A110371:300 (IHC)
AntibodyGoat Anti-Rabbit Secondary Antibody, HRP, PolyclonalInvitrogenCat #: 314601: 5000 (WB)
AntibodyGoat Anti-Mouse Secondary Antibody, HRP, PolyclonalInvitrogenCat #: 62–65201: 5000 (WB)
Commercial assay or kitLactate Fluorescence Assay kitAbcamCat #: ab65331
Commercial assay or kitcAMP complete ELISA kitAbcamCat #: ab133051
Commercial assay or kitQIAamp DNA Mini KitsQIAGENCat #: 1725270
Commercial assay or kitSsoAdvanced Universal SYBR Green SupermixBio-RadCat #: 1725270
Commercial assay or kitRIPA BufferSigma-AldrichCat #: 20-188
Commercial assay or kitPhosphatase and protease inhibitor cocktailSigma-Aldrich
Commercial assay or kitBradford assayBio-RadCat #: 5000205
Commercial assay or kitWestern Bright ECL HRP substrateAdvanstaCat #: K12045-D20
Chemical compound, drugClozapine-N-oxide (CNO) dihydrochlorideHello BioCat #: HB6149
Chemical compound, drugNaClSigma-AldrichCat #: S3014-1kg
Chemical compound, drugL-lactateSigma-AldrichCat #: L-7022
Chemical compound, drugD-(-)-2-Amino-5-Phosphonopentanoic acid (D-APV)Sigma-AldrichCat #: A8054
Chemical compound, drugArtificial cerebrospinal fluid (ACSF)Harvard ApparatusCat #: 597316
Chemical compound, drugDorminal 20%Alfasan International BVCat #: 013003
Chemical compound, drugUrethaneSigma-AldrichCat #: U2500-500G
Software, algorithmFIJI ImageJNational Institutes of Health, Bethesda, MD, USA
Software, algorithmPrism GraphPadGraphPad Software, San Diego, CA, USAVersion 10
Software, algorithmExcelMicrosoft
OtherMicrodialysis guide cannula (CMA 11 elite) and probe (3 mm membrane)CMA IncUsed to collect ECF from ACC for L-lactate and cAMP assay
OtherStainless steel guide cannulae, OD 0.41 mm-27G/CRWD Life ScienceCat #: 62069Used in drug and ODN delivery into ACC
OtherDummy cannulaeRWD Life ScienceCat #: 62169Used in drug and ODN delivery into ACC
OtherBrain slicerBraintree Scientific, Braintree, MA, USAUsed to collect ACC from whole brain
OtherStereotaxic frameRWDUsed to fix head of rats during surgeries
Other33-Gauge metal needle, 10 μl micro-syringeHamilton, NV, USAUsed in AAV and drug delivery
OtherMicroinjection pumpWorld Precision Instruments, USAUsed in AAV delivery

Additional files

Supplementary file 1

Comparison of performance index of control vs. hM4Di-CNO group.

https://cdn.elifesciences.org/articles/85751/elife-85751-supp1-v1.docx
Supplementary file 2

Comparison of performance index of control vs. rescue group.

https://cdn.elifesciences.org/articles/85751/elife-85751-supp2-v1.docx
Supplementary file 3

Primer sequences and preparation of 20 μl reaction mixture for real-time PCR.

https://cdn.elifesciences.org/articles/85751/elife-85751-supp3-v1.docx
MDAR checklist
https://cdn.elifesciences.org/articles/85751/elife-85751-mdarchecklist1-v1.docx

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