Astrocyte and L-lactate in the anterior cingulate cortex modulate schema memory and neuronal mitochondrial biogenesis
- Department of Neuroscience, City University of Hong Kong, China
- Department of Biomedical Sciences, City University of Hong Kong, China
- Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, China
- Centre for Biosystems, Neuroscience, and Nanotechnology, City University of Hong Kong, China
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 …
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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 …
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Figure 2—source data 1
Zip file containing data for Figure 2A–O, Figure 2—figure supplement 2A–D, and Figure 2—figure supplement 3E–G in GraphPad Prism file format.
- https://cdn.elifesciences.org/articles/85751/elife-85751-fig2-data1-v1.zip
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. …
Figure 3
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 …
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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
Figure 4
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 …
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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 …
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Figure 5—source data 1
Zip file containing data for Figure 5B–C and Figure 5—figure supplement 1B in GraphPad Prism file format.
- https://cdn.elifesciences.org/articles/85751/elife-85751-fig5-data1-v1.zip
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 …
Figure 6
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 resource | Designation | Source or reference | Identifiers | Additional 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-mCherry | Shanghai Taitool Bioscience Co. Ltd | ||
| Transfected construct (Rattus norvegicus) | AAV8-GFAP-mCherry | Shanghai Taitool Bioscience Co. Ltd | ||
| Sequence-based reagent | Rat MCT2 antisense oligodeoxynucleotide (ODN), 200 nmol (HPLC purified) | Integrated DNA Technologies (IDT) | Cat #: 107968967 | |
| Sequence-based reagent | Rat Relative scrambled ODN, 200 nmol (HPLC purified) | Integrated DNA Technologies (IDT) | Cat #: 107969138 | |
| Sequence-based reagent | Rat D-loop Forward and Reverse Primer | Integrated DNA Technologies (IDT) | Cat #: 107056074 and Cat #: 107056075 | Used to measure mtDNA by real-time PCR |
| Sequence-based reagent | Rat β-actin Forward and Reverse Primer | Integrated DNA Technologies (IDT) | Cat #: 107056076 and Cat #: 107056077 | Used to measure nDNA by real-time PCR |
| Antibody | Anti-GFAP (Mouse Monoclonal) | Abcam | Cat #: ab4648 | 1:500 (IHC) |
| Antibody | Anti-NeuN (Rabbit Polyclonal) | Merck Millipore | Cat #: AB978 | 1:500 (IHC) |
| Antibody | Anti-mCherry (Chicken Polyclonal) | Abcam | Cat #: ab205402 | 1:1000 (IHC) |
| Antibody | Anti-cAMP (Rabbit Monoclonal) | Abcam | Cat #: ab134901 | 1:500 (IHC) |
| Antibody | Anti-SIRT3 (Rabbit Polyclonal) | Sigma-Aldrich | Cat #: SAB5700222 | 1: 250 (IHC) |
| Antibody | Anti-PGC-1α (Rabbit Polyclonal) | Abcam | Cat #: ab191838 | 1: 500 (IHC) |
| Antibody | Anti-ATPB (Mouse Monoclonal) | Abcam | Cat #: ab14730 | 1: 500 (IHC) |
| Antibody | Anti-MCT2 (Rabbit Polyclonal) | Merck Millipore | Cat #: AB3542 | 1: 500 (WB) |
| Antibody | Anti-β-actin (Mouse Monoclonal) | Immunoway | Cat #: YM3028 | 1: 5000 (WB) |
| Antibody | Alexa Flour 488 (Goat Anti-Mouse Polyclonal) | Thermo Fisher Scientific | Cat #: A11001 | 1:300 (IHC) |
| Antibody | Alexa Flour 594 (Goat Anti-Mouse Polyclonal) | Thermo Fisher Scientific | Cat #: A11032 | 1:300 (IHC) |
| Antibody | Alexa Flour 488 (Goat Anti-Rabbit Polyclonal) | Thermo Fisher Scientific | Cat #: A11034 | 1:300 (IHC) |
| Antibody | Alexa Flour 594 (Goat Anti-Mouse Polyclonal) | Thermo Fisher Scientific | Cat #: A11037 | 1:300 (IHC) |
| Antibody | Goat Anti-Rabbit Secondary Antibody, HRP, Polyclonal | Invitrogen | Cat #: 31460 | 1: 5000 (WB) |
| Antibody | Goat Anti-Mouse Secondary Antibody, HRP, Polyclonal | Invitrogen | Cat #: 62–6520 | 1: 5000 (WB) |
| Commercial assay or kit | Lactate Fluorescence Assay kit | Abcam | Cat #: ab65331 | |
| Commercial assay or kit | cAMP complete ELISA kit | Abcam | Cat #: ab133051 | |
| Commercial assay or kit | QIAamp DNA Mini Kits | QIAGEN | Cat #: 1725270 | |
| Commercial assay or kit | SsoAdvanced Universal SYBR Green Supermix | Bio-Rad | Cat #: 1725270 | |
| Commercial assay or kit | RIPA Buffer | Sigma-Aldrich | Cat #: 20-188 | |
| Commercial assay or kit | Phosphatase and protease inhibitor cocktail | Sigma-Aldrich | ||
| Commercial assay or kit | Bradford assay | Bio-Rad | Cat #: 5000205 | |
| Commercial assay or kit | Western Bright ECL HRP substrate | Advansta | Cat #: K12045-D20 | |
| Chemical compound, drug | Clozapine-N-oxide (CNO) dihydrochloride | Hello Bio | Cat #: HB6149 | |
| Chemical compound, drug | NaCl | Sigma-Aldrich | Cat #: S3014-1kg | |
| Chemical compound, drug | L-lactate | Sigma-Aldrich | Cat #: L-7022 | |
| Chemical compound, drug | D-(-)-2-Amino-5-Phosphonopentanoic acid (D-APV) | Sigma-Aldrich | Cat #: A8054 | |
| Chemical compound, drug | Artificial cerebrospinal fluid (ACSF) | Harvard Apparatus | Cat #: 597316 | |
| Chemical compound, drug | Dorminal 20% | Alfasan International BV | Cat #: 013003 | |
| Chemical compound, drug | Urethane | Sigma-Aldrich | Cat #: U2500-500G | |
| Software, algorithm | FIJI ImageJ | National Institutes of Health, Bethesda, MD, USA | ||
| Software, algorithm | Prism GraphPad | GraphPad Software, San Diego, CA, USA | Version 10 | |
| Software, algorithm | Excel | Microsoft | ||
| Other | Microdialysis guide cannula (CMA 11 elite) and probe (3 mm membrane) | CMA Inc | Used to collect ECF from ACC for L-lactate and cAMP assay | |
| Other | Stainless steel guide cannulae, OD 0.41 mm-27G/C | RWD Life Science | Cat #: 62069 | Used in drug and ODN delivery into ACC |
| Other | Dummy cannulae | RWD Life Science | Cat #: 62169 | Used in drug and ODN delivery into ACC |
| Other | Brain slicer | Braintree Scientific, Braintree, MA, USA | Used to collect ACC from whole brain | |
| Other | Stereotaxic frame | RWD | Used to fix head of rats during surgeries | |
| Other | 33-Gauge metal needle, 10 μl micro-syringe | Hamilton, NV, USA | Used in AAV and drug delivery | |
| Other | Microinjection pump | World Precision Instruments, USA | Used in AAV delivery |
Additional files
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Supplementary file 1
Comparison of performance index of control vs. hM4Di-CNO group.
- https://cdn.elifesciences.org/articles/85751/elife-85751-supp1-v1.docx
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Supplementary file 2
Comparison of performance index of control vs. rescue group.
- https://cdn.elifesciences.org/articles/85751/elife-85751-supp2-v1.docx
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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
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MDAR checklist
- https://cdn.elifesciences.org/articles/85751/elife-85751-mdarchecklist1-v1.docx
