Lactate from astrocytes fuels learning-induced mRNA translation in excitatory and inhibitory neurons methods

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    "name": "Lactate from astrocytes fuels learning-induced mRNA translation in excitatory and inhibitory neurons methods",
    "description": "Evidence-backed execution summary for Lactate from astrocytes fuels learning-induced mRNA translation in excitatory and inhibitory neurons methods from Lactate from astrocytes fuels learning-induced mRNA translation in excitatory and inhibitory neurons.",
    "totalTime": "PT9120M",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "Pyruvate or B3HB rescues memory loss caused by MCT1/MCT4 but not MCT2 knockdown",
        "text": "We previously showed that a bilateral dHP injection of AS-ODNs against MCT1 and/or MCT4 one hour before training down-regulates levels of the corresponding transporter relative to scrambled ODN (SCR-ODN) controls. Here, using the same ODN sequences and experimental schedule, we found that a bilateral hippocampal injection of AS-ODN against either MCT1 or MCT4 persistently disrupted long-term memory, and this effect was fully reversed by bilateral dHP injection of pyruvate 15 min prior to training (Fig.; MCT1 AS: F 2,21 = 8.45, p = 0.002; MCT4 AS: F 2,32 = 10.63, p < 0.001; MCT1 and 4 AS: F 2,32 = 9.58, p < 0.001, two-way RM ANOVA, followed by SNK post-hoc analyses). As when injected alone, neither pyruvate nor B3HB affected memory retention when co-administered with SCR-ODN controls against MCT..."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "Pyruvate or B3HB rescues memory loss caused by MCT1/MCT4 but not MCT2 knockdown",
        "text": "If this hypothesis is correct, disrupting the expression of MCT2 should impede long-term memory rescue by pyruvate or B3HB. Previously, we showed that a bilateral injection of MCT2-AS-ODN into the dHP one hour before training, compared to SCR-ODN, downregulates MCT2 protein levels. Here we used the same ODNs and approach and found that knockdown of MCT2 with AS-ODN persistently disrupted long-term IA memory at 2 and 7 days after training, compared to SCR-ODN (Fig.; F 4,51 = 15.39, p < 0.001, two-way RM ANOVA, followed by SNK post-hoc analysis). In contrast to what we found with the knockdown of MCT1 and MCT4, neither pyruvate nor B3HB could rescue memory impairment induced by MCT2-AS-ODN (pyruvate, p = 0.916; B3HB, p = 0.898). Furthermore, as with MCT1 and MCT4 SCR-ODNs, co-injection of pyruvate with MCT2 SCR-ODN did not chan..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Learning-dependent translation in excitatory and inhibitory neurons requires lactate",
        "text": "To this end, we assessed the relative levels of active translation in the dHP of rats by performing in vivo SU rface SE nsing of T ranslation (SUnSET), a technique that measures active protein synthesis. This technique assesses, via western blot or immunohistochemistry, the levels of puromycin incorporation into elongating peptide chains, and has been widely used to measure active in vivo translation -."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Learning-dependent translation in excitatory and inhibitory neurons requires lactate",
        "text": "Long-term memory formation requires temporally regulated translation of specific mRNAs -. To establish a link between the learning-induced mRNA translation in neurons, for which glycogenolysis and astrocytic lactate are necessary, and a target mRNA that is rapidly translated in excitatory neurons following learning, we analyzed the expression of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), an immediate early gene that plays a critical role in memory formation. Arc/Arg3.1 mRNA is rapidly translated in the soma and dendrites of activated neurons; Arc/Arg3.1 transcription, mRNA transport to synapses and translation in brain regions including the hippocampus are necessary for memory formation. Previously, using western blot analyses, we showed that Arc/Arg3.1 induction requires glycogenolysis and lactate. Here, we sought to increase our understanding of the s..."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Cannula implants and drug injection",
        "text": "Cannula implants were performed as described previously. Briefly, rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg), and guide cannulae (C313G-SPC; 22 gauge; Plastics One) were implanted toward the dorsal hippocampus (4 mm posterior to bregma, 2.6 mm lateral to midline, 2 mm ventral to skull surface) using a stereotaxic apparatus (Kopf Instruments). Rats were administered meloxicam (3 mg/kg, sub-cutaneous, once pre-surgery), and recovered for at least 10 days before undergoing behavioral experiments. Drugs were delivered in 1.0 µl over 3 min via injection through the cannula (28 gauge, extending 1.5 mm beyond the guide) attached to polyethylene tubing (PE50) connected to a 10-µl Hamilton syringe and controlled by a microinfusion pump (Harvard Apparatus). DAB (300 pmol; Sigma), L-lactate (100 n..."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "Inhibitory avoidance (IA)",
        "text": "IA was carried out as described previously,. The IA chamber (Med Associates) consisted of a rectangular Plexiglas box divided into a safe compartment and a shock compartment. The safe compartment was white and illuminated by a light fixture fastened to the compartment wall. The shock compartment was black and unilluminated. Foot shocks were delivered to the grid floor of this chamber via a constant current scrambler circuit. The two compartments were separated by an automatically operated sliding door. During training sessions, each rat was placed in the safe compartment with its head facing away from the door. After 10 s (s) the door automatically opened, allowing the rat access to the shock chamber. The door closed 1 s after the rat entered the shock chamber, and a brief foot shock (0.9 mA for 2 s) was administered. Latency to enter the shock compartment wa..."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Western blot analysis",
        "text": "Whole protein extracts and western blots were performed, as described previously. Dorsal hippocampi were dissected rapidly in cold dissection buffer (in mM: 2.6 KCl, 1.23 sodium phosphate monobasic, 26 sodium bicarbonate, 5 kynurenic acid, 212 sucrose, 10 dextrose, 0.5 CaCl 2, and 1 MgCl 2 ), followed by homogenization in buffer containing 0.2 M NaCl, 10 mM HEPES, 2 mM EDTA, 2 mM EGTA, 0.5 mM DTT, 2 mM NaF, 1 µM microcystin, 1 mM benzamidine, and phosphatase and protease inhibitor mixtures (Sigma-Aldrich). Tissues were homogenized and centrifuged at max speed in 4°C for 30 min, after which the supernatant was removed and the remaining pellet was discarded. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad). For Western blot analyses, equal amounts of protein (25 µg) to which 10%..."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "Immunohistochemistry",
        "text": "Rats were heavily anesthetized with choral hydrate (750 mg/kg), and perfused via the ascending aorta with 0.01 M phosphate-buffered saline (PBS; pH 7.4) immediately followed by ice cold 4% paraformaldehyde (PFA) in PBS. Brains were removed and post-fixed over-night in 4% PFA, and then submerged in 30% sucrose in PBS at 4 °C for at least 48 h. Thirty 30 µm thick brain sections containing the dorsal hippocampus (-2.3 mm to -4.52 from Bregma) were cut using a cryostat. Three non-serial brain slices per animal were blocked with 5% goat serum, 1% BSA, and 0.4% triton in PBS at room temperature, and then incubated in either rabbit Arc/Arg3.1 primary antibody for 24 h rocked gently at 4 ˚C (1:2000, Synaptic Systems, cat #156 003), and/or mouse anti-CaMKIIα (1:400, Millipore, cat #05-532), or mouse anti-parvalbumi..."
      }
    ],
    "tool": [
      {
        "@type": "HowToTool",
        "name": "Cannula implants and drug injection"
      },
      {
        "@type": "HowToTool",
        "name": "Inhibitory avoidance (IA)"
      },
      {
        "@type": "HowToTool",
        "name": "Western blot analysis"
      },
      {
        "@type": "HowToTool",
        "name": "Immunohistochemistry"
      },
      {
        "@type": "HowToTool",
        "name": "Statistics and reproducibility"
      }
    ],
    "supply": [
      {
        "@type": "HowToSupply",
        "name": "Pyruvate or B3HB rescues memory loss caused by MCT1/MCT4 but not MCT2 knockdown"
      },
      {
        "@type": "HowToSupply",
        "name": "Pyruvate or B3HB rescues memory loss caused by MCT1/MCT4 but not MCT2 knockdown"
      },
      {
        "@type": "HowToSupply",
        "name": "Learning-dependent translation in excitatory and inhibitory neurons requires lactate"
      },
      {
        "@type": "HowToSupply",
        "name": "Cannula implants and drug injection"
      },
      {
        "@type": "HowToSupply",
        "name": "Western blot analysis"
      },
      {
        "@type": "HowToSupply",
        "name": "Immunohistochemistry"
      },
      {
        "@type": "HowToSupply",
        "name": "In vivo SUnSET"
      }
    ],
    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "Lactate from astrocytes fuels learning-induced mRNA translation in excitatory and inhibitory neurons",
      "datePublished": "2019",
      "author": [
        {
          "@type": "Person",
          "name": "Giannina Descalzi"
        },
        {
          "@type": "Person",
          "name": "Virginia Gao"
        },
        {
          "@type": "Person",
          "name": "Michael Q. Steinman"
        },
        {
          "@type": "Person",
          "name": "Akinobu Suzuki"
        },
        {
          "@type": "Person",
          "name": "Cristina M. Alberini"
        }
      ],
      "identifier": "10.1038/s42003-019-0495-2"
    }
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