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Neuroscience

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Published: June 28th, 2019

DOI:

10.3791/58503

1Section on Neuroadaptation and Protein Metabolism, National Institute of Mental Health, National Institutes of Health

Protein synthesis is a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is an important biological process required for long-term adaptive change in the nervous system1. Inhibiting protein synthesis blocks long-term memory storage in both invertebrates and vertebrates2. Protein synthesis is essential for maintenance of the late phases of some forms of long-term potentiation (LTP) and long-term depression (LTD)3, neuronal survival during development4, and for general maintenance of the neuron and its synaptic connections5. Measurement of rates of brain protein synthesis may be an important tool with....

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Note: All animal procedures were approved by the National Institute of Mental Health Animal Care and Use Committee and were performed according with the National Institutes of Health Guidelines on the Care and Use of Animals.

An overview of the protocol is presented in Figure 1.

1. Surgically implant catheters in a femoral vein and artery for administration of the tracer and collection of timed arterial blood samples, respectively. Comple.......

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Here we show a representative experiment demonstrating the effects of prior administration of a protein synthesis inhibitor on rCPS. Anisomycin in normal saline was administered to an adult C57/BL6 male wild-type mouse subcutaneously (100 mg/kg) 30 min prior to initiation of rCPS determination. Effects of anisomycin treatment compared to a vehicle-treated control animal show that rCPS is almost undetectable in the anisomycin-treated mouse (Figure 4). These da.......

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We present a quantitative method for determination of regional rates of cerebral protein synthesis (rCPS) in vivo in experimental animals. This method has considerable advantages over existing methods: 1. Measurements are made in the awake behaving animal, so they reflect ongoing processes in the functioning brain. 2. Measurements are made by means of quantitative autoradiography affording the ability to determine rCPS in all regions and subregions of the brain simultaneously. 3. The kinetic model of the method takes int.......

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The authors would like to acknowledge Zengyan Xia for the genotyping of the mice, Tom Burlin for the processing of amino acids and films, and Mei Qin for performing some of the rCPS experiments. This research was supported by the Intramural Research Program of the NIMH, ZIA MH00889. RMS was also supported by an Autism Speaks Postdoctoral Fellowship 8679 and a FRAXA Postdoctoral Fellowship.

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Name Company Catalog Number Comments
Mice The Jackson Laboratory 003024 Fmr1 knockout breeding pairs
Anisomycin Tocris Bioscience 1290
Microhematocrit Tubes Drummond Scientific 1-000-3200-H capillary tubes
Critoseal Capillary Tube Sealant Leica Microsystems 39215003 sealant putty
Glass vial inserts Agilent 5183-2089 used to collect blood samples
Digi-Med Blood Pressure Analyzer Micro-Med Inc. BPA-400 blood pressure analyzer
Bayer Breeze 2 Blood Glucose Monitoring System Bayer Breeze 9570A glucose meter
Gastight syringe Hamilton Co. 1710 tuberculin glass syringe
HeatMax HotHands-2 Hand Warmers HeatMax Model HH2 warming pads
Heparin Lock Flush Solution Fresenius Kabi USA, LLC 504505 heparin saline
Clear animal container Instech MTANK/W animal enclosure
Spring tether Instech PS62 catheter tube/rodent attachment
Swivel Instech 375/25 hooks to spring tether
Swivel arm and mount Instech SMCLA hooks to swivel and animal enclosure
Tether button Instech VAB62BS/22 attaches to bottom of spring tether
Stainless steel tube Made in-house N/A used to snake catheters through mouse
Matrx VIP 3000 Matrx 91305430 isoflurane vaporizer
Isoflurane Stoelting Co. 50207 isoflurane/halothane adsorber
Clippers Oster Finisher Model 59
Surgical skin hooks Made in-house (??) N/A (??)
0.9% Sodium Chloride Saline APP Pharmaceuticals LLC 918610
Forceps Fine Science Tools 11274-20
Surgical scissors Fine Science Tools 14058-11
Microscissors Fine Science Tools 15000-00
UNIFY silk surgical sutures AD Surgical #S-S618R13 6-0 USP, non-absorbable
PE-8 polyethylene tubing SAI Infusion Technologies PE-8-25
Syringe Becton Dickinson and Co. 309659 1cc/mL
PE-10 polyethylene tubing Clay Adams 427400
MCID Analysis Imaging Research Inc. Version 7.0 optical density analysis
Gelatin-coated slides (75x25mm) FD Neurotechnologies PO101
Cryostat Leica CM1850
Super RX-N medical x-ray film Fuji 47410-19291
Hypercassettes (8x10 in) Amersham Pharmacia Biotech 11649
[1-14C]leucine Moravek MC404E
Microcentrifuge tube Sarstedt Aktiengesellschaft & Co. 72.692.005 used to deproteinize blood samples
Glass pasteur pipette Wheaton 357335
Glass wool Sigma-Aldrich 18421
Nitrogen NIH Supply Center 6830009737285
Scintillation fluid CytoScint 882453
Liquid scintilllation counter Packard Tri-Carb 2250CA
Amino acid analyzer Pickering Laboratories Pinnacle PCX
HPLC unit Agilent Technologies 1260 Infinity include 1260 Bio-Inert Pump
Surgical microscope Wild Heerbrugg M650
Sulfosalicylic acid Sigma-Aldrich MKBS1634V 5-sulfosalicylic acid dihydrate
Norleucine Sigma N8513
1.0 N HCl Sigma-Aldrich H9892
[H3]leucine Moraevk MC672
Falcon tube Thermo Scientific 339652 50 mL conical centrifuge tubes
Stopwatch Heuer Microsplit Model 1000 1/100 min
Euthanasia Solution Vet One H6438
Northern Light Precision Illuminator Imaging Research Inc. Model B95 fluorescent light box
Micro-NIKKOR 55mm f/2.8 Nikon 1442 CDD camera

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