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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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. Complete surgery at least 22 h prior to administration of the tracer. Surgery requires about 1 h to complete.

1. Gather necessary materials: ster....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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