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 which to study adaptive changes as well as neurodevelopmental disorders and disorders related to learning and memory.

We have developed a method to quantify rates of cerebral protein synthesis in vivo in an awake animal that offers inherent advantages over other techniques that estimate rates in ex vivo or in vitro preparations of brain tissue⁶. Foremost is the applicability to measurements in the intact brain in an awake animal. This is a key consideration because it allows measurements with synaptic structure and function in place and without concerns about post mortem effects. Moreover, the quantitative autoradiographic approach that we employ achieves a high degree of spatial localization. Whereas the energy of ¹⁴C is such that we cannot localize the tracer at the subcellular or cellular level, we can measure rates in cell layers and small brain regions such as hypothalamic nuclei, with approximately a 25 µm resolution⁷.

One challenge of in vivo measurements with radiotracers is to ensure that radiolabel measured is in the product of the reaction of interest rather than unreacted labeled precursor or other extraneous labeled metabolic products⁶. We chose L-[1-¹⁴C]-leucine as the tracer amino acid because it is either incorporated into protein or rapidly metabolized to ¹⁴CO_2, which is diluted in the large pool of unlabeled CO₂ in brain resulting from the high rate of energy metabolism⁸. Moreover, any ¹⁴C not incorporated into protein exists primarily as free [¹⁴C]-leucine, which over the 60 min experimental period, is almost entirely cleared from the tissue⁶. Proteins are then fixed to tissue with formalin and subsequently rinsed with waterto remove any free [¹⁴C]-leucine before autoradiography.

Another important consideration is the issue of the dilution of the specific activity of the precursor amino acid pool by unlabeled amino acids derived from tissue proteolysis. We have shown that in adult rat and mouse, about 40% of the precursor leucine pool for protein synthesis in the brain comes from amino acids derived from protein breakdown⁶. This must be included in the computation of regional rates of cerebral protein synthesis (rCPS) and must be confirmed in studies in which this relationship may change. The theoretical basis and the assumptions of the method have been presented in detail elsewhere⁶. In this paper, we focus on the procedural issues of the application of this methodology.

This method has been employed for the determination of rCPS in ground squirrels⁹, sheep¹⁰, rhesus monkeys¹¹, rats^{12,13,14,15,16,17,18,19,20,21}, a mouse model of Tuberous Sclerosis complex²², a mouse model of fragile X syndrome^23,24,25,26, fragile X premutation mice²⁷, and a mouse model of phenylketonuria²⁸. In this manuscript, we present the procedures for measurement of rCPS with the in vivo autoradiographic L-[1-¹⁴C]-leucine method. We present rCPS in brain regions of an awake control mouse. We also demonstrate that in vivo administration of anisomycin, an inhibitor of translation, abolishes protein synthesis in the brain.

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: sterile surgical instruments (surgical scissors, micro-scissors, forceps, three surgical skin hooks), equipment for isoflurane anesthesia (isoflurane vaporizer, active gas scavenger, sealed anesthesia chamber, anesthesia nose cone), sterile surgery stage, fur clippers, 70% ethanol, betadine, sterile gauze, surgical tape, commercial hand warmers, surgical microscope, sterile 0.9% sodium chloride (saline), sterile heparin 100 USP units/mL in 0.9% sodium chloride (heparinized saline), sterile five 20-cm strips of 6-0 absorbable suture, sterile 25-cm strands of PE-8 and PE-10 polyethylene catheters with one end cut at 45^o, sterile 1 cc syringes, sterile 32 gauge needles, cautery equipment, sterile 15-20 cm hollow stainless steel rod (2.5 mm inside diameter, 3 mm outside diameter), local anesthetics (bupivacaine and lidocaine ointment), and an animal enclosure with swivel appendage setup (30 cm spring tether with button, swivel, swivel mount and arm, 20 X 13 cm clear cylindrical container).
2. Prepare animal for surgery.
3. Assure that proper aseptic and sterile techniques are used as required by your institution.
   1. Weigh the animal. The animal must be at least 25 g for successful surgery.
   2. Place the animal inside a sealed plexiglass chamber and connect the chamber to the isoflurane anesthesia apparatus. Set flow rate to 2.5 L/min for males and 3.0 L/min for females of 1.5% isoflurane in O₂. After roughly 2 min, ensure the mouse is appropriately sedated by lack of a withdrawal reflex with a toe pinch.
   3. Once sedated, remove the mouse from the chamber and lay it in a prone position with its face inside the anesthesia nose cone. Set up nose cone to receive gas from the vaporizer and to return gas to the gas scavenger. The scavenger will capture isoflurane in a charcoal filter.
   4. Use clippers to shave fur between the shoulder blades. Make sure to properly sterilize the shaved region, alternating three times between betadine and ethanol scrubs.
   5. Flip the mouse over into a supine position keeping the face in the nose cone. Tape down the left leg onto the surgery stage and use clippers to shave fur from the left inner thigh to the upper left abdomen. Make sure to properly sterilize the shaved region, alternating three times between betadine and ethanol scrubs.
   6. Slide an activated commercially available handwarmer, wrapped in gauze, under the mouse. Tape down the right leg onto the surgery stage.
4. Insert catheter into the left femoral vein.
   1. With the aid of a surgical microscope, use surgical scissors to make a 1 cm incision from the upper medial portion of the left thigh rostrally towards the midline, revealing the femoral artery and vein.
   2. Retract loose skin with surgical skin hooks to further expose the vesicles.
   3. Apply sterile 0.9% sodium chloride to exposed area to maintain adequate moisture.
   4. Use forceps to blunt dissect, separating connective tissue around a small section of the femoral artery and vein. Carefully, separate the artery and vein (Figure 2).
   5. Use forceps to thread one strand of absorbable suture (Strand A) under both the femoral vein and artery at the most lateral point of the incision. Pull the suture halfway through so the ends are even.
   6. At a more proximal point to the groin, use forceps to thread a second suture (Strand B) under only the femoral vein. Gently tie a half knot that will be used to restrict blood flow.
   7. At a point between Strand A and Strand B, use forceps to thread a third suture (Strand C) under only the femoral vein. Gently tie a full knot that will be used to restrict blood flow. Be careful not to tear the vein.
   8. Gently tug on Strand B to restrict blood flow. Use a hemostat to gently pull Strand B to maintain restricted blood flow.
   9. Cut a small hole in the restricted area of the femoral vein with microscissors and carefully insert the angled end of the PE-8 tubing (previously flushed with heparin saline) towards Strand B. Once inserted, release Strand B’s tension and guide the catheter further up the vein. Tighten Strand B around the vein containing the catheter.
   10. Using Strand C, tie an additional knot around the catheter. Make sure this knot does not capture the femoral artery.
   11. Gently pull back on the syringe barrel to partially fill the tubing with blood to ensure that the catheter has been implanted properly.
5. Following the same procedure, insert a PE-10 catheter into the left femoral artery.
6. Complete surgical procedure.
   1. Once both femoral vein and artery catheters have been secured, tie Strand A into a knot around both catheters.
   2. Cut all excess sutures and remove skin hooks. Flush the arterial catheter with heparinized saline to prevent clotting. Cauterize the ends of both catheters to create a seal.
   3. Place the mouse in the prone position and make a small incision at the base of the neck and apply saline to the exposed area.
   4. Insert hollow metal rod subdermally from the neck incision to the femoral incision. Snake the catheters through the hollow rod and out of the neck incision. Remove the hollow rod. Implanting catheters subdermally will prevent mice from damaging the catheters.
   5. Apply bupivacaine to the sides of the wound. Close the femoral incision with suture. Add lidocaine ointment over closed, sutured wound.
   6. Snake the catheters through a 30-cm flexible hollow tube (spring tether) and suture the button of the spring tether under the skin. Apply bupivacaine to the sides of the wound. Suture the button of the spring tether under the skin. Add lidocaine ointment over closed, sutured wound.
   7. Move the mouse into a clear cylindrical container (20 cm high, 13 cm diameter) with a swivel mount and arm to house the animal during the recovery period. Place a hand warmer under the container to keep the animal warm.
   8. Screw the top of the spring tether to a swivel and secure the swivel to the swivel arm attached to the cylindrical container. Make sure the mouse has full range of motion and the catheters can be accessed.
   9. Place a slotted lid that does not interfere with the swivel mechanism over the animal enclosure. Refer to Figure 3 for the full setup.
   10. Allow the mouse to recover. At least 22 h is recommended.

2. Prepare L-[1-¹⁴C]leucine solution for injection and 16% (w/v) 5-sulfosalicylic acid (SSA) dihydrate solution for deproteinizing plasma samples. In the SSA solution, also include 0.04 mM norleucine and 1 µCi/mL [H³]leucine as internal standards for amino acid analysis and analysis of tracer concentration in the acid-soluble plasma fractions, respectively. Store the SSA up to two months at 4 ˚C.

1. Purchase commercially available L-[1-¹⁴C]leucine (50-60 mCi/mmol), which is sold as a solution in 2% ethanol or 0.1 N HCl. Blow dry a known activity of the tracer under a gentle stream of nitrogen and reconstitute in a solution of sterile normal saline made up to a concentration of 100 μCi/mL.

3. Administer L-[1-¹⁴C]leucine intravenously and collect arterial blood samples.

1. Gather necessary materials: 18 1.5 mL microtubes for deproteinizing plasma samples (add 70 mL of deionized water to each tube), 17 250mL glass vial inserts (15 inserts for collection of arterial blood samples and 2 inserts for collection of dead space blood to be reinjected. To limit the collection of extra and unnecessary blood, small, thin glass vial inserts with tapered bottoms that allow for accessible pipetting of supernatant plasma is recommended), 2 microcapillary tubes (32 X 0.8mm, for hematocrit measurement), 1 heparin and lithium fluoride-coated microcentrifuge tube (to prevent clotting and glycolysis, respectively), hemostats (cover the tips with tygon tubing so that clamps will not damage PE tubing), blood glucose monitor, blood pressure transducer, 1 mL sterile syringes (for saline flushes), and commercially available euthanasia solution (diluted 1:1 in deionized water (for mice)).
2. Ensure the animal is in a normal physiological state at the outset of the experiment.
   1. Clamp the arterial tubing about 2 cm from the end and cut off the tip, creating an opening for blood to flow. Then unclamp tubing and collect dead space blood ((c. 30 mL) to collect any residual saline and/or blood from previous draws), and, in a separate tube, collect a control sample (approx. 30 µL), hematocrit samples (about half of the capillary tube volume), and a glucose sample (approx. 20 µL).
   2. Measure hematocrit by plugging one end with sealant putty and centrifuge for 1 min at 4500 x g. Measure the ratio of the volume of red cells to the total blood volume. If an animal has a hematocrit below 30%, do not continue the study.
   3. Use a commercially available blood glucose monitor to measure the glucose level in a drop of blood.
   4. Centrifuge control sample for 2 min at 18,000 x g to separate plasma. Deproteinize plasma samples as follows: add 5 µL of plasma to 70 µL of deionized water in a 1.5 mL microtube, add 25 µL of the 16% SSA solution and vortex. Place on ice for 30 min before freezing on dry ice.
   5. Return dead space blood to the animal through the venous line, followed by a heparinized saline flush to prevent excess blood loss.
   6. Connect the arterial line to a blood pressure transducer to measure mean arterial blood pressure.
   7. After taking the samples be sure to re-clamp the arterial line and to flush the line with a small (50 mL) volume of heparinized saline.
3. Administer tracer intravenously and collect timed arterial blood samples.
   1. Use a Y-connector to attach one syringe with the tracer (100µCi/kg) and one syringe with 50 mL sterile saline to flush the venous line after injection of tracer. Connect Y-connector to the venous line.
   2. Initiate the study by simultaneously starting a stop watch and injecting the tracer. Flush the venous line with saline (c. 100mL) immediately following injection.
   3. Collect blood samples 1-7 continuously throughout the first 2 min of the experiment in the same manner. After collecting the 7th sample, collect 30 µL dead-space blood before each remaining sample. Samples 8-14 are collected at 3, 5, 10, 15, 30, 45, and 60 min, respectively.
   4. Process blood samples immediately after collection, as was described for the control sample. If there is a delay, place the samples on ice. Carefully reinject dead space blood into the artery via the arterial catheter and flush with heparin saline.
   5. At some point during the experiment, process three internal standards by adding 25 µL 16% SSA, 0.04 mM norleucine, and 1 mCi/mL [³H]leucine to 75 µL water, vortex and place on ice.
   6. After collecting the 14th sample at 60 min, inject approximately 0.2 mL of B-euthanasia-D into the venous line to euthanize the animal. Record the time of death.
   7. Unscrew the animal from the swivel mount and remove from the animal enclosure. Carefully remove the brain, place on aluminum foil, and freeze on dry ice. Do not freeze brains with liquid nitrogen as brains may crack. Store brain, samples, and internal standards at -80 ˚C until ready for processing. Processing can be performed at any point afterwards.

4. Analyze concentrations of leucine and L-[1-¹⁴C]leucine in plasma samples.

1. Thaw samples and internal standards on ice, vortex, and centrifuge 18,000 x g for 5 min at 2 ˚C. The supernatant fraction will contain the free labeled and unlabeled leucine.
2. Transfer 40 µL of the supernatant to a liquid scintillation vial and add scintillation cocktail. Quantify disintegrations per min (DPM) of ³H and ¹⁴C by means of liquid scintillation counting and a quench curve designed for simultaneous double-label (³H and ¹⁴C) counting.
3. To quantify plasma leucine concentrations, use an HPLC system with a sodium cation exchange column and post-column derivatization with o-phthaldehyde and fluorometric detection.
   1. Set HPLC to the following specifications: fluorometer excitation of 330 nm and emission of 465 nm. The mobile phase consists of sodium eluant, pH 7.40, and sodium eluant + 5% sulfolane, pH 3.15. Set the buffer flow rate of 0.400 mL/min and the derivatization instrument flow rate to 0.300 mL/min. Set the column temperature to 48 ˚C and reactor temperature to 45 ˚C.
   2. Calibrate the system with a range of amino acid concentrations (including norleucine) between 30 and 500 pmol/10mL. The calibration curve is linear. The amino acid concentrations of the tested 10 mL injection samples fall within the ranges of this calibration curve.

5. Perform quantitative autoradiography.

1. Prepare brain sections 20 µm in thickness for autoradiography. Section brain by means of a cryostat at -20 ˚C.
2. Thaw mount serial brain sections on gelatin-coated slides. Air dry.
3. Wash slides in five changes of 10% formalin for 30 min per change, followed by a continuous flow of deionized water for 1 h. Cover the slides loosely with foil to avoid dust and allow to dry for 24 h.
4. Arrange slides in an X-ray film cassette (cassettes that fit 20X25 cm mammography films are recommended) along with a set of [¹⁴C]methylmethacrylate standards, which were previously calibrated against tissue of known ¹⁴C concentrations as described²⁹. Standards can be commercially purchased but ensure that they cover a range of 2-300 mCi/g of tissue and are calibrated against 20 µm tissue thickness. Under red safelight, place a piece of mammography film, emulsion side down, on top of the sections.
5. Seal the cassettes and place in a black changing bag and store in a cabinet for 40-45 days.
6. Develop films according to manufacturer’s directions. Note: Automated film development is not recommended because the background may be uneven and can affect quantification.

6. Analyze images.

Note: A commercially available program for image analysis coupled with a CCD camera and a fluorescent light box with even illumination is recommended. The relative optical densities in the illuminated film are detected by the CCD camera.

1. Construct a calibration curve of optical density (OD) v. tissue ¹⁴C concentration based on the ODs of the set of calibrated standards on the film. Fit these data (including the blank or background) to a polynomial equation. Either a second or third degree polynomial equation fits very well.
2. To analyze specific brain regions, locate the region of interest (ROI) in six to eight sections by comparison with a brain atlas. Record the ODs of the pixels within a ROI in all sections and, based on the calibration curve, compute the tissue ¹⁴C concentration in each pixel. Compute the average tissue ¹⁴C concentration in the ROI.

7. Computation of rCPS. Compute rCPS in each ROI by means of the following equation:

Where P*(T) is the weighted average tissue concentration of ¹⁴C in the ROI, C_p(t) and C^*_p(t) are the arterial plasma concentrations of unlabeled and labeled leucine at time, t, T is the time that the animal died (about 60 min), and λ is the fraction of leucine in the tissue precursor pool that comes from the plasma. Evaluation of λ is carried out in a separate experiment ⁶. λ has been evaluated in WT, Fmr1 knockout, Tsc^+/-, and PKU mice ^6,22,25,28. If an experiment involves either genetic or pharmacological changes that might affect rates of protein synthesis, degradation, or metabolism of leucine, λ should be evaluated under the new conditions.

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