Name: quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo
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Description: Watch this Scientific Journal Video about Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo at JoVE.com

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.

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...

<ol>
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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...

Protein synthesis is&#160;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 &#181;m 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 14CO2, 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, rats12,13,14,15,16,17,18,19,20,21, a mouse model of Tuberous Sclerosis complex22, a mouse model of fragile X syndrome23,24,25,26, 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.

<table>
	<tbody>
		<tr>
			<td>Mice</td>
			<td>The Jackson Laboratory</td>
			<td>003024</td>
			<td>Fmr1 knockout breeding pairs</td>
		</tr>
		<tr>
			<td>Anisomycin</td>
			<td>Tocris Bioscience</td>
			<td>1290</td>
			<td></td>
		</tr>
		<tr>
			<td>Microhematocrit Tubes</td>
			<td>Drummond Scientific</td>
			<td>1-000-3200-H</td>
			<td>capillary tubes</td>
		</tr>
		<tr>
			<td>Critoseal Capillary Tube Sealant</td>
			<td>Leica Microsystems</td>
			<td>39215003</td>
			<td>sealant putty</td>
		</tr>
		<tr>
			<td>Glass vial inserts</td>
			<td>Agilent</td>
			<td>5183-2089</td>
			<td>used to collect blood samples</td>
		</tr>
		<tr>
			<td>Digi-Med Blood Pressure Analyzer</td>
			<td>Micro-Med Inc.</td>
			<td>BPA-400</td>
			<td>blood pressure analyzer</td>
		</tr>
		<tr>
			<td>Bayer Breeze 2 Blood Glucose Monitoring System</td>
			<td>Bayer Breeze</td>
			<td>9570A</td>
			<td>glucose meter</td>
		</tr>
		<tr>
			<td>Gastight syringe</td>
			<td>Hamilton Co.</td>
			<td>1710</td>
			<td>tuberculin glass syringe</td>
		</tr>
		<tr>
			<td>HeatMax HotHands-2 Hand Warmers</td>
			<td>HeatMax</td>
			<td>Model HH2</td>
			<td>warming pads</td>
		</tr>
		<tr>
			<td>Heparin Lock Flush Solution</td>
			<td>Fresenius Kabi USA, LLC</td>
			<td>504505</td>
			<td>heparin saline</td>
		</tr>
		<tr>
			<td>Clear animal container</td>
			<td>Instech</td>
			<td>MTANK/W</td>
			<td>animal enclosure</td>
		</tr>
		<tr>
			<td>Spring tether</td>
			<td>Instech</td>
			<td>PS62</td>
			<td>catheter tube/rodent attachment</td>
		</tr>
		<tr>
			<td>Swivel</td>
			<td>Instech</td>
			<td>375/25</td>
			<td>hooks to spring tether</td>
		</tr>
		<tr>
			<td>Swivel arm and mount</td>
			<td>Instech</td>
			<td>SMCLA</td>
			<td>hooks to swivel and animal enclosure</td>
		</tr>
		<tr>
			<td>Tether button</td>
			<td>Instech</td>
			<td>VAB62BS/22</td>
			<td>attaches to bottom of spring tether</td>
		</tr>
		<tr>
			<td>Stainless steel tube</td>
			<td>Made in-house</td>
			<td>N/A</td>
			<td>used to snake catheters through mouse</td>
		</tr>
		<tr>
			<td>Matrx VIP 3000</td>
			<td>Matrx</td>
			<td>91305430</td>
			<td>isoflurane vaporizer</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Stoelting Co.</td>
			<td>50207</td>
			<td>isoflurane/halothane adsorber</td>
		</tr>
		<tr>
			<td>Clippers</td>
			<td>Oster Finisher</td>
			<td>Model 59</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical skin hooks</td>
			<td>Made in-house (??)</td>
			<td>N/A (??)</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride Saline</td>
			<td>APP Pharmaceuticals LLC</td>
			<td>918610</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11274-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>UNIFY silk surgical sutures</td>
			<td>AD Surgical</td>
			<td>#S-S618R13</td>
			<td>6-0 USP, non-absorbable</td>
		</tr>
		<tr>
			<td>PE-8 polyethylene tubing</td>
			<td>SAI Infusion Technologies</td>
			<td>PE-8-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Becton Dickinson and Co.</td>
			<td>309659</td>
			<td>1cc/mL</td>
		</tr>
		<tr>
			<td>PE-10 polyethylene tubing</td>
			<td>Clay Adams</td>
			<td>427400</td>
			<td></td>
		</tr>
		<tr>
			<td>MCID Analysis</td>
			<td>Imaging Research Inc.</td>
			<td>Version 7.0</td>
			<td>optical density analysis</td>
		</tr>
		<tr>
			<td>Gelatin-coated slides (75x25mm)</td>
			<td>FD Neurotechnologies</td>
			<td>PO101</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td></td>
		</tr>
		<tr>
			<td>Super RX-N medical x-ray film</td>
			<td>Fuji</td>
			<td>47410-19291</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypercassettes (8x10 in)</td>
			<td>Amersham Pharmacia Biotech</td>
			<td>11649</td>
			<td></td>
		</tr>
		<tr>
			<td>[1-14C]leucine</td>
			<td>Moravek</td>
			<td>MC404E</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Sarstedt Aktiengesellschaft &#38; Co.</td>
			<td>72.692.005</td>
			<td>used to deproteinize blood samples</td>
		</tr>
		<tr>
			<td>Glass pasteur pipette</td>
			<td>Wheaton</td>
			<td>357335</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td>Sigma-Aldrich</td>
			<td>18421</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen</td>
			<td>NIH Supply Center</td>
			<td>6830009737285</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation fluid</td>
			<td>CytoScint</td>
			<td>882453</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid scintilllation counter</td>
			<td>Packard Tri-Carb</td>
			<td>2250CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Amino acid analyzer</td>
			<td>Pickering Laboratories</td>
			<td>Pinnacle PCX</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC unit</td>
			<td>Agilent Technologies</td>
			<td>1260 Infinity</td>
			<td>include 1260 Bio-Inert Pump</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Wild Heerbrugg</td>
			<td>M650</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfosalicylic acid</td>
			<td>Sigma-Aldrich</td>
			<td>MKBS1634V</td>
			<td>5-sulfosalicylic acid dihydrate</td>
		</tr>
		<tr>
			<td>Norleucine</td>
			<td>Sigma</td>
			<td>N8513</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 N HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>[H3]leucine</td>
			<td>Moraevk</td>
			<td>MC672</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tube</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td>50 mL conical centrifuge tubes</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>Heuer Microsplit</td>
			<td>Model 1000</td>
			<td>1/100 min</td>
		</tr>
		<tr>
			<td>Euthanasia Solution</td>
			<td>Vet One</td>
			<td>H6438</td>
			<td></td>
		</tr>
		<tr>
			<td>Northern Light Precision Illuminator</td>
			<td>Imaging Research Inc.</td>
			<td>Model B95</td>
			<td>fluorescent light box</td>
		</tr>
		<tr>
			<td>Micro-NIKKOR 55mm f/2.8</td>
			<td>Nikon</td>
			<td>1442</td>
			<td>CDD camera</td>
		</tr>
	</tbody>
</table>

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.

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...

Watch this Scientific Journal Video about Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo at JoVE.com

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

Protein synthesis is&#160;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 ...

Measurement of regional rates of brain protein synthesis can trace the response of the brain to long term changes such that occur during development and neuroplasticity. Our method has the advantages that measurements are fully quantitative, and they are made in the awake behaving animal. The quantitative autoradiographic technique permits measurements in all brain regions simultaneously.Demonstrating the procedure will be Anita Torossian, a post-baccalaureate fellow in my laboratory, and Tianjian Huang, our animal surgeon. Begin this procedure with preparation for surgery as detailed in the text protocol. Once on the surgery stage, use surgical scissors to make a one centimeter incision from the upper medial portion of the left thigh rostrally towards the midline revealing the femoral artery and vein.Retract loose skin with surgical skin hooks above and on either side of the incision. Secure the skin hooks by taping them to the surgery stage. Apply sterile 0.9%sodium chloride to the exposed area to maintain adequate moisture.Use forceps to blunt dissect, separating the connective tissue around a small section of the femoral artery and vein. Carefully separate the artery and vein. Now use forceps to thread one strand of absorbable suture 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. At a more proximal point to the groin, use forceps to thread a second suture under only the femoral vein. Gently tie a half-knot that will be used to restrict blood flow.At a point between strand A and strand B, use forceps to thread a third suture 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.Gently tug on strand B to restrict blood flow. Use a hemostat to gently tug strand B to maintain blood restriction. Now, connect the non-cut end of the PE tubing to a 32 gauge needle and a one millimeter syringe filled with heparinized saline.Flush the catheter to remove air bubbles. Cut a small hole in the restricted area of the femoral vein with micro scissors, and carefully insert the angled end of the flushed PE eight tubing toward 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.Using strand C, tie an additional knot around the catheter. Make sure this knot does not capture the femoral artery. Gently pull back on the syringe barrel to partially fill the tubing with blood to ensure that the catheter has been implanted properly before inserting a PE 10 catheter into the left femoral artery using the same procedure.Once both the femoral vein and artery catheters have been secured, tie strand A into a knot around both catheters. After cutting all excess sutures and removing skin hooks, flush the arterial catheter with heparinized saline to prevent clotting. Cauterize the ends of both catheters to create a seal.Place the mouse in the prone position and make a small incision at the base of the neck applying saline to the exposed area. Insert a 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.After removing the hollow rod, close the femoral incision with suture followed by post-surgical analgesia. Snake the catheters through a 30 centimeter flexible hollow tube to make the spring tether before suturing the button of the spring tether under the skin followed by post-surgical analgesia. Move the mouse into a clear cylindrical container with a swivel mount and arm to house the mouse during the recovery period.Place a hand warmer under the container to keep the mouse warm. Ensure that the mouse is in a normal physiological state at the outset of the experiment by taking samples as detailed in the text protocol. To administer the tracer intravenously, use a Y-connector with a syringe holding the C 14 labeled leucine tracer connected to one arm, and a syringe with 100 to 200 microliters of sterile saline to flush the venous line connected to the other arm.Connect the Y-connector to the venous line. Initiate the study by simultaneously starting a stop watch, injecting the tracer, and collecting timed arterial blood samples. Flush the venous line with saline immediately following injection.Collect blood samples one through seven continuously throughout the first two minutes of the experiment in the same manner. After collecting the seven samples, collect 30 microliters of dead space blood before each remaining sample. Samples eight through 14 are collected at three, five, 10, 15, 30, 45, and 60 minutes respectively.At some point during the experiment, process three tubes for internal standards containing tritiated leucine and norleucine as detailed in the text protocol. To quantify plasma leucine concentrations, use an HPLC system with a sodium cation exchange column and post column derivatization with orthophthalaldehyde and flourometric detection. The area under the leucine curve is proportional to the concentration of leucine in the sample.Use comparison with standards to quantify leucine concentrations in the samples. Then use a liquid scintillation counter to quantify disintegrations per minute of tritium and C 14 in the plasma samples. Use these concentrations to construct the clearance curve of C 14 labeled leucine from the circulation and the time course of its specific activity in the arterial plasma.From the graph, calculate the integrated C 14 labeled leucine specific activity in the arterial plasma. To perform quantitative autoradiography, prepare brain sections 20 microns in thickness. Section brain by means of a cryostat at minus 20 degrees celsius.Thaw mount sections on gelatin coated slides. After fixation of slides, arrange slides in an X-ray film cassette along with a set of previously calibrated C 14 labeled methyl methacrylate standards. In a dark room and under safe light, place a piece of X-ray film, emulsion-side down over the sides and standards.Seal the cassette and place it in a black changing bag. Develop the films according to the manufacturer's instructions. Automated film development is not recommended because the background may be uneven and can affect quantification.Construct a calibration curve of optical density versus tissue C 14 concentration based on the optical density values of the set of calibrated standards on the film. Fit these data to a polynomial equation. Either a second or third degree polynomial equation fits very well.To analyze specific brain regions, locate the region of interest or ROI in six to eight sections by comparison with a brain atlas. Record the optical density of the pixels within an ROI in all sections. Based on the calibration curve, compute the tissue C 14 concentration in each pixel.Finally, compute the regional rates of cerebral protein synthesis from the average tissue C 14 concentration in the ROI in the integral of the ratios of arterial plasma concentrations of unlabeled and labeled leucine at times t and lamba. The fraction of leucine in the tissue precursor pool that comes from the plasma. Shown here are representative images from a vehicle treated animal compared with an animal treated with anisomycin, an inhibitor of protein synthesis.Rates of protein synthesis are proportional to the level of darkness in the image. Anisomycin drastically reduces the measured rates of brain protein synthesis indicating the specificity of this method. Here, digitized autoradiograms are shown from an awake behaving mouse at the level of the hippocampus and hypothalamus.The darker regions have higher regional rates of cerebral protein synthesis. Shown here is a digitized autoradiogram from an awake behaving control mouse at the level of the dorsal hippocampus. Rates of cerebral protein synthesis are color-coated in the images according to the color bar.While attempting this procedure, it's important to be sure that animals are in a normal physiological state during the measurements. Our methodology has already demonstrated disregulation of protein synthesis in neurodevelopmental disorders like Fragile X syndrome. It may also be a useful marker for degenerative changes and conditions like Alzheimer's disease.The protein synthesis method can be used in conjunction with immune histochemistry in alternating sections to correlate changes in proteins synthesis with regional changes in specific proteins. In summary, the quantitative autoradiographic L-1 C 14 leucine method is ideal for accurate determination of regional rates of protein synthesis in vivo. It offers considerable advantages in terms of accuracy and its applicability to in vivo conditions.

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