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. Complete surgery at least 22 h prior to administration of the tracer. Surgery requires about 1 h to complete.
<ol>
	<li>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 45o, 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).</li>
	<li>Prepare animal for surgery.</li>
	<li>Assure that proper aseptic and sterile techniques are used as required by your institution.
	<ol>
		<li>Weigh the animal. The animal must be at least 25 g for successful surgery.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Slide an activated commercially available handwarmer, wrapped in gauze, under the mouse. Tape down the right leg onto the surgery stage.</li>
	</ol>
	</li>
	<li>Insert catheter into the left femoral vein.
	<ol>
		<li>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.</li>
		<li>Retract loose skin with surgical skin hooks to further expose the vesicles.</li>
		<li>Apply sterile 0.9% sodium chloride to exposed area to maintain adequate moisture.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Gently tug on Strand B to restrict blood flow. Use a hemostat to gently pull Strand B to maintain restricted blood flow.</li>
		<li>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&#8217;s tension and guide the catheter further up the vein. Tighten Strand B around the vein containing the catheter.</li>
		<li>Using Strand C, tie an additional knot around the catheter. Make sure this knot does not capture the femoral artery.</li>
		<li>Gently pull back on the syringe barrel to partially fill the tubing with blood to ensure that the catheter has been implanted properly.</li>
	</ol>
	</li>
	<li>Following the same procedure, insert a PE-10 catheter into the left femoral artery.</li>
	<li>Complete surgical procedure.
	<ol>
		<li>Once both femoral vein and artery catheters have been secured, tie Strand A into a knot around both catheters.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Apply bupivacaine to the sides of the wound. Close the femoral incision with suture. Add lidocaine ointment over closed, sutured wound.</li>
		<li>Snake the catheters through a 30-cm flexible hollow tube (spring tether)&#160;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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Place a slotted lid that does not interfere with the swivel mechanism over the animal enclosure. Refer to Figure 3 for the full setup.</li>
		<li>Allow the mouse to recover. At least 22 h is recommended.</li>
	</ol>
	</li>
</ol>
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 &#181;Ci/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 &#730;C.
<ol>
	<li>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 &#956;Ci/mL.</li>
</ol>
3. Administer L-[1-14C]leucine intravenously and collect arterial blood samples.
<ol>
	<li>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)).</li>
	<li>Ensure the animal is in a normal physiological state at the outset of the experiment.
	<ol>
		<li>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 &#181;L), hematocrit samples (about half of the capillary tube volume), and a glucose sample (approx. 20 &#181;L).</li>
		<li>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.</li>
		<li>Use a commercially available blood glucose monitor to measure the glucose level in a drop of blood.</li>
		<li>Centrifuge control sample for 2 min at 18,000 x g to separate plasma. Deproteinize plasma samples as follows: add 5 &#181;L of plasma to 70 &#181;L of deionized water in a 1.5 mL microtube, add 25 &#181;L of the 16% SSA solution and vortex. Place on ice for 30 min before freezing on dry ice.</li>
		<li>Return dead space blood to the animal through the venous line, followed by a heparinized saline flush to prevent excess blood loss.</li>
		<li>Connect the arterial line to a blood pressure transducer to measure mean arterial blood pressure.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Administer tracer intravenously and collect timed arterial blood samples.
	<ol>
		<li>Use a Y-connector to attach one syringe with the tracer (100&#181;Ci/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.</li>
		<li>Initiate the study by simultaneously starting a stop watch and injecting the tracer. Flush the venous line with saline (c. 100mL) immediately following injection.</li>
		<li>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 &#181;L dead-space blood before each remaining sample. Samples 8-14 are collected at 3, 5, 10, 15, 30, 45, and 60 min, respectively.</li>
		<li>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.</li>
		<li>At some point during the experiment, process three internal standards by adding 25 &#181;L 16% SSA, 0.04 mM norleucine, and 1 mCi/mL [3H]leucine to 75 &#181;L water, vortex and place on ice.</li>
		<li>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.</li>
		<li>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 &#730;C until ready for processing. Processing can be performed at any point afterwards.</li>
	</ol>
	</li>
</ol>
4. Analyze concentrations of leucine and L-[1-14C]leucine in plasma samples.
<ol>
	<li>Thaw samples and internal standards on ice, vortex, and centrifuge 18,000 x g for 5 min at 2 &#730;C. The supernatant fraction will contain the free labeled and unlabeled leucine.</li>
	<li>Transfer 40 &#181;L 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.</li>
	<li>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.
	<ol>
		<li>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 &#730;C and reactor temperature to 45 &#730;C.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Perform quantitative autoradiography.
<ol>
	<li>Prepare brain sections 20 &#181;m in thickness for autoradiography. Section brain by means of a cryostat at -20 &#730;C.</li>
	<li>Thaw mount serial brain sections on gelatin-coated slides. Air dry.</li>
	<li>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.</li>
	<li>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 &#181;m tissue thickness. Under red safelight, place a piece of mammography film, emulsion side down, on top of the sections.</li>
	<li>Seal the cassettes and place in a black changing bag and store in a cabinet for 40-45 days.</li>
	<li>Develop films according to manufacturer&#8217;s directions. Note: Automated film development is not recommended because the background may be uneven and can affect quantification.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
7. Computation of rCPS. Compute rCPS in each ROI by means of the following equation:
<img alt="Equation" src="/files/ftp_upload/58503/58503eq1.jpg" />
Where P*(T) is the weighted average tissue concentration of 14C in the ROI, Cp(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 &#955; is the fraction of leucine in the tissue precursor pool that comes from the plasma. Evaluation of &#955;&#160;is carried out in a separate experiment 6. &#955; 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, &#955; should be evaluated under the new conditions.

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<li>Sare, R. M., Huang, T., Burlin, T., Loutaev, I., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+rates+of+cerebral+protein+synthesis+measured+in+vivo+in+a+mouse+model+of+Tuberous+Sclerosis+Complex%3A+unexpected+consequences+of+reduced+tuberin%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Decreased rates of cerebral protein synthesis measured in vivo in a mouse model of Tuberous Sclerosis Complex: unexpected consequences of reduced tuberin.</a> Journal of Neurochemistry. 145, 417-425 (2018).</li>
<li>Liu, Z. H., Huang, T., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lithium+reverses+increased+rates+of+cerebral+protein+synthesis+in+a+mouse+model+of+fragile+X+syndrome%5BTitle%5D)+AND+%22Neurobiology+of+Disease%22%5BJournal%5D)">Lithium reverses increased rates of cerebral protein synthesis in a mouse model of fragile X syndrome.</a> Neurobiology of Disease. 45, 1145-1152 (2012).</li>
<li>Qin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+cerebral+protein+synthesis+in+fragile+X+syndrome%3A+studies+in+human+subjects+and+knockout+mice%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%3A+Official+Journal+of+the+International+Society+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">Altered cerebral protein synthesis in fragile X syndrome: studies in human subjects and knockout mice.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 33, 499-507 (2013).</li>
<li>Qin, M., Kang, J., Burlin, T. V., Jiang, C., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postadolescent+changes+in+regional+cerebral+protein+synthesis%3A+an+in+vivo+study+in+the+FMR1+null+mouse%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%3A+the+Official+Journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Postadolescent changes in regional cerebral protein synthesis: an in vivo study in the FMR1 null mouse.</a> The Journal of Neuroscience: the Official Journal of the Society for Neuroscience. 25, 5087-5095 (2005).</li>
<li>Qin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((R-Baclofen+Reverses+a+Social+Behavior+Deficit+and+Elevated+Protein+Synthesis+in+a+Mouse+Model+of+Fragile+X+Syndrome%5BTitle%5D)+AND+%22The+International+Journal+of+Neuropsychopharmacology%22%5BJournal%5D)">R-Baclofen Reverses a Social Behavior Deficit and Elevated Protein Synthesis in a Mouse Model of Fragile X Syndrome.</a> The International Journal of Neuropsychopharmacology. 18, pyv034(2015).</li>
<li>Qin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebral+protein+synthesis+in+a+knockin+mouse+model+of+the+fragile+X+premutation%5BTitle%5D)+AND+%22ASN+Neuro%22%5BJournal%5D)">Cerebral protein synthesis in a knockin mouse model of the fragile X premutation.</a> ASN Neuro. 6, (2014).</li>
<li>Smith, C. B., Kang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebral+protein+synthesis+in+a+genetic+mouse+model+of+phenylketonuria%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Cerebral protein synthesis in a genetic mouse model of phenylketonuria.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 11014-11019 (2000).</li>
<li>Reivich, M., Jehle, J., Sokoloff, L., Kety, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+regional+cerebral+blood+flow+with+antipyrine-14C+in+awake+cats%5BTitle%5D)+AND+%22Journal+of+Applied+Physiology%22%5BJournal%5D)">Measurement of regional cerebral blood flow with antipyrine-14C in awake cats.</a> Journal of Applied Physiology. 27, 296-300 (1969).</li>
</ol>

The authors have no conflicts of interest to disclose.

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 into account the possibility of recycling of unlabeled amino acids derived from tissue protein degradation and its effect on the precursor pool for protein synthesis6.
The primary limitation of this method is that it is time-consuming and demanding. Whereas it is tempting to employ simpler and higher throughput methods, the limitations of data obtained must be acknowledged.
Because of the complexity of measuring rCPS in an intact mouse, problems with maintenance of a normal physiological state, collecting adequate blood samples, and avoiding possibly interfering conditions may be encountered. Surgical implantation of the venous and arterial catheters is challenging. As with any surgical procedure, especially with the handling of delicate vasculature, there is an inherent risk for mortality of the animal. For us, it is rare (about 1%). During the ensuing 22 h recovery period, occasionally (about 4%) an animal will pull a catheter out. During the measurement, it is important that catheters are patent and that animals are in a normal physiological state. In our recent experience, arterial blood cannot be collected in about 2% of animals and about 1% of animals had a low hematocrit (&#60; 40%) or low arterial blood pressure (&#60; 85 mm Hg), suggesting blood loss during surgery and/or recovery.
In the preparation of brain sections for autoradiography, it is important to ensure that section thickness is 20 &#181;m because that is the section thickness to which [14C]methylmethacrylate standards have been calibrated. Use care to ensure good quality sections, i.e., without tears, folds, or bubbles as these imperfections will interfere with the autoradiographic analysis. We develop autoradiographic films by hand rather than in an automated film processor because we find that background optical density can be uneven following automated processing, and this can affect the quantification.
In the equation for rCPS, we include a factor, lambda (&#955;), that is the fraction of leucine that comes from the arterial plasma, the remainder comes from the recycling of amino acids derived from tissue protein degradation6. We have evaluated &#955; in separate experiments in WT and Fmr1 KO (fragile X model) C57Bl/6J mice and shown that its value is 0.603. The value of &#955; may vary depending on species, genetic background, or the presence of a genetic mutation. Therefore, if designing protein synthesis experiments for other models, one will need to evaluate &#955; before an accurate measurement can be obtained.
Our work in genetic mouse models of neurodevelopmental disorders demonstrates that this methodology reveals changes in rCPS in these models and in some cases responses to pharmacological treatments22,23,25,26. It is also conceivable that the rCPS measurement may also monitor degenerative changes in brain in conditions such as models of Alzheimer&#39;s disease, Parkinson&#39;s disease, fragile X tremor ataxia syndrome, traumatic brain injury, etc. In these models, it might be possible to track early degenerative changes and possibly also responses to early interventions. The rCPS method can be used together with&#160;immunohistochemistry in parallel sections to further examine specific brain changes25. In summary, the quantitative autoradiographic L-[1-14C]-leucine method is ideal for accurate determination of rCPS values in vivo. It offers considerable advantages in terms of accuracy and applicability to in vivo conditions over existing methods.

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>&lt;em&gt;Fmr1 &lt;/em&gt;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-&lt;sup&gt;14&lt;/sup&gt;C]leucine</td><td>Moravek</td><td>MC404E</td><td></td></tr><tr><td>Microcentrifuge tube</td><td>Sarstedt Aktiengesellschaft &amp;amp; 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>[H&lt;sup&gt;3&lt;/sup&gt;]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 data represent a validation that the in vivo autoradiographic L-[1-14C]-leucine method measures rates of protein synthesis in brain.
We present a figure of L-[1-14C]-leucine autoradiograms at four levels of the brain to demonstrate the resolution of the method (Figure 5). Illustrated are the cell layers in the olfactory bulb (Figure 5A and B), the hippocampus (Figure 5C), and the cerebellum (Figure 5G). Nuclei in the hypothalamus (Figure 5D), the pons (Figure 5E and F), and the brain stem (Figure 5H) are also clearly seen in the autoradiograms. We also show the quantitative regional rates of protein synthesis in the frontal cortex (5.88 nmol/g/min) (Figure 6A) and dorsal hippocampus (5.35 nmol/g/min) (Figure 6B) of a typical control animal.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58503/58503fig1.jpg" /> 
Figure 1: Schematic representing the steps of the entire rCPS protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58503/58503fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58503/58503fig2.jpg" /> 
Figure 2: Image of exposed femoral artery and femoral vein. Laying parallel to one another, the femoral artery is shown above the femoral vein. The femoral vein also has a deeper red color than the femoral artery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58503/58503fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58503/58503fig3.jpg" /> 
Figure 3: Image of recommended animal enclosure set-up for rCPS experiment. It utilizes a clear cylindrical animal enclosure with swivel appendage connected to a spring tether. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58503/58503fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58503/58503fig4.jpg" /> 
Figure 4: Representative images from a vehicle-treated animal (A) compared with an animal treated with anisomycin (100 mg/kg, subcutaneously) 30 min prior to administration of tracer (B). Rates of protein synthesis are proportional to the level of darkness in the image. Anisomycin drastically reduces the measured rates of protein synthesis indicating the specificity of this method. The scale bar in the upper right of A represents 1 mm and applies to both images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58503/58503fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58503/58503fig5.jpg" /> 
Figure 5: Digitized autoradiograms from an awake behaving mouse at the level of the olfactory bulb (A, B), hypothalamus (C, D), pons (E, F), cerebellum (G), and brain stem (G, H). The darker regions have higher rCPS. The scale bar in panel G applies to panels A, C, E, and G. Autoradiograms on the right (B, D, F, and H) are enlarged images from the areas designated on the images on the left and the scale bar in panel H applies to panels B, D, F, and H. Abbreviations are as follows: FrA, frontal association cortex; OB, olfactory bulb; AO, anterior olfactory nucleus; Gl, glomerular layer; EPl, external plexiform layer; BLA, basolateral amygdala; py, pyramidal cell layer; dHi, dorsal hippocampus; DG, dentate gyrus; MHb, medial habenula; Rt, thalamic reticular nucleus; VMH, ventral medial hypothalamic nucleus; Arc, arcuate nucleus; EW, Edinger-Westphal nucleus; R, red nucleus; PN, pontine nucleus; ML, molecular layer; GL, granular layer; Pc, Purkinje cell layer; Cu, cuneate nucleus; AP, area postrema; 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58503/58503fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58503/58503fig6.jpg" /> 
Figure 6: Digitized autoradiograms from an awake behaving control mouse at the level of the frontal cortex (A) and dorsal hippocampus (B). Rates of cerebral protein synthesis are color coded in the images according to the color bar shown on the right. The scale bar in the lower left of A represents 1 mm and applies to both images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58503/58503fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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JoVE Journal

Neuroscience

7.0K Views.  National Institutes of Health. Meting van regionale tarieven van de hersenen eiwitsynthese kan de reactie van de hersenen op veranderingen op lange termijn zodanig dat optreden tijdens de ontwikkeling en neuroplasticiteit te traceren. Onze methode heeft de voordelen dat metingen volledig kwantitatief zijn, en ze worden gemaakt in de wakker gedragende dier. De kwantitatieve autoradiografische techniek maakt metingen in alle hersengebieden tegelijk mogelijk.Het aantonen van de procedure zal Anita Torossian, een post-b...

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

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Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

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Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

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Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to nociceptive stimuli. Although some studies have shown that neuropathic pain may involve the dorsolateral prefrontal cortex, rostral anterior cingulate cortex, amygdala, hippocampus, periaqueductal gray, rostral ventromedial medulla, and medial thalamus, the neural substrates and their connections that mediate CPSP remain unclear. [14C]-Iodoantipyrine (IAP) uptake can be measured to evaluate spontaneously active pain. It can be used to assess the activation of neural substrates that may be involved in CPSP in an animal model. The [14C]-IAP method in rats is less expensive to perform compared with other brain mapping techniques. The present [14C]-IAP protocol is used to measure the activation of neural substrates that are involved in CPSP that is induced by lesions of the ventral basal nucleus (VB) of the thalamus in a rodent model.

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to ...

Medicine

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

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

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