This study was funded by the National Natural Science Foundation of China (Project Code no. 81373557; no. 81403327).

This study was approved by the ethics committee at the China Academy of Chinese Medical Sciences (reference number 20160014). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996). Four adult male rats (weight 250-280 g) were used in this study. All animals were housed in a 12 h light/dark cycle with controlled temperature and humidity, and allowed free access to food and water. The instruments a...

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L., Mey, J., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+in+neuroanatomical+tracing.">Current concepts in neuroanatomical tracing.</a> Prog Neurobiol. 62, 327-351 (2000).</li><li>Vercelli, A., Repici, M., Garbossa, D., Grimaldi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+techniques+for+tracing+pathways+in+the+central+nervous+system+of+developing+and+adult+mammals.">Recent techniques for tracing pathways in the central nervous system of developing and adult mammals.</a> Brain Res Bull. 51, 11-28 (2000).</li><li>Liao, C. C., Reed, J. L., Kaas, J. H., Qi, H. 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The authors have nothing to disclose.

Selecting a proper tracer is a critical step for a successful neural tracing experiment. In the family of BDA, high molecular weight BDA (10,000 molecular weight) was recommended to be preferentially transported through the anterograde neural pathway in contrast to low molecular weight BDA (3,000 molecular weight)2,3,11,12,13. However, many studies also sugges...

High molecular weight BDA (10,000 molecular weight), a highly sensitive tracer, has been used for tracing neural pathways in the central nervous system for over 20 years1. Although the use of the BDA is a common neural tract tracing technique, the quality of BDA labeling can be affected in animals by various factors1,2,3. Our recent study indicated that the optimal structure of BDA labeling is not only associated with a proper post-injection survival time, but also correlated with the staining method4. Until now, conventional avidin-biotin-peroxidase complex (ABC), streptavidin-fluorescein isothiocyanate, and streptavidin-AF594 staining methods were used for revealing the BDA labeling in previous studies2,3,4,5. In comparison, fluorescent staining for BDA can be easily performed.
In order to extend the application of high molecular weight BDA, a refined protocol was introduced in the present study. Following the injection of BDA into the VPM of the thalamus in the rat brain, BDA labeling was revealed by the regular method of standard ABC staining as well as by double fluorescent staining, which was carried out for observing the correlation of BDA labeling and basic neural elements or interneurons in the cortical target with streptavidin-AF594 and fluorescent Nissl histochemistry or PV-immunochemistry, respectively. Through the reciprocal neural pathways between VPM and the primary somatosensory cortex (S1)6,7,8, we focused our observation on BDA labeling in the thalamocortical projected axons and corticothalamic projected cell somas in the S1. By this process, we expected to provide not only a detailed protocol for obtaining the high quality of neural labeling with high molecular weight BDA, but also a refined protocol on the combination of fluorescent BDA labeling and other fluorescent neural markers with histochemistry or immunochemistry. This approach is preferable to study the local neural circuits and their chemical characteristics under a confocal laser scanning microscopy.

<table>
	<tbody>
		<tr>
			<td>Biotinylated dextran amine (BDA)</td>
			<td>Molecular Probes</td>
			<td>D1956</td>
			<td>10,000 molecular weight</td>
		</tr>
		<tr>
			<td>Streptavidin-Alexa Fluor 594</td>
			<td>Molecular Probes</td>
			<td>S32356</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>500/525 green fluorescent Nissl stain</td>
			<td>Molecular Probes</td>
			<td>N21480</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Brain stereotaxis instrument</td>
			<td>Narishige</td>
			<td>SR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Thermo</td>
			<td>Microm International GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal imaging</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Drill</td>
			<td>Saeyang Microtech</td>
			<td>Marathon-N7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences</td>
			<td>SCKX (JUN) 2012-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>superfrost plus microscope slides</td>
			<td>Thermo</td>
			<td>#4951PLUS-001</td>
			<td>25x75x1mm</td>
		</tr>
		<tr>
			<td>Photoshop and Illustration</td>
			<td>Adobe</td>
			<td>CS5</td>
			<td></td>
		</tr>
	</tbody>
</table>

improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Survival of 10 days post injection of BDA into the VPM was sufficient for producing intense neural labeling on the corresponding cortical areas ipsilateral to the injection side (Figure 2). Both conventional ABC and fluorescent staining procedures for BDA revealed the similar pattern of neural labeling on the S1, including anterogradely labeled thalamocortical axons and retrogradely labeled corticothalamic neurons (Figure 2C<stro...

Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

The overall goal of this video is to demonstrate a refined protocol for the application of high molecular weight BDA for studying the optimal neural labeling in the central nervous system. This measure can help answer key questions in the neural networking field. By visualizing the frame structure of neural labeling through neural circuits.The main advantage of this technique is that it provides the opportunity to start a local neural circuits and their chemical characteristics. Prepare one microliter microsyringe, equipped with a glass micropipet, and test it by drawing up liquid paraffin. After anesthetizing the rat using an approved protocol, confirm deep anesthesia by the absence of reaction to a tail pinch.Then shave the top of the animal's head with an electric razor and scrub the surgical site with 10%povidone iodine, followed by 70%ethanol. Secure the rat into the stereotaxic device by inserting blunt ear bars into the ears and placing the rat's upper incisors into the mouth holder. Apply an ophthalmic ointment on the eyes.Use sterile surgical gloves and drapes to maintain sterile conditions. Clean the head skin of the surgical site again using 70%ethanol. After making a sagittal incision in the skin with a scalpel, along the sagittal suture, use sterile cotton-tipped applicators to scrape the muscle and periosteum away from the skull.Using pre-defined coordinates from an atlas, determine the location for the craniotomy. Perform a craniotomy using a burr drill with a round tip bit to drill to about a one millimeter depth until the meninges are visible. Excise the dura mater using micro forceps to expose the cerebral cortex over the injection site.Expel the liquid paraffin from the microsyringe and then draw up 10%BDA solution. Mount the syringe into the microinjection apparatus and connect the micropump. Under a stereomicroscope, manipulate the microinjection apparatus.Insert the filled glass micropipet into the VPM through the cortical surface of the brain, to a depth of 5.8 millimeters. Then, use the micropump to pressure-inject 100 nanoliters of 10%BDA into the VPM, over a period of three minutes. After five minutes, slowly withdraw the pipet.Suture the wound with sterile thread, then place the rat in a warm recovery area, until it regains consciousness and is fully recovered. After euthanizing the rat, use scissors and forceps to open the thoracic cavity of the rat and access the heart. Insert an intravenous catheter into the left ventricle, toward the aorta, and then open the right auricle.First, perfuse with 0.9%saline at physiological temperature, for about one to two minutes, until the blood exiting from the heart is clear. And then continue with 250 to 300 milliliters of 4%paraformaldehyde, in 0.1 molar phosphate buffer. Post-fix the dissected brain in 4%paraformaldehyde for two hours at room temperature.Then transfer the brain to 30%sucrose in 0.1 molar PBS and cryoprotect for three days at four degrees Celsius. After three days, when the brain has sunk in the solution, divide the brain into three blocks in the coronal direction with the aid of a brain matrix. The central block contains the ventral posteromedial nucleus of thalamus in the primary somatosensory cortex.Mount the central block of the brain on the freezing stage of the sliding microtome for sectioning in the coronal plane and section at 40 microns when frozen. Collect the sections in a six-well dish, containing 0.1 molar PBS. Begin staining by rinsing the sections in 0.1 molar PBS for about one minute with rocking.Then transfer the sections to a mixed solution of streptavidin-AF594 in AF500/525 green fluorescent Nissl stain in 0.1 molar PBS containing 0.3%Triton X-100, and incubate for two hours at room temperature. After staining, wash the sections three times in 0.1 molar PB with rocking. Mount the sections on the microscope slides using standard histochemical techniques.Air dry the sections for about an hour. Apply 50%glycerin in distilled water to the thorescent sections and cover slip before imaging the slides. This representative photomicrograph shows the injection site of BDA in red of the ventral posteromedial nucleus of thalamus on the background of green Nissl staining.Thorescent BDA-labeling is shown here, including thalamyl corticol axons in layer four and cortical thalamic neurons in layers five and six. For comparison, this photomicrograph demonstrates that conventional BDA-labeling with a standard avidin-biotin peroxidase procedure reveals a similar pattern of neural labeling to thorescent BDA-labeling. This higher magnification view of a thorescent BDA-labeled section shows the anterogradely labeled axonal arbors in detail, in red.The retrogradely labeled neuronal cell body, dendrites and dendritic spines are shown in detail here. Once mastered, craniotomy and stereotaxic injection can be done in 20 to 30 minutes, if it's performed properly. After watching this video, you should have a good understanding of how to apply high molecular weight BDA to revive neuronal connections and detailed structure of neural labeling.

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8.8K vistas.  China Academy of Chinese Medical Sciences. The overall goal of this video is to demonstrate a refined protocol for the application of high molecular weight BDA for studying the optimal neural labeling in the central nervous system. This measure can help answer key questions in the neural networking field. By visualizing the frame structure of neural labeling through neural circuits.The main advantage of this technique is that it provides the opportunity to start a local neural circuits and their chemical characteristics. Prepar...