Name: assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method
Uploaded: 2017-06-22T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method at JoVE.com

This work was supported by a pilot grant under NIH P20 GM109005 (A.R.A.) and by the Center for Translational Neuroscience IDeA program award P30 GM110702.

Experiments were conducted in accordance with the ethical standards approved by the Institutional Animal Care and Use Committee at UAMS.
1. Animals and 5-Fu Injection Paradigm
<ol>
	<li>Purchase 6-month-old male C57Bl6/J wild-type mice and house them under a constant 12 h light/dark cycle until they reach 1 year of age.</li>
	<li>Dilute the 5-Fu in 0.9% sterile saline. Use 60 mg/kg as the required dose per mouse.</li>
	<li>Give the intraperitoneal injections of the 5-Fu (once per wee...

     

<ol>
	<li>Stuart, G. J., Spruston, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+integration:+60+years+of+progress.">Dendritic integration: 60 years of progress.</a> Nat Neurosci. 18 (12), 1713-1721 (2015).</li><li>Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+out:+mechanisms+of+dendritic+arborization.">Branching out: mechanisms of dendritic arborization.</a> Nat Rev Neurosci. 11 (5), 316-328 (2010).</li><li>Kulkarni, V. A., Firestein, B. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+dendritic+arborization+mediated+by+CaM-kinase+I+activation+and+enhanced+CREB-dependent+transcription+of+Wnt-2.">Activity-dependent dendritic arborization mediated by CaM-kinase I activation and enhanced CREB-dependent transcription of Wnt-2.</a> Neuron. 50 (6), 897-909 (2006).</li><li>Bourne, J. N., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+structure+and+function+at+hippocampal+dendritic+spines.">Balancing structure and function at hippocampal dendritic spines.</a> Ann Rev Neurosci. 31, 47-67 (2008).</li><li>Lai, K. O., Ip, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+plasticity+of+dendritic+spines:+the+underlying+mechanisms+and+its+dysregulation+in+brain+disorders.">Structural plasticity of dendritic spines: the underlying mechanisms and its dysregulation in brain disorders.</a> Biochim Biophys Acta. 1832 (12), 2257-2263 (2013).</li><li>Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+development,+and+plasticity+of+dendritic+spines.">Structure, development, and plasticity of dendritic spines.</a> Current Op Neurobiol. 9 (3), 343-348 (1999).</li><li>Harris, K. M., Kater, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spines:+cellular+specializations+imparting+both+stability+and+flexibility+to+synaptic+function.">Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function.</a> Ann Rev Neurosci. 17, 341-371 (1994).</li><li>Leuner, B., Shors, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress,+anxiety,+and+dendritic+spines:+what+are+the+connections.">Stress, anxiety, and dendritic spines: what are the connections.</a> Neuroscience. 251, 108-119 (2013).</li><li>Harris, K. M., Fiala, J. C., Ostroff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+at+dendritic+spine+synapses+during+long-term+potentiation.">Structural changes at dendritic spine synapses during long-term potentiation.</a> Philos Trans R Soc Lond B Biol Sci. 358 (1432), 745-748 (2003).</li><li>Kasai, H., Matsuzaki, M., Noguchi, J., Yasumatsu, N., Nakahara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-stability-function+relationships+of+dendritic+spines.">Structure-stability-function relationships of dendritic spines.</a> Trends Neurosci. 26 (7), 360-368 (2003).</li><li>Das, G., Reuhl, K., Zhou, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Golgi-Cox+method.">The Golgi-Cox method.</a> Methods Mol Biol. 1018, 313-321 (2013).</li><li>Koyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unending+fascination+with+the+Golgi+method.">The unending fascination with the Golgi method.</a> OA Anat. 1 (3), 24 (2013).</li><li>Pasternak, J. F., Woolsey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+"selectivity"+of+the+Golgi-Cox+method.">On the "selectivity" of the Golgi-Cox method.</a> J Comp Neurol. 160 (3), 307-312 (1975).</li><li>Friedland, D. R., Los, J. G., Ryugo, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+Golgi+staining+protocol+for+use+in+the+human+brain+stem+and+cerebellum.">A modified Golgi staining protocol for use in the human brain stem and cerebellum.</a> J Neurosci Methods. 150 (1), 90-95 (2006).</li><li>Rosoklija, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Golgi+methods+for+impregnation+of+brain+tissue+from+humans+and+monkeys.">Optimization of Golgi methods for impregnation of brain tissue from humans and monkeys.</a> J Neurosci Methods. 131 (1-2), 1-7 (2003).</li><li>de Castro, F., Lopez-Mascaraque, L., De Carlos, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cajal:+lessons+on+brain+development.">Cajal: lessons on brain development.</a> Brain Res Rev. 55 (2), 481-489 (2007).</li><li>Gabbott, P. L., Somogyi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"single"+section+Golgi-impregnation+procedure:+methodological+description.">The "single" section Golgi-impregnation procedure: methodological description.</a> J Neurosci Methods. 11 (4), 221-230 (1984).</li><li>Zaqout, S., Kaindl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golgi-Cox+staining+step+by+step.">Golgi-Cox staining step by step.</a> Front Neuroanat. 10 (38), (2016).</li><li>. Vibroslice NVSL &amp; Vibroslice NVSLM123 Available from: <a target="_blank" href="https://www.wpiinc.com/clientuploads/pdf/NVSL_NVSLM1_IM.pdf">https://www.wpiinc.com/clientuploads/pdf/NVSL_NVSLM1_IM.pdf</a> (2000)</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurolucida 11.03. , (2017).</li><li>Sholl, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+organization+in+the+neurons+of+the+visual+and+motor+cortices+of+the+cat.">Dendritic organization in the neurons of the visual and motor cortices of the cat.</a> J Anat. 87 (4), 387-406 (1953).</li><li>Pillai, A. 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Compared to more modern techniques, the Golgi-Cox method has several advantages that make it the preferred method for examining spine morphology: 1) The staining can be used for essentially any tissue, 2) A basic light microscope setup is all that is needed to acquire Golgi-based images, 3) The Golgi-Cox imaging is faster than confocal imaging, and 4) Golgi stained sections are viable for several months to years longer than samples that are fluorescently-labeled. Even with these advantages, the Golgi-Cox method still has...

Dendrites are the largest portion of neurons that receive and process presynaptic input1. Their dendritic processes have a complex geometry, where the proximal branches have a larger diameter than the distal branches. As dendrites develop, they form several connections with other neurons in a process referred to as dendritic arborization. The extent and pattern of this branching determines the amount of synaptic inputs that a dendrite can adequately process2.
Dendritic arborization is a necessary process for activity-dependent plasticity and proper development of neuronal circuits. Extension, retraction, branching, and synaptogenesis are intricate processes that include intrinsic genetic programs and influences from extrinsic factors. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation3,4. The alterations in dendritic complexity are associated with pathophysiological and behavioral changes5. Abnormalities are related to several disease states, including Fragile X Syndrome and Down Syndrome6.
Dendritic spines are the specialized subcellular compartments of the dendritic arbors that receive excitatory input within the central nervous system. There are three morphological classes of dendritic spines, with the name of each class based on their size and shape: 1) mushroom spines, which have complex postsynaptic densities with more glutamate receptors than other spines7; 2) stubby spines, which lack a stem; and 3) thin spines, which consist of a protracted, narrow stem and a globular head8. The dendritic spine volume is used in part to define them, with thin spines generally smaller (0.01 &#181;m3) compared to mushroom spines (0.8 &#181;m3)9,10. The spines stabilize with maturation. For example, the thin spines either retract after a few days or develop into mushroom spines. Alternatively, the mushroom spines are relatively stable and can survive for an extended period.The strength of the neuronal connections is thought to be based on the number of spines and/or their volume11,12,13.
The classical Golgi staining method and its more modern variations have all been useful for examining the dendritic spine morphology and density. One unique aspect of the Golgi staining is that it randomly stains about 5% of the total neurons, which allows for the tracing of individual neurons14,15. Although the exact mechanism in which the Golgi method stains individual neurons is still unknown, the principle of the method is based on the crystallization of silver chromate (Ag2CrO4)16,17. There are three main types of the Golgi method: the rapid Golgi, the Golgi-Cox, and the Golgi-Kopsch18,19. All three methods start with an initial incubation phase in chromium salts for several days to months, but there are certain key differences between them. The rapid Golgi uses osmium tetroxide in the first step, whereas the Golgi-Kopsch includes paraformaldehyde. The staining in both the rapid-Golgi and the Golgi-Kopsch is followed by an incubation in a 1-2% silver nitrate solution for about 7 days. The Golgi-Cox method uses mercuric chloride and potassium dichromate instead of silver nitrate and has an impregnation time of 2-4 weeks. The tissues are then sectioned and quickly placed in a diluted ammonia solution, followed by a photographic fixer to remove salts. Of the three types, the Golgi-Cox method is thought to be the best at staining the dendritic arbors without much background interference, in part, because the crystal artifacts do not occur on the surface of the tissue (unlike in the rapid Golgi method)17,20,21.
The present method is a slight variation of the protocol provided with a commercial Golgi staining kit, and was designed to assess how a relatively low dose of the 5-Fu would affect the dendritic morphological characteristics and the spine density. Any data acquired could provide further insight into how chemotherapeutic treatment affects the neuronal circuitry.

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			<td height="21" style="height:21px;width:216px;">C57Bl/6 Male Mice&#160;</td>
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assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

The effects of the 5-Fu treatment on the dendritic arborization and complexity in the hippocampus of Golgi-stained brain sections were quantified and traced using a commercially available imaging software. After tracing, the dendritic arborization, the spine density, and the spine morphology were analyzed using Sholl analysis and the dendritic complexity index (DCI). Sholl analysis is a quantitative analytical method that can be used to determine the dendritic arbor morphology<sup class="...

Watch this Scientific Journal Video about Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method at JoVE.com

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

The overall goal of this methodology is to properly Golgi stain brain tissue in order to examine and quantify dendritic morphology and complexity. This method can help answer key questions in neuroscience. Since Golgi staining only stains a small subset of cells, this technique could be used to trace the structure of neurons.The main advantage of this technique is that brain sections can be easily stained without risk to them drying out or falling off during any of the washing steps. Demonstrating the procedure will be Fred Kiffer, Tyler Alexander and Julie Anderson, graduate students in Dr.Allen's laboratory. Begin by immersing freshly harvested, unfixed rodent half brains in five milliliters or mercuric chloride-based solution.As mercuric chloride solution is light sensitive, cover the tube with foil. Store the samples in the dark at room temperature. The following day, slowly decant the solution into a temporary container before properly disposing of it in a biohazard waste container.Add five milliliters of fresh mercuric chloride solution and ensure that the brains are immersed. Return the samples to the dark at room temperature for 13 more days. After 14 days, prepare post-impregnation buffer by adding 30 grams of powered post-impregnation buffer to 90 milliliters of distilled or deionized water, DH2O.Fill each well of the six-well plate with 6.5 milliliters of the post-impregnation buffer, one well per brain. Then, remove the mercuric chloride solution from the brain tissue as before. Rinse the tissue with DH2O, then transfer the tissue to the six-well plates with post-impregnation buffer.Cover the plates with foil and store at room temperature in the dark. The next day, aspirate and replace the post-impregnation buffer. Re-cover the plate and store in a four degree Celsius fridge.Prepare two or three 12-well plates per brain by first labeling the plates on their lids with numbers in ascending order from left to right and up to down. Add the identification number of the brain sectioned. Pipette two milliliters of 1X PBS into each well of each plate, then set up the stage and turn on the vibratome.Then, put tape on the tissue block of the specimen holder and add a little cyanoacrylate glue on top. Remove a brain from the six-well plate and place it on a plastic or glass dish. Use a stainless steel double edged blade to cut the cerebellum caudally, making sure that the portion of the cerebellum remaining on the brain is flat.Then, place the flat surface of the remaining cerebellum on the glue. After the glue is dry, place the specimen holder with the tissue into the specimen bath. Place the specimen bath on the vibratome and cover the tissue sample in PBS.Then, attach the blade to the blade holder of the vibratome and slowly lower the blade into the specimen bath until it is fully submerged and slightly below the top of the tissue. Next, set the vibratome speed to seven, the amplitude to six and the cutting angle to 12 degrees. Start the vibration and cut 200-micron sections.Use a large paint brush to move the tissue sections from the specimen bath into each designated well of the 12-well plates. Continue to cut the tissue until the desired number of tissue sections is reached. After sectioning, use a transfer pipette to aspirate the PBS from the wells of the 12-well plate, then add two milliliters of PBS-T and wash for 30 minutes with agitation.Close to the end of the wash period, dilute three parts ammonium hydroxide solution with five parts DH2O. Then, after removing the PBS-T, add the diluted ammonium hydroxide solution. Protect from light using foil and stain the sections for 19 to 21 minutes.Then, after safely removing the ammonium hydroxide solution, add post-staining buffer. Protect with foil and stain for another 19 to 21 minutes. Finally, rinse the sections in PBS-T three times for five to 10 minutes each time.Mount the sections on 1%gelatin coated slides with the large paint brush. Allow them to dry for 20 to 30 minutes at room temperature, then place them in a Coplin Jar and leave overnight. Remove the slides from the Coplin Jar and place them in a plastic rack in the fume hood.Place in a staining dish containing 100%ethanol for five minutes. Dehydrate the sections through two further five-minute ethanol washes. Next, place the slides in a glass rack and use a metal handle to lower the rack into a glass staining dish containing 99%xylene.Wash the slides in two changes of xylene for five minutes each. After the time for the second wash has elapsed, remove the slides from the xylene one at a time and quickly cover the tissue with approximately 0.25 milliliters of mounting media. Next, take a slide cover and lay it over the media.Push any trapped air bubbles underneath the slide covers to the edge with a blunt object, such as the end of paint brush. Place the slides in an area out of direct sunlight and allow them to dry for two to three days. After drying, proceed to image acquisition using a microscope and analysis with the appropriate software.This bright field image shows a Golgi-stained neuron that meets the criteria for imaging and further analysis. All three spine types can easily be seen. Staining is consistent across the entire length of the dendrite and the dendrite is isolated from other neurons.This figure shows that Golgi stained neurons within the Dentate Gyrus showed decreased length at the fourth and sixth order following 5-fluorouracil treatment compared to the saline-treated group. Golgi staining can be used to detect significant differences in spine density. In the CA1 apical pyramidal dendrites, 5-fluorouracil administration to live mice significantly decreased spine density.In the basal dendrites, there were no significant changes in the overall density of spines. Sholl Analysis of CA1 apical pyramidal dendrites revealed that 5-fluorouracil treatment significantly decreased dendritic arborization at distances between 40 to 160 microns away from the soma. In the basal dendrites, the 5-fluorouracil treatment decreased the dendritic arborization at 30 to 110 microns away from the soma.Once mastered, this technique, accounting for both the Golgi staining process and mounting, cleaning and covering can be done in three hours per brain if performed properly. After its development, this technique paved the way for researchers in the field of neurobiology to explore neuroanatomical connections in a murine model. While attempting this procedure, it's important to remember to keep the sections in ammonium hydroxide solution for only 19 to 21 minutes as to not overstain the neurons.After watching this video, you should have a good understanding of how to properly stain, section and rinse brain tissue. Don't forget that working with the mercuric chloride-based solution, ammonium hydroxide solution and xylene can be extremely hazardous, so take precautions and wear lab gloves and work in a fume hood when performing this procedure.

Research

JoVE Journal

Neuroscience

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