This work was supported by Sacred Heart University Undergraduate Research InitiativeGrants.

All experimental procedures are approved by the Sacred Heart University Institutional Animal Care and Use Committee and are in accordance with the NIH Guide for the Care and Use of Animals.
1. Isolation and infiltration of brain tissue
<ol>
	<li>Premix solutions A and B of the Golgi staining kit 24 h prior to use and keep in dark bottles and/or in dark. Make approximately 80 mL of solution A and B mix which is sufficient to change the solution after 24 h. Store in airtight bottles. 
	NOTE: Perfusion with either saline or paraformaldehyde is not necessary.</li>
	<li>Sacrifice rats by guillotine following carbon dioxide euthanasia and remove brains within seconds. Rinse brains in saline if required but is not necessary.
	<ol>
		<li>Place the brain, cortex down so that the hypothalamus is visible because the cuts are made anterior and posterior to it, on a non-porous surface. Cut into an anterior (contains the prefrontal cortex) and posterior (contains the hippocampus) block as shown in Figure 1.</li>
	</ol>
	</li>
	<li>Place blocks into the premixed solutions of A and B, making sure that they are well immersed in the solution. Ensure that the volume of solutions A and B is sufficient to immerse the blocks. Store blocks in either brown bottles or clear bottles covered with foil (to keep light out) in the dark at room temperature.</li>
	<li>Replace solution after 24 h and keep in the dark for another 13 days at room temperature. 
	NOTE: If used with mouse brains, there is no need to block, and the entire brain can be placed in the solution. If staining brain areas, which are different in size, the time in solutions A and B may have to be determined by trial and error.</li>
	<li>After two weeks the tissue is infiltrated well with solutions A and B, transfer the blocks to the cryoprotectant solution (solution C in the Golgi staining kit), and leave them for 48-72 h at 4 &#176;C. 
	NOTE: After cryoprotection, blocks may be frozen until further processing. Also, note that all solutions from the kit are collected and disposed of as hazardous waste.</li>
	<li>Freeze brain, cortex down, on a glass slide on dry ice. Once frozen either cut on the cryostat or store at -80 &#176;C until sectioning using a cryostat. 
	&#8203;NOTE: Do not freeze in liquid nitrogen as this produces cracks in the tissue.</li>
</ol>
2. Sectioning of brain tissues
<ol>
	<li>Place a small amount of tissue medium on a pre-cooled cryostat chuck. Mount blocks on cryostat chucks by thawing one side of the block slightly in hand (gloved) and placing it on the tissue medium. 
	NOTE: It is not necessary to embed the tissue. The block containing the hippocampus is somewhat easier to cut than the block with the prefrontal cortex because the latter is anterior to the corpus callosum and the two halves are separate.</li>
	<li>Cut 100 &#181;m sections on a cryostat at -22 &#176;C and mount onto subbed slides. Sections up to 150 &#181;m of thickness can be used. Try to mount 3-4 coronal sections per slide as this decreases the number of slides that require processing. Use one of the following techniques to keep the sections frozen till they get on the slide. 
	NOTE: These sections are not fixed in the usual way and therefore they tend to melt quickly. Allow the sections to melt on the slide. If they begin to melt before being placed on the slide, it is impossible to get them to be flat on the slide. There are several techniques to do this.
	<ol>
		<li>Use a freezing spray on the knife and the block. Depending on the temperature and humidity in the room this may be necessary for every section. Use either the antiroll plate or a brush to keep the section flat while cutting. 
		NOTE: Make sure to have enough freezing spray. Several cans can be used when cutting 20 blocks.</li>
		<li>Thaw mount, if possible, by using a room temperature slide and quickly appose it to the section. With practice, one can mount several sections at once. If this doesn&#39;t work, keep slides in the cryostat so they are very cold and transfer the section with cold forceps or a cold paintbrush and then thaw mount.</li>
	</ol>
	</li>
	<li>Clean the knife with paper wipes between slices. If the knife requires more cleaning, use 100% ethanol (EtOH), and let it dry before cutting the next slice.</li>
	<li>Once mounted onto the slide, place the slide flat on cardboard slide trays and allow sections to dry at room temperature (for several hours to a maximum of 48 h) in the dark. Do not cover the slide tray. Ensure that the sections are completely dry before Golgi impregnation. Store flat, in the dark, on slide trays until Golgi impregnation. 
	&#8203;NOTE: Drying the sections does not cause any damage.</li>
</ol>
3. Staining and dehydration of brain tissue
<ol>
	<li>Perform staining in glass staining dishes. Mix solutions D and E immediately prior to staining. As per the protocol, add one part of solution D, one part of solution E, and two parts of distilled water. For glass staining dishes, make a 200 mL solution to completely submerge the sections. For Koplin jars, this requires less volume.</li>
	<li>Place slides in racks spaced far enough apart to allow solution access to sections.</li>
	<li>Place sections in distilled water for 4 min (2x) before placing them into the Golgi impregnation staining solution for 10 min. Change the staining solution every 70 sections. Change the distilled water when it turns yellow. 
	NOTE: The timing for the staining step is critical. Too short does not allow enough staining and too long causes over staining, making the dendrites hard to separate when doing analysis. Once out of the staining solution the timing is less critical.</li>
	<li>Dehydrate the sections as follows: 70% EtOH (5 min), 95% EtOH (5 min; 2x), 100% EtOH (5 min; 2x), clearing agent (5 min; 3x). Change all solutions often, especially the 100% EtOH and clearing agent to ensure the sections remain anhydrous. 
	NOTE: It is not necessary to go through a 50% EtOH step. Counterstaining is not needed for cell visualization because the Golgi impregnation provides sufficient contrast. The use of other clearing agents has not worked as well as the one used here (see Table of materials).</li>
	<li>Coverslip with glass coverslips that are 60 mm long with a generous amount of mounting medium. Make sure that there are, as few air bubbles as possible. If needed, carefully remove, and redo the coverslip (can be done even weeks later). Do this very slowly in order not to damage the section (can be done because of the large amount of mounting medium). 
	NOTE: A large amount of mounting medium is somewhat messy but still necessary because enough mounting medium must be used to cover the thick 100 &#181;m sections.</li>
	<li>To ensure that only one coverslip is placed on the sections, separate the coverslips in advance of the process.</li>
	<li>Once cover slipped, dry slides flat on any non-porous paper for 3-5 days, moving them slightly, especially after the first day, to avoid sticking. After 3-5 days transfer the slides to slide holders and, ideally, dry slides for at least 3 weeks before examining them. Keep slides flat to decrease the possibility of air bubbles forming.</li>
</ol>
4. Determination of dendritic spine density
<ol>
	<li>For analysis of dendritic spine density in pyramidal neurons of both the mPFC and the CA1 region of the hippocampus, examine the most lateral secondary basal dendrites and the most lateral tertiary apical dendrites as described in step 4.1.1 (Figure 2).
	<ol>
		<li>Choose a dendrite, measure the length of the dendrite using an image analysis program, count the spines on the dendrites using a hand counter, and record both length and number of spines.</li>
	</ol>
	</li>
	<li>Study and analyze six cells per region (mPFC, CA1) per brain. Quantify a minimum of six brains per group as previously described7,8. Choose neurons that meet the following criteria for analysis: cell bodies and dendrites are well impregnated; dendrites are distinguishable from adjacent cells and are continuous.</li>
	<li>Count the spines at 1000x (oil-immersion) by hand counting with a light microscope and measure dendritic length using an image analysis program. Calculate spine density by dividing the spine number by the length of the dendrite and express data as the number of spines/ 10 &#181;m dendrite. 
	NOTE: There are far more sophisticated methods for differentiating spine subtypes and dendritic architecture that can be used, but hand counting with a light microscope at 1000x can give a rapid result that can then determine if further investigation is necessary. Although every effort is made to consistently sample similar dendrites, there are variations in thickness that may affect the counting.</li>
</ol>

<ol>
	<li>Pannese, E. <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+Stain:+invention,+diffusion+and+impact+on+neurosciences.">The Golgi Stain: invention, diffusion and impact on neurosciences.</a> Journal of the History of the Neurosciences. 8 (2), 132-140 (1999).</li><li>Bentivoglio, M., 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=The+Original+Histological+Slides+of+Camillo+Golgi+and+His+Discoveries+on+Neuronal+Structure.">The Original Histological Slides of Camillo Golgi and His Discoveries on Neuronal Structure.</a> Frontiers in Neuroanatomy. 13, 3 (2019).</li><li>Swanson, L. W., Newman, E., Araque, A., Dubinsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Beautiful Brain: The Drawings of Santiago Ramon y Cajal. , 208 (2017).</li><li>Dall'Oglio, A., Ferme, D., Brusco, J., Moreira, J. E., Rasia-Filho, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&amp;#34;single-section&amp;#34;+Golgi+method+adapted+for+formalin-fixed+human+brain+and+light+microscopy.">The &amp;#34;single-section&amp;#34; Golgi method adapted for formalin-fixed human brain and light microscopy.</a> Journal of Neuroscience Methods. 189 (1), 51-55 (2010).</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> Journal of Neuroscience Methods. 11 (4), 221-230 (1984).</li><li>Gould, E., Frankfurt, M., Westlind-Danielsson, A., McEwen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+forebrain+astrocytes+are+sensitive+to+thyroid+hormone.">Developing forebrain astrocytes are sensitive to thyroid hormone.</a> Glia. 3 (4), 283-292 (1990).</li><li>Gould, E., Woolley, C. S., Frankfurt, M., McEwen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadal+steroids+regulate+dendritic+spine+density+in+hippocampal+pyramidal+cells+in+adulthood.">Gonadal steroids regulate dendritic spine density in hippocampal pyramidal cells in adulthood.</a> Journal of Neuroscience. 10 (4), 1286-1291 (1990).</li><li>Woolley, C. S., Gould, E., Frankfurt, M., McEwen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+fluctuation+in+dendritic+spine+density+on+adult+hippocampal+pyramidal+neurons.">Naturally occurring fluctuation in dendritic spine density on adult hippocampal pyramidal neurons.</a> Journal of Neuroscience. 10 (12), 4035-4039 (1990).</li><li>Frankfurt, M., Salas-Ramirez, K., Friedman, E., Luine, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine+alters+dendritic+spine+density+in+cortical+and+subcortical+brain+regions+of+the+postpartum+and+virgin+female+rat.">Cocaine alters dendritic spine density in cortical and subcortical brain regions of the postpartum and virgin female rat.</a> Synapse. 65 (9), 955-961 (2011).</li><li>Frankfurt, M., Luine, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+role+of+dendritic+spines+and+memory:+Interaction(s)+with+estradiol.">The evolving role of dendritic spines and memory: Interaction(s) with estradiol.</a> Hormones Behavior. 74, 28-36 (2015).</li><li>Bowman, R. E., Luine, V., Khandaker, H., Villafane, J. J., Frankfurt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adolescent+bisphenol-A+exposure+decreases+dendritic+spine+density:+role+of+sex+and+age.">Adolescent bisphenol-A exposure decreases dendritic spine density: role of sex and age.</a> Synapse. 68 (11), 498-507 (2014).</li><li>Bowman, R. E., 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=Bisphenol-A+exposure+during+adolescence+leads+to+enduring+alterations+in+cognition+and+dendritic+spine+density+in+adult+male+and+female+rats.">Bisphenol-A exposure during adolescence leads to enduring alterations in cognition and dendritic spine density in adult male and female rats.</a> Hormones Behavior. 69, 89-97 (2015).</li><li>Eilam-Stock, T., Serrano, P., Frankfurt, M., Luine, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol-A+impairs+memory+and+reduces+dendritic+spine+density+in+adult+male+rats.">Bisphenol-A impairs memory and reduces dendritic spine density in adult male rats.</a> Behavioral Neuroscience. 126 (1), 175-185 (2012).</li><li>Inagaki, T., Frankfurt, M., Luine, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-induced+memory+enhancements+are+blocked+by+acute+bisphenol+A+in+adult+female+rats:+role+of+dendritic+spines.">Estrogen-induced memory enhancements are blocked by acute bisphenol A in adult female rats: role of dendritic spines.</a> Endocrinology. 153 (7), 3357-3367 (2012).</li><li>Jacome, L. F., 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=Gonadal+Hormones+Rapidly+Enhance+Spatial+Memory+and+Increase+Hippocampal+Spine+Density+in+Male+Rats.">Gonadal Hormones Rapidly Enhance Spatial Memory and Increase Hippocampal Spine Density in Male Rats.</a> Endocrinology. 157 (4), 1357-1362 (2016).</li><li>Frankfurt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol-A:+a+plastic+manufacturing+compound+disrupts+critical+brain+structures+and+impairs+memory.">Bisphenol-A: a plastic manufacturing compound disrupts critical brain structures and impairs memory.</a> Research Features. , (2021).</li><li>Wallace, M., Luine, V., Arellanos, A., Frankfurt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovariectomized+rats+show+decreased+recognition+memory+and+spine+density+in+the+hippocampus+and+prefrontal+cortex.">Ovariectomized rats show decreased recognition memory and spine density in the hippocampus and prefrontal cortex.</a> Brain Research. 1126 (1), 176-182 (2006).</li><li>Wallace, M., Frankfurt, M., Arellanos, A., Inagaki, T., Luine, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+recognition+memory+and+decreased+prefrontal+cortex+spine+density+in+aged+female+rats.">Impaired recognition memory and decreased prefrontal cortex spine density in aged female rats.</a> Annals of the New York Academy of Science. 1097, 54-57 (2007).</li><li>Bowman, R. E., Hagedorn, J., Madden, E., Frankfurt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+adolescent+Bisphenol-A+exposure+on+memory+and+spine+density+in+ovariectomized+female+rats:+Adolescence+vs+adulthood.">Effects of adolescent Bisphenol-A exposure on memory and spine density in ovariectomized female rats: Adolescence vs adulthood.</a> Hormones Behavior. 107, 26-34 (2019).</li><li>Novaes, L. S., Dos Santos, N. B., Perfetto, J. G., Goosens, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+prevents+acute+restraint+stress-induced+anxiety-related+behavior+but+not+changes+in+basolateral+amygdala+spine+density.">Environmental enrichment prevents acute restraint stress-induced anxiety-related behavior but not changes in basolateral amygdala spine density.</a> Psychoneuroendocrinology. 98, 6-10 (2018).</li><li>Trzesniewski, J., Altmann, S., J&#228;ger, L., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+Purkinje+cell+size+is+compatible+with+near+normal+morphology+and+function+of+the+cerebellar+cortex+in+a+mouse+model+of+spinocerebellar+ataxia.">Reduced Purkinje cell size is compatible with near normal morphology and function of the cerebellar cortex in a mouse model of spinocerebellar ataxia.</a> Experimental Neurology. 311, 205-212 (2019).</li><li>Zemmar, 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=Oligodendrocyte-+and+Neuron-Specific+Nogo-A+Restrict+Dendritic+Branching+and+Spine+Density+in+the+Adult+Mouse+Motor+Cortex.">Oligodendrocyte- and Neuron-Specific Nogo-A Restrict Dendritic Branching and Spine Density in the Adult Mouse Motor Cortex.</a> Cerebral Cortex. 28 (6), 2109-2117 (2018).</li></ol>

The authors have nothing to disclose.

The present protocol describes a method of Golgi impregnation that allows for rapid simultaneous processing of many sections. It is an improvement over previously described5 more labor-intensive methods and consistently yields impregnated neurons for analysis. In addition, there is less exposure to toxic chemicals used in Golgi impregnation. The most challenging part of the process is getting the sections to be flat on the slides, which takes considerable practice. Keeping everything as cold as po...

The method of using potassium dichromate and silver nitrate to label neurons was first described by Camillo Golgi1,2 and subsequently used by Santiago Ramon y Cajal to produce an immense body of work differentiating neuronal and glial subtypes. A recently published book with his illustrations is now available3. Following Ramon y Cajal&#39;s studies, which were published more than 100 years ago, very little Golgi impregnation was used. Golgi impregnation is a laborious process that allows three-dimensional visualization of neurons with a light microscope. There have been numerous modifications of the Golgi method over the years to make the method easier and the staining more consistent4. In 1984, Gabbott and Somogyi5 described the single section Golgi impregnation procedure which allowed for more rapid processing. This Golgi impregnation method requires perfusion with 4% paraformaldehyde and 1.5% picric acid, post-fixation followed by vibratome sectioning into a bath of 3% potassium dichromate. Sections are mounted onto glass slides, the four corners of coverslips glued so that when immersed in silver nitrate, diffusion is gradual. Coverslips are then popped off, sections are dehydrated, and eventually cover slipped permanently with mounting medium. This technique was successfully used to label neurons and glia6,7,8 in the hippocampus. The rapid Golgi method described here is an improvement because there is far less exposure to both potassium dichromate and silver nitrate and no paraformaldehyde and picric acid are used. In addition, although cells that were impregnated using modifications of the Gabbott and Somogyi5 method could be analyzed, often the sections were over-or under-exposed or fell off the slides during the dehydration step and generally, several experiments had to be pooled to have enough cells for analysis.
The present protocol describes the use of the Golgi staining kit (see Table of materials) to label dendrites and dendritic spines in the hippocampus and medial prefrontal cortex (mPFC) of the rat. The advantages of this method over previous ones are that it is rapid, there is less exposure to noxious chemicals for the researcher and there is consistent staining of neurons. The protocol described below has been used with minor modifications to assess dendritic spine density in the hippocampus and mPFC of the rat in many studies9,10,11,12,13,14,15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cardboard slides trays</td>
			<td>Fisher Scientific</td>
			<td>12-587-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 24 x 60mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-M</td>
			<td></td>
		</tr>
		<tr>
			<td>FD Rapid GolgiStain kit</td>
			<td>FD Neurotechnologies</td>
			<td>PK 401</td>
			<td>Stable at RT in the dark for months; Golgi staining kit</td>
		</tr>
		<tr>
			<td>Freezing Spray</td>
			<td>Fisher Scientific</td>
			<td>23-022524</td>
			<td></td>
		</tr>
		<tr>
			<td>HISTO-CLEAR</td>
			<td>Fisher Scientific</td>
			<td>50-899-90147</td>
			<td>clearing agent</td>
		</tr>
		<tr>
			<td>NCSS Software</td>
			<td>Kaysville, UT, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP-15-100</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek CTYO OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td>Used to mount brains on cryostat chuck</td>
		</tr>
	</tbody>
</table>

rapid golgi stain for dendritic spine visualization in hippocampus and prefrontal cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

Using the rapid Golgi method, cells are consistently well impregnated so that there are plenty of cells to analyze. This is a marked improvement over prior methods where experiments had to be pooled to have enough data for analysis. Therefore, more samples can be processed at once and brains can be stored frozen until processing. Examples of Golgi impregnated cells in the CA1 region of the hippocampus are shown at low and high power in Figure 3. Counting of spines in a given region yields co...

Watch this Scientific Journal Video about Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex at JoVE.com

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial ...

rapid-golgi-stain-for-dendritic-spine-visualization-hippocampus

Microsoft Bing

Research

JoVE Journal

Neuroscience

5.8K Views.  Donald and Barbara Zucker School of Medicine at Hofstra/Northwell. The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.

Video: Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

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.

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 ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find preventive and curative measures. The most effective way to achieve this is through direct examination of diseased and healthy brain tissue. The authors present a protocol to obtain, process, characterize and store good quality brain tissue donated by individuals registered in an antemortem brain donation program. The donation program includes a face-to-face empathic approach to people, a collection of complementary clinical, biological, social and lifestyle information and serial multi-dimensional assessments over time to track individual trajectories of normal aging and cognitive decline. Since many neurological diseases are asymmetrical, our brain bank offers a unique protocol for slicing fresh specimens. Brain sections of both hemispheres are alternately frozen (at -80 &#176;C) or fixed in formalin; a fixed slice on one hemisphere corresponds to a frozen one on the other hemisphere. With this approach, a complete histological characterization of all frozen material can be obtained, and omics studies can be performed on histologically well-defined tissues from both hemispheres thus offering a more complete assessment of neurodegenerative disease mechanisms. Correct and definite diagnosis of these diseases can only be achieved by combining the clinical syndrome with the neuropathological evaluation, which often adds important etiological clues necessary to interpret the pathogenesis. This method can be time consuming, expensive and limited as it only covers a limited geographical area. Regardless of its limitations, the high degree of characterization it provides can be rewarding. Our ultimate goal is to establish the first Italian Brain Bank, all the while emphasizing the importance of neuropathologically verified epidemiological studies.

This protocol describes a method to track individual brain-aging trajectories through a brain donation program and proper characterization of brains. Brain donors are involved in a long-term longitudinal study including serial multi-dimensional assessments. The protocol contains a detailed description of brain processing and an accurate diagnostic methodology.

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

LFP Recording in Mouse Brain for Cognitive Research and Drug Analysis

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal populations. It serves as a crucial tool in cognitive research, particularly in brain regions like the hippocampus and prefrontal cortex. Dual LFP recordings between these areas are of particular interest as they allow the exploration of interregional signal communication. However, methods for performing these recordings are rarely described, and most commercial recording devices are either expensive or lack adaptability to accommodate specific experimental designs. This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of antipsychotic drugs and potassium channel modulators on LFP properties in these areas. The technique enables the measurement of LFP properties, including power spectra within each brain region and coherence between the two. Additionally, a low-cost, custom-designed recording device has been developed for these experiments. In summary, this protocol provides a means to record signals with high signal-to-noise ratios in different brain regions, facilitating the investigation of interregional information communication within the brain.

Author Spotlight: Studying Drug Impacts on Brain Signals Using Dual LFP Recording Protocol in Mice

This protocol outlines the use of a custom-designed recording device and electrodes to record local field potentials and investigate information flow in the mouse hippocampus and prefrontal cortex.

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal ...