Name: rapid golgi stain for dendritic spine visualization in hippocampus and prefrontal cortex
Uploaded: 2021-12-03T14:00:00
Description: Watch this Scientific Journal Video about Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex at JoVE.com

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 b...

<ol>
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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 ...

Although relatively simple, the Golgi impregnation technique does have some tricky steps and failure to do them properly results in cells that are unfit for analysis. This technique provides rapid, reproducible labeling of Golgi impregnated neurons. And in addition, the small standard error of the mean allows for comparison between experiments.The most challenging part about this technique is cutting the cryostat sections, because there are an unusual consistency and fixation, and therefore getting them on the slide flat is a bit of a challenge takes practice. First, place a small quantity of tissue medium on a pre-cool cryostat chuck. Mount the brain blocks on cryostat chucks by thawing one side of the block slightly in a gloved hand and placing it on the tissue medium.Use a freezing spray on the knife and the block. Use either the anti-roll plate or brush to keep the section flat while cutting. Cut 100 micrometer sections on a cryostat at minus 22 degrees Celsius.Thaw mount, if possible, by using a room temperature slide and quickly oppose it to the section. Alternatively, keep slides in the cryostat, so they are freezing. Transfer the section with cold forceps or a cold paint brush and then thaw mount.Mount three to four coronal sections onto subsides. Clean the knife with paper wipes between slices. If the knife requires further cleaning, use 100%ethanol and dry before cutting the next slice.Place slides in racks, spaced far enough to allow solution access to sections. Place sections in distilled water twice for four minutes before placing them into the Golgi impregnation solution for 10 minutes. Separate the cover slips to ensure only one cover slip is placed on the sections.Cover the glass slides with a generous amount of mounting medium before placing a glass cover slip on the slide. Once cover slipped, dry slides flagged on any non-porous paper for three to five days, moving them slightly, especially after the first day to avoid sticking. Later transfer the slides to slide holders and ideally, dry slides for at least three weeks before examining them.To analyze dendritic spine density in pyramidal neurons of both the medial prefrontal cortex and the CA1 region of the hippocampus, measure the dendritic length using in image analysis program. Count the spines on the dendrites using a hand counter and record both length and number of spines. For analysis, choose neurons with cell bodies and dendrites while impregnated, and dendrites that are continuous and distinguishable from the adjacent cells.The figure shows examples of Golgi impregnated cells in the CA1 region of the hippocampus is shown at low and high power. The figure illustrates an experiment in which basal dendritic spine density was increased on pyramidal cells in adolescent male and female rats after environmental enrichment in CA1 and the medial prefrontal cortex. It's challenging to keep the sections cold enough when cutting and we use a great deal of freezing spray in order to do that.And then subsequently, it is difficult to get the sections onto the slides and have them dry flat. This technique is used to examine neuroanatomical characteristics. It can be used alone or in combination with other neuroanatomical tracing methods.

Research

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