This work was funded by NIH grants HD043680, MH106392, DA013137, and NS100624.

All animal protocols were reviewed and approved by the Animal Care and Use Committee at the University of South Carolina (federal assurance number: D16-00028).
1. Preparation of DiI/Tungsten bead tubing
<ol>
	<li>Dissolve 100 mg of polyvinylpyrrolidone (PVP) with 10 mL of ddH2O. Vortex the PVP solution lightly.</li>
	<li>Fill the tubing with the PVP solution (see Table of Materials) and leave it for 20 min. Then, expel the PVP solution through the other end of the...

<ol>
	<li>Gan, W. B., Grutzendler, J., Wong, W. T., Wong, R. O., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+"DiOlistic"+labeling+of+the+nervous+system+using+lipophilic+dye+combinations.">Multicolor "DiOlistic" labeling of the nervous system using lipophilic dye combinations.</a> Neuron. 27, 219-225 (2000).</li><li>Gan, W. B., Grutzendler, J., Wong, R. O., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ballistic+delivery+of+dyes+for+structural+and+functional+studies+of+the+nervous+system.">Ballistic delivery of dyes for structural and functional studies of the nervous system.</a> Cold Spring Harbor Protocol. 2009 (4), 5202 (2009).</li><li>Seabold, G. K., Daunais, J. B., Rau, A., Grant, K. A., Alvarez, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiOLISTIC+labeling+of+neurons+from+rodent+and+non-human+primate+brain+slices.">DiOLISTIC labeling of neurons from rodent and non-human primate brain slices.</a> Journal of Visualized Experiments. (41), (2010).</li><li>Spacek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+Golgi+method:+a+time-lapse+study+of+the+early+stages+of+impregnation+in+single+sections.">Dynamics of the Golgi method: a time-lapse study of the early stages of impregnation in single sections.</a> Journal of Neurocytology. 18 (1), 27-38 (1989).</li><li>McLaurin, K. A., Li, H., Booze, R. M., Mactutus, C. 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In this protocol, we describe a versatile labeling technique for neurons from both rat brain and those grown in vitro. Furthermore, we report the methodology for utilizing neuronal reconstruction software and neuronal reconstruction quantitative analysis software to assess neuronal morphology and dendritic spines. The assessment of neuronal morphology and dendritic spines provides an opportunity to determine alterations in dendritic branching complexity, neuronal arbor complexity, dendritic spine morphology, and synaptic...

In 2000, Gan et al. described a rapid labeling technique for individual neurons and glia in the nervous system that combined various lipophilic dyes, allowing for the simultaneous labeling of many brain cells with different colors1,2. More recently, a ballistic labeling technique was described by Seabold et al.3 that introduced fluorescent dyes (Dil) into the neurons of brain slices. A versatile staining technique, ballistic labeling is appreciated for its ability to be utilized in multiple animal species and across a wide range of ages. Furthermore, it can be combined with immunostaining to identify subpopulations of brain cells3. Compared with traditional techniques (e.g., Golgi-Cox silver impregnation, microinjection)4, ballistic labeling affords an opportunity to more clearly distinguish morphological characteristics, including dendritic spines, a feature that is critical for drawing inferences about neuronal complexity and synaptic connectivity5.
Excitatory pyramidal neurons are characterized by a single, large apical dendrite, multiple shorter basal dendrites, and thousands of dendritic spines6. Pyramidal neurons are found in multiple brain regions related to higher order cognitive processing, including the prefrontal cortex (PFC) and hippocampus. In the PFC, pyramidal neurons are observed in layers II/III and layer V, with each exhibiting unique morphology. Specifically, pyramidal neurons in layer II/III of the PFC have a shorter apical dendrite and less branching than pyramidal neurons in layer V6. Within the hippocampus, pyramidal neurons are located in both the CA1 and CA3 regions, with each displaying distinct morphologies. Specifically, pyramidal neurons in the CA1 region exhibit a more distinctive apical dendrite, with branching occurring farther from the soma, relative to the CA3 region6.
Dendritic spines on pyramidal neurons in both the PFC and hippocampus are the primary site of excitatory synapses7. Morphological characteristics of dendritic spines, which are classically characterized into three primary categories (i.e., thin, stubby, or mushroom8), have been related to the size of the excitatory synapse9. Thin spines, characterized by a long, thin neck, small bulbous head, and smaller postsynaptic densities, are more unstable and develop weaker connections. However, mushroom spines, which have a larger dendritic spine head, are recognized for forming stronger synaptic connections, an effect resulting from their larger size. In sharp contrast, stubby spines are devoid of a spine neck, exhibiting an approximately equal head and neck volume ratio8. Within the hippocampus, branched spines may also be observed, whereby the spine has multiple heads that emerge from the same dendritic spine neck10. Therefore, the morphological changes of dendritic spines could reflect functionality and structural capacity. Furthermore, studies have demonstrated that the size and shape of dendritic spines relates to their structural plasticity, leading to the idea that small spines are involved in learning and attention, whereas larger, more stable spines, are involved in long-term processes, including memory11. Additionally, the distribution of dendritic spines along the dendrite may be associated with synaptic connectivity5,12.
Thus, the present methodological paper has three goals: 1) Present our protocol for ballistic labeling, which has been utilized with a success rate (i.e., neurons meeting selection criteria and appropriate for analysis) of 83.3%5,12,13 and across multiple brain regions (i.e., PFC, nucleus accumbens, hippocampus); 2) Demonstrate the generalizability of the technique and its application to neurons grown in vitro; 3) Detail the methodology utilized in neuronal reconstruction software and the inferences that can be drawn from such data.

<table border="0" cellpadding="0" cellspacing="0" style="width:868px;" width="869">
	<colgroup>
		<col />
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">20Gx25mm PrecisionGlide needle</td>
			<td style="width:263px;">BD</td>
			<td style="width:143px;">305175</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well cell culture plate</td>
			<td>Costar</td>
			<td>3562</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Glass Bottom Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-20-C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic-Antimycotic solution</td>
			<td>Cellgro</td>
			<td>30004CI</td>
			<td>100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 supplement</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>50X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barrel liner</td>
			<td>BIO-RAD</td>
			<td>165-2417</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borax</td>
			<td>Sigma</td>
			<td>B9876</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Boric acid</td>
			<td>Sigma</td>
			<td>B0252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cartridge holder</td>
			<td>BIO-RAD</td>
			<td>165-2426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Confocal imaging software</td>
			<td style="width:263px;">Nikon</td>
			<td style="width:143px;">EZ-C1</td>
			<td>version 3.81b</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Confocal microscope</td>
			<td style="width:263px;">Nikon</td>
			<td style="width:143px;">TE-2000E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass</td>
			<td>VWR</td>
			<td>637-137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DilC18(3)</td>
			<td>Fisher Scientific</td>
			<td>D282</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12 medium</td>
			<td>Life Technologies</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14095</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #7 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">F344 rat</td>
			<td style="width:263px;">(Harlan Laboratories, Indianapolis, IN)</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>VWR</td>
			<td>101174Y</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Sigma</td>
			<td>H4641</td>
			<td>10X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Helios diffusion screens</td>
			<td>BIO-RAD</td>
			<td>165-2475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Helios gene gun kit</td>
			<td>BIO-RAD</td>
			<td>165-2411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Helios gene gun system</td>
			<td>BIO-RAD</td>
			<td>165-2431</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Helium hose assembly</td>
			<td>BIO-RAD</td>
			<td>165-2412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris Forceps</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris Scissors</td>
			<td>World Precision Instruments</td>
			<td>500216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene chloride</td>
			<td>Fisher Scientific</td>
			<td>D150-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Neurolucida 360 software</td>
			<td style="width:263px;">mbf bioscience</td>
			<td style="width:143px;"></td>
			<td>dendritic spine analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P9155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvinylpyrrolidone</td>
			<td>Fisher Scientific</td>
			<td>5295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
			<td>Fisher Scientific</td>
			<td>P36930</td>
			<td>mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat brain matrix, 300 - 600g, Coronal, 0.5mm</td>
			<td>Ted Pella</td>
			<td>15047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Life Technologies</td>
			<td>25080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe kit</td>
			<td>BIO-RAD</td>
			<td>165-2421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tefzel tubing</td>
			<td>BIO-RAD</td>
			<td>165-2441</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubing cutter</td>
			<td>BIO-RAD</td>
			<td>165-2422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubing Prep station</td>
			<td>BIO-RAD</td>
			<td>165-2418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tungsten M-25 Microcarrier 1.7 &#181;m</td>
			<td>BIO-RAD</td>
			<td>165-2269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas Scissors</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
	</tbody>
</table>

ballistic labeling of pyramidal neurons in brain slices and in primary cell culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

In Figure 2A, the typical pyramidal neurons in the hippocampal region in the rat brain sections were identified by ballistic labeling technology, characterized by one large apical dendrite and several smaller basal dendrites around the soma. Figure 2B shows the neuron in the neuronal reconstruction quantitative analysis software after the soma was detected, dendritic branches were traced, and spines were detected. Subsequently, the data were analyzed using neuro...

Watch this Scientific Journal Video about Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture at JoVE.com

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

This protocol makes it possible to evaluate potential morphological alterations in neurons and dendritic spines that may underline neurochemical and behavioral abnormalities. It is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction. Begin by preparing PVP solution according to manuscript directions.Fill the tubing with PVP and leave it for 20 minutes. And expel it through the other end with a 10 milliliter syringe. Combine 170 milligrams of tungsten microcarrier beads with 250 microliters of methylene chloride.Then thoroughly vortex the suspension. Next, add 6 milligrams of lipophilic DilC18(3)dye to 300 microliters of methylene chloride and vortex. Pipet 250 microliters of the tungsten bead suspension onto a glass slide.Wait for the suspension to air dry and add 300 microliters of the dye solution. Once dried, use a razor to split the mixture into two 1.5 milliliter centrifuge tubes and fill the tubes with water. Sonicate the mixture in a water bath until homogenous.Making sure that the sonicator tip lies directly on the tubes. Combine the two mixtures in a 15 milliliter conical tube. And sonicate for another three minutes, making sure that no large clumps of dye coated beads remain.After sonication, draw the mixture into the PVP coated tubing with a 10 milliliter syringe. And feed the tubing into the preparation station. Rotate the tubing for one minute.Carefully remove all the water using a syringe. Turn on the nitrogen gas and adjust the nitrogen flow to approximately 0.5 liters per minute. Rotate the tubing in the preparation station and dry it with nitrogen for 30 minutes.Remove the tubing from the station and cut it into 13 millimeter segments with a tubing cutter. Make sure that the rat is not responsive to noxious stimuli and reflexes are absent. Then secure it in the supine position.Make an incision along the thoracic midline. Separate the diaphragm and open the chest with scissors. Then insert a 20 gauge 25 millimeter needle into the left ventricle.Immediately cut the right atrium with scissors and perfuse 15 milliliters of 100 millimolar PBS with a 5 millimeters per minute flow rate. Then perfuse 100 milliliters of 4%PFA buffered in PBS. After perfusion, remove the entire rat brain.And postfix it with 4%PFA for 10 minutes. Use a rat brain matrix to cut 500 micrometer thick coronal section. Make a fist cut and keep the blade in place.Then make a second cut with a second blade and vertically remove the first blade, keeping the tissue on the blade surface. Place the brain slices in the 24-well plate with 1 milliliter of PBS in each well. And repeat the process until all slices have been cut.Remove PBS from each targeted well. Load the cartilage with piece of Dil/tungsten tubing and place it into the applicator. Put a piece of filter paper between the two mesh screens.And connect the applicator to the helium hose. Then adjust the output pressure of helium to 90 pounds per square inch. Place the applicator vertically on the center of the targeted well 1.5 centimeters between the sample and the mesh screen.Then fire the Dil/tungsten tubing. Load the cartilage with the next tubing and continuously fire the beads from the tubing on the remaining slices. Fill the 24-well plate with 100 millimolar PBS.And wash the slices three times with 500 microliters of fresh PBS. Making sure that the slices do not flip over during the wash. When finished, add 500 microliters of fresh PBS to the slices.And incubate them for three hours at four degree Celsius in the dark. After the incubation, use a fine brush to transfer the brain slices onto glass slides. And immediately add one milliliter of antifade mounting medium to each section.Place a 22 by 15 millimeter coverslip over the sections and dry the slides in the dark. Turn on the confocal microscope system and switch to a 60X objective. Adjust the imaging settings according to manuscript directions.And obtain Z-stack images for the targeted neuron type based on brain region boundaries and morphological characteristics of neurons. Isolate primary cortical neuron from F344/N rats at postnatal day one and culture them in a 35 millimeter glass bottom dish for one week. Refreshing half of the medium on the third day after isolation.Wash the dish twice with 1 milliliter of 100 millimolar PBS. And fix the cells with 4%PFA for 15 minutes at room temperature. Ballistically label the cells as previously described.And wash them three more times with 1 milliliter of PBS. Add 500 microliters of PBS to the cells and incubate them for three hours at four degree Celsius in the dark. Then add 200 microliters of antifade mounting medium.And obtain Z-stack images for each targeted neuron. Typical pyramidal neurons in hippocampal region in the rat brain sections were identified with ballistic labeling technology characterized by one large apical dendrite and several smaller basal dendrites around the soma. Neuronal reconstruction quantitative analysis software was used to trace dendritic branches and detect spines.Subsequently, the software was used to assess the dendritic branching complexity and neuronal arbor complexity. Morphological changes in dendritic spines were assessed using length, volume, and head diameter. Then spines were classified into thin, stubby, and mushroom spines.And the relative frequency of the number of the spines between each radius was examined. Further more the ballistic labeling technique was validated on a primary pyramidal neuron in cell culture. Pyramidal neurons were identified based on the triangle shape of the soma and large apical dendrite.Then neuronal reconstruction software was used to analyze the distribution of thin dendritic spines and dendritic spine length. When attempting this protocol, it is important to avoid large clumps or clusters of ballistic dye coated tungsten beads starting preparation. Clumps would not allow individual neurons to be distinguished.Combined with neuronal reconstruction software this method allows us to examine neuronal and dendritic spine morphology in hippocampal pyramidal neurons. Neuronal reconstruction software utilize an algorithm to offer automatical assisted classification of dendritic spines. The quantification of multiple neuronal parameters offers an opportunity to better understand the mechanism underlying neuronal cognitive dysfunction.

ballistic-labeling-pyramidal-neurons-brain-slices-primary-cell

Research

JoVE Journal

Neuroscience

6.5K Views.  University of South Carolina. Questo protocollo consente di valutare potenziali alterazioni morfologiche nei neuroni e nelle spine dendritiche che possono sottolineare anomalie neurochimiche e comportamentali. È unico e utile per visualizzare i neuroni in diverse regioni cerebrali nei ratti, che in combinazione con sofisticato software di ricostruzione consente ai ricercatori di chiarire i possibili meccanismi alla base della disfunzione neurocognitiva. Inizia preparando la soluzione PVP secondo le indicazioni manoscritt...