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

Summary

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.

Abstract

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.

Introduction

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 colors^1,2. 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 immunostaini....

Protocol

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

1. Dissolve 100 mg of polyvinylpyrrolidone (PVP) with 10 mL of ddH₂O. Vortex the PVP solution lightly.
2. 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 tubing using a 10 mL syringe.
3. Combine 170 mg of tungsten microcarrier beads with 250 µL of methylene chloride. Vortex the tungsten bea....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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