Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Summary

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Abstract

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

Introduction

Histological stains have traditionally been used to aid in the identification of cortical areas in various species by revealing differences in architectonic features. The combined use of histochemical techniques such as for Nissl substance, cytochrome oxidase (CO) reactivity, or myelin can prove fruitful as they reveal similar areal boundaries in the adult brain. However, these histochemical stains do not always adequately reveal clear boundaries between cortical areas and layers in the immature brain.

In the central nervous system, zinc has several critical functions that include stabilizing DNA structure, acting as an enzyme cofactor, par....

Protocol

The following protocol follows the animal care guidelines established by the Institutional Animal Care and Use Committee (IACUC) at The City College of New York, which conform to all appropriate state and federal guidelines. Anesthesia is appropriate for ferrets, and should be modified according to species studied.

Figure 1: Flowchart outlining the major....

Results

The major steps involved in this protocol to stain brain sections for synaptic zinc are presented in a flowchart in Figure 1. The protocol can be divided into three phases: 1) Perfusion and tissue collection, 2) Tissue preparation and staining, and 3) Zinc histochemistry. Briefly, during the first phase of the protocol, the animal is anesthetized and injected IP with the appropriate dose of sodium selenite. After a sufficient time period (ideally 60 - 90 min).......

Discussion

The current study employs a histochemical technique based on a modified version of the Danscher (1982) method¹⁰, whereby synaptic zinc localization may be detected and visualized in the brain. This method essentially works by injecting the animal with the zinc chelator sodium selenite (Na₂SeO₃) (15 mg/kg). Following injection, the selenite travels to the brain and binds to free zinc that is localized to presynaptic vesicles of zinc containing neurons. Zinc ions bound to molec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Euthasol (Euthanasia solution)	Henry Schein	710101
Sodium selenite	Sigma-Aldrich	214485
Ketamine (Ketaved)	Henry Schein	48858	100 mg/ml injectables
Xylazine (Anased)	Henry Schein	33198	100 mg/ml injectables
Paraformaldehyde	Sigma-Aldrich	F8775	Dilute to 4%
Gum arabic	Sigma-Aldrich	G9752-500G
Citric acid	Sigma-Aldrich	C1909
Sodium citrate	Sigma-Aldrich	W302600
Hydroquinone	Sigma-Aldrich	H9003
Silver lactate	Sigma-Aldrich	85210
Fish gelatine	Sigma-Aldrich	G7765
Cytochrome c	Sigma-Aldrich	C2506	(Type III, from equine heart)
Catalse	Sigma-Aldrich	C10
Sucrose	Domino
Xylene	Fisher Scientific	X5P-1GAL
Permount	Fisher Scientific	SP15-500
100% Ethanol	Fisher Scientific	A406-20	Used for dehydration prior to slide mounting
Coverslips	Brain Research Laboratories	#3660-1
Frosted unsubbed slides	Brain Research Laboratories	#3875-FR
Microtome	American Optical Company	860
Microscope	Olympus	BX-60
Adope Photoshop	Adobe Systems, San Jose, CA		To assemble images
ImageJ	Free software can be downloaded at http://rsb.info.nih.gov/ij/		For densometric measurements
Plastic tray	Any standard plastic tray may be used		to immerse slides in developer solution
Hot plate	Any standard hotplate may be used