Visualization of Cortical Modules in Flattened Mammalian Cortices

Summary

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

Abstract

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

Introduction

Some of the most significant changes in the brain structure across phylogeny can be observed in the cerebral cortex. Despite significant differences, the cortex of animals follows a common pattern and can be broadly divided in two distinct ways, by layers and areas¹. Cortical layers lie parallel to the surface of the brain and vary in number from 3 layers in reptilian cortices² to 6 layers in mammalian cortices¹. Cortical areas on the other hand are distinct regions of the cortex which largely correspond to distinct functionalities, e.g., the somatosensory cortex is involved in the sensat....

Protocol

All experimental procedures were performed according to the German guidelines on animal welfare under the supervision of local ethics committees (LaGeSo). Human and bat brain data were derived from Naumann et al.⁵ The following procedure is performed on a male adult Wistar rat (strain: RJHan:WI).

1. Perfusion and Brain Extraction

NOTE: In order to obtain a homogenously fixed and blood-free brain, transcardial perfusion of the animal is highly encouraged, as residual blood increases unspecific background signal during staining. Nevertheless, it is also possible to obtain flattened se....

Results

We obtained flattened cortical sections of the somatosensory cortex in a variety of brains, and processed them for cytochrome oxidase histochemistry to visualize the somatotopic modules representing different body parts. This comparative approach allows studying the evolutionary forces that shape cortex, e.g., showing highly conserved representation of mystacial vibrissae in rodents and lagomorpha as barrels²¹ (Figure 2). In c.......

Discussion

Modularity in the cerebral cortex has been identified using a variety of techniques. The earliest studies typically identified cortical modules by either visualizing cell dense regions, or an absence of fibers¹. Subsequent methods have utilized the presence of dendritic bundles²⁴, afferents from a particular region²⁵, or enrichment of neurotransmitters²⁶. Here we demonstrate two techniques, (i) cytochrome oxidase histochemistr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cytochrome oxidase staining
Cytochrome c from equine heart	Sigma-Aldrich	C2506
3,3'Diaminobenzidine tetrahydrochloride hydrate	Sigma-Aldrich	D5637
D(+)-Saccharose	Carl Roth	4621.1
Ammonium nickel(II) sulfate hexahydrate	Sigma-Aldrich	A1827
HEPES	Carl Roth	9105.4
Name	Company	Catalog Number	Comments
Antigen retrieval
Trisodium citrate dihydrate	Sigma-Aldrich	S1804
Citric acid monohydrate	Sigma-Aldrich	C1909
Name	Company	Catalog Number	Comments
Phosphate buffer/phosphate-buffered saline/prefix/PFA
Potassium dihydrogen phosphate	Carl Roth	3904.2
Sodium chloride	Carl Roth	9265.1
Di-Sodium hydrogen phosphate dihydrate	Carl Roth	4984.3
Paraformaldehyde	Carl Roth	0335.3
TRITON-X 100	Carl Roth	3051.3
Name	Company	Catalog Number	Comments
Immunohistochemistry
Calbindin D-28k puriefied from chicken gut, Mouse monoclonal	Swant	RRID: AB_10000347
Calbindin D-28k from recombinant rat calbindin D-28k, Rabbit polyclonal	Swant	RRID: AB_10000340
Albumin Fraction V, biotin free	Carl Roth	0163.4
Name	Company	Catalog Number	Comments
Mounting or freezing media
Fluoromount (immunofluorescence)	Sigma-Aldrich	F4680
Eukitt (histochemistry)	Sigma-Aldrich	03989
Tissue freezing medium	Leica Biosystems	NC0696746
Name	Company	Catalog Number	Comments
Alcohol dehydration
Ethanol 100%	Carl Roth	9065.3
Ethanol 96%	Carl Roth	P075.3
2-Propanol	Carl Roth	6752.4
Xylene substitute	Fluka	78475
Name	Company	Catalog Number	Comments
Devices/tools
Microm HM 650V	Thermo Scientific
Jung RM2035	Leica Biosystems
Dumont #55 Forceps - Inox	Fine Science Tools	11255-20
Dumont #5 Forceps - Inox Biology Tip	Fine Science Tools	11252-30
Dumont #5SF Forceps - Inox Super Fine Tip	Fine Science Tools	11252-00
Bone Shears - 24 cm	Fine Science Tools	16150-24
Friedman Rongeur	Fine Science Tools	16000-14
Blunt Scissors	Fine Science Tools	14000-18
Surgical Scissors - Large Loops	Fine Science Tools	14101-14
Surgical Scissors - Sharp-Blunt	Fine Science Tools	14001-13
Fine Iris Scissors	Fine Science Tools	14094-11