The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Summary

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Abstract

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

Introduction

The c-fos gene was identified for the first time at the beginning of 1980^1,2 and its product was characterized in 1984 as a nuclear protein having gene-activator properties^3,4. It participates in long-term mechanisms associated with neuron stimulation. Indeed, changes in neuronal activity lead to second messenger signaling cascades that induce the expression of the immediate early gene c-fos, which induces the production of the transcription factor c-FOS. The latter initiates the expression of late genes and thus participates in adaptive responses of the nervous system to many different types of stimuli⁴. Th....

Protocol

Note: c-FOS detection is a standardized procedure involving several steps (Figure 1). All experiments were performed on rats or mice. Experimental protocols were approved by the Ethics Committee in Animal Experiment Charles Darwin (Ce5/2011/05), done in accordance with the European Communities Council Directive of September 22, 2010 (2010/63/EU) for animal care, and conducted in accordance with French laws for animal care.

1. Preparation of Solutions

1. Prepare 0.2 M Sodium phosphate buffer: add 6.24 g NaH₂PO₄*2H₂O and 22.56 g Na₂HPO₄*H₂O to 1 L dist....

Results

The c-FOS detection is a useful tool that allows identifying groups of activated cells under specific conditions such as hypoxia and hypercapnia in vivo (Figure 2A) or in situations that mimic these conditions ex vivo (Figure 2B). In vivo, newborn, young, or adult rodents were placed in an airtight box in which the gaseous environment is continually renewed by a gas mixture with a composition precisely defined for 30 to 180 min<.......

Discussion

C-fos is an immediate early gene, and the detection of its product, the c-FOS protein, is classically used to identify neuronal populations involved in specific respiratory responses in vivo^11,13,25,28 and ex vivo^{16-18,27,32,33}.

Critical Steps Within the Protocol

Be careful during the perfusion step. The 4% PFA solution must be well prepared and the fixation and post-fixation steps m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture plate 12-well	Costa	35/3
15 mm Netwell inserts with mesh polyester membrane	Corning	3477	The 15mm diameter well inserts have 74µm polyester mesh bottoms attached to polystyrene inserts
Primary antibody (rabbit polyclonal antibody against the c-Fos protein)	Santa Cruz Biotechnology	sc-52
Vectastain Elite ABC KIT	Vector laboratories	PK-6101
(Rabbit IgG-secondary antibody)
NaH2PO4*2H2O	Sigma	71505
Na2HPO4	Sigma	S7907
Paraformaldehyde	Sigma	P6148
NaOH 0.1N	Sigma	43617
Polyvinyl-Pyrrolidone	Sigma	PVP-360
Sucrose	Sigma	S7903
NaCl	Sigma	S7653
Ethylene-glycol	Sigma	33068
Triton X100	Sigma	T8787
Trisma HCl	Sigma	T5941
Trisma Base	Sigma	T1503
3.3-diaminobenzidine tetrahydrochloride	Rockland	DAB50
Nickel ammonium sulphate	Alfa Aesar	12519
H2O2	Sigma	H1009
Xylene	Sigma	33817
Entellan Neo	Merck Millipore	107961
Slide	Thermo-scientific	1014356190	Superfrost ultraplus
Cover glass	Thermo-scientific	Q10143263NR1	24 x 60mm
BSA	Sigma	A2153