In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

Summary

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

Abstract

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.

Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

Introduction

The development of dendritic and axonal structures is an important facet of circuit regulation in the nervous system, including in the cerebral cortex. It plays a critical role during the selective wiring of the diverse neuronal subpopulations. A number of recent reports have shown that, in addition to connectivity, the molecular diversity of neurons is reflected by the acquisition of highly specific modes of firing. However, the mechanisms determining the excitability and connectivity of the distinct neuronal subtypes during development, as well as their degree of coordination, are still poorly understood^1,2.....

Protocol

All animal procedures were approved by the Community of Madrid Animal Care and Use Committee, in compliance with national and European legislation (PROEX 118/14; PROEX 331/15). Maintain sterile conditions during the procedure.

1. In Utero Electroporation

NOTE: This protocol for IUE is adapted from others that have been previously published^5,6,7. This manuscript descr.......

Discussion

This protocol describes in detail how to label neurons of the somatosensory cortex of C75BL/6 mice in order to analyze their connectivity and their excitability. With respect to existing methods, it visualizes discriminating aspects of connectivity, such as the number of axonal branches per neuron, their precise topography, and their anatomical location. By altering the position of the electrodes, it is possible to target other neuron populations, such as the cingulate cortex (keep the same angle between the electrodes a.......

Materials

Name	Company	Catalog Number	Comments
pCAG-Cre	Addgene	13775
pCALNL-GFP	Addgene	13770
pCAG-DsRed2	Addgene	15777
pCAG-GFP	Addgene	11150
Fast Green	Carl Roth	301.1
EndoFree Plasmid Maxi Kit	QIAGEN	12362
Carprofen (Rimadyl)	Pfizer GmbH	1615 ESP
Isoflurane (IsoFlo)	Abbott (Esteve)	1385 ESP
Ketamine (Imalgene)	Merial	2528-ESP
Xylazine (Xilagesic)	Calier	0682-ESP
Povidone Iodine	Meda	694109.6
Eye Ointment (Lipolac)	Angelini	65.277
Hanks' Balanced Salt Solution (HBSS)	Gibco by Life Technologies	24020-091
Penicillin-Streptomycin	Sigma -Aldrich	P4333
Scalpel Handle #3 - 12cm	Fine Science Tools	10003-12
Scalpel Blades #10	Fine Science Tools	10010-00
Adson Forceps-Serrated - Straight 12 cm	Fine Science Tools	1106-12
Hardened Fine Scissors - Straight   11 cm	Fine Science Tools	14090-11
Scissors Mezenbaum-Nelson Curved L=14,5cm	Teleflex	PO143281
Thin curved tips - Style 7 Dumoxel	Dumont	0303-7-PO
Dumont #5 Forceps-Inox	Fine Science Tools	11251-20
Mathieu Needle Holder - Serrated	Fine Science Tools	12010-14
AutoClip Applier	Braintree scientific, Inc	ACS APL
9mm AutoClips	MikRon Precision, Inc.	205016
Sutures - Polysorb 6-0	Covidien	UL-101
Electric Razor	Panasonic	ER 240
Borosilicate glass capillaries (100mm, 1.0/0.58 Outer/Inner diameter)	Wold Precision Instrument Inc.	1B100F-4
Aspirator tube assemblies for calibrated microcapillary pipettes	Sigma -Aldrich	A5177-5EA
Gauze (Aposan)	Laboratorios Indas, S.A.U.	C.N. 482232.8
Cotton Swabs (Star Cott)	Albasa	-
Needle 25G (BD Microlance 3)	Becton, Dickinson and Company	300600
Sucrose	Sigma -Aldrich	S0389
Paraformaldehyde	Sigma -Aldrich	158127
OCT Compound	Sakura	4583
Tissue Culture Dish 100 x 20 mm	Falcon	353003
GFP Tag Polyclonal Antibody	Thermo Fisher Scientific	A-11122
Secondary Antibody, Alexa Fluor 488 conjugate	Thermo Fisher Scientific	A-11008
DAPI	Sigma-Aldrich	D9542
Fetal Bovine Serum	Thermo Fisher Scientific	10270106
Triton X-100	Sigma-Aldrich	X100-500ML
Electroporator ECM 830	BTX Harvard Apparatus	45-0002
Platinum electrodes 650P 7 mm	Nepagene	CUY650P7
Microscope for Fluorescent Imaging - MZ10F	Leica	-
VIP 3000 Isofluorane Vaporizer	Matrx	-
TCS-SP5 Laser Scanning System	Leica	-
Axiovert 200 Microscope	Zeiss	-
Cryostat - CM 1950	Leica	-
P-97 Micropette Puller	Sutter Instrument Company	P-97
Patch clamp analysis softwarw (p-Clamp Clampfit 10.3)	Molecular Devices	-
Acquisition software (MultiClamp 700B Amplifier)	Molecular Devices	DD1440A
Motorized Micromanipulator + Rotating Base	Sutter Instrument	MP-225
Air Table	Newport	-
Miniature Peristaltic Pumps	WPI	-