Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Summary

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

Abstract

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Introduction

Dorsal root ganglia (DRG) are comprised of soma from afferent neurons innervating various parts of the body, and important insights can be gained from studying sensory neurons with different innervation targets¹. Various indirect or invasive methods are used to isolate and identify afferent neurons for e.g. injecting the nerve with markers like fluorogold¹, injecting dye into the muscles by surgical method². Of the different dyes that have been successfully used to label neurons and neuronal tracts, fluorescent dyes like DiI are widely used in neuroanatomical tracings owing to their rapid uptake by neuron terminals, long ....

Protocol

Note: All animal work must be carried out according to institutional and animal care guidelines.

1. Preparation of Animal and Labeling

1. Anesthetize rats: Anesthetize a rat (typically 150-400 g) using an intraperitoneal injection of a mixture of ketamine (50 mg/kg body weight), xylazine (5 mg/kg) and acepromazine (1 mg/kg). Check for reflexes by pinching the foot, wait till there is no response. Since the anesthesia response varies between animals, it is best to determine the effect of the anesthetics by testing pain reflexes.
2. DiI Solution: Weigh DiI powder and dissolve in sterile 100% DMSO to achieve a 1.5% solution (15....

Results

Using the method described here, we identified muscle afferent neurons in DRG and went on to determine the voltage-gated sodium channels (NaV) expressed by these neurons. DiI labeled muscle afferent neurons (Figure 1C) comprised a small fraction of the total population of neurons in the DRG with an average of 16% of neurons (167/1047) labeled. The quantification was done by first measuring the fluorescence using ImageJ and plotting histograms of the intensity data to determine the threshold for positivel.......

Discussion

Muscle afferents originating from triceps surae muscles (e.g. gastrocnemius) have been used in multiple studies to investigate the exercise pressor reflex (EPR)¹³. Given the interest in studying sensory neurons mediating the EPR, the gastrocnemius muscles were selected for dye injection. DiI was chosen due to its lipophilic nature and since it is not lost from the neuron by diffusion through the intact plasma membrane^3,14. Using this method as a guide, one can inject the dye into any target.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaV 1.8	Abcam	Ab93616
NaV 1.1 / 1.6/ 1.7 and 1.9	Alamone Labs, Jerusalem, Israel	ASC- 001/ 009 /008 /017
Alexa Fluor secondary antibodies	Invitrogen
Normal goat serum
Fluoro-Gel	Electron Microscopy Sciences	17985