Name: identification of specific sensory neuron populations for study of expressed ion channels
Uploaded: 2013-12-24T13:00:00
Description: Watch this Scientific Journal Video about Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels at JoVE.com

This work was funded by National Institutes of Health Grant AR059397 (KSE).

Note: All animal work must be carried out according to institutional and animal care guidelines.
1. Preparation of Animal and Labeling
<ol>
	<li>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 ...

<ol>
	<li>Hu, P., McLachlan, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+reactions+of+cutaneous+and+muscle+afferent+neurons+to+peripheral+nerve+transection+in+rats.">Selective reactions of cutaneous and muscle afferent neurons to peripheral nerve transection in rats.</a> J. Neurosci. 23, 10559-10567 (2003).</li><li>Wang, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+reactive+oxygen+species+modulates+voltage-gated+sodium+channels+in+dorsal+root+ganglia+of+rats.">Endogenous reactive oxygen species modulates voltage-gated sodium channels in dorsal root ganglia of rats.</a> J. Appl. Physiol. 110, 1439-1447 (2011).</li><li>K&#246;bbert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+in+neuroanatomical+tracing.">Current concepts in neuroanatomical tracing.</a> Prog. Neurobiol. 62, 327-351 (2000).</li><li>Matsubayashi, Y., Iwai, L., Kawasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+double-labeling+with+carbocyanine+neuronal+tracing+and+immunohistochemistry+using+a+cholesterol-specific+detergent+digitonin.">Fluorescent double-labeling with carbocyanine neuronal tracing and immunohistochemistry using a cholesterol-specific detergent digitonin.</a> J. Neurosci. Methods. 174, 71-81 (2008).</li><li>Ikeda, S. R., Schofield, G. G., Weight, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na++and+Ca2++currents+of+acutely+isolated+adult+rat+nodose+ganglion+cells.">Na+ and Ca2+ currents of acutely isolated adult rat nodose ganglion cells.</a> J. Neurophysiol. 55, 527-539 (1986).</li><li>Yarotskyy, V., Elmslie, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roscovitine+Inhibits+CaV3.1+(T-Type)+Channels+by+Preferentially+Affecting+Closed-State+Inactivation.">Roscovitine Inhibits CaV3.1 (T-Type) Channels by Preferentially Affecting Closed-State Inactivation.</a> J. Pharmacol. Exp.Ther. 340, 463-472 (2012).</li><li>Ganapathi, S. B., Kester, M., Elmslie, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-dependent+block+of+HERG+potassium+channels+by+R-roscovitine:+implications+for+cancer+therapy.">State-dependent block of HERG potassium channels by R-roscovitine: implications for cancer therapy.</a> Am. J. Physiol. Cell Physiol. 296, 701-710 (2009).</li><li>Jones, S. W., Baker, G. B., Vanderwolf, C. H., Boulton, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuromethods Vol. 14 Neurophysiological Techniques: Basic Methods and Concepts. , 143-192 (1990).</li><li>Penner, R., Sakmann, B., Neher, E. r. w. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording. 1, 3-30 (1995).</li><li>Marty, A. a. N., Erwin, B., Sakmann, E. r. w. i. n., Neher, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording. 2, 31-52 (1995).</li><li>Ramachandra, R., McGrew, S. Y., Baxter, J. C., Kiveric, E., Elmslie, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodotoxin-resistant+voltage-dependent+sodium+(NaV)+channels+in+identified+muscle+afferent+neurons.">Tetrodotoxin-resistant voltage-dependent sodium (NaV) channels in identified muscle afferent neurons.</a> J. Neurophysiol. , (2012).</li><li>Ramachandra, R., McGrew, S. Y., Baxter, J. C., Howard, J. R., Elmslie, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NaV1.8+channels+are+expressed+in+large,+as+well+as+small,+diameter+sensory+afferent+neurons.+.">NaV1.8 channels are expressed in large, as well as small, diameter sensory afferent neurons. .</a> Channels. 7, 0 (2013).</li><li>Smith, S. A., Mitchell, J. H., Garry, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+exercise+pressor+reflex+in+health+and+disease.">The mammalian exercise pressor reflex in health and disease.</a> Exp. Physiol. 91, 89-102 (2006).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dil+and+diO:+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing.">Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends Neurosci. 12, 333-335 (1989).</li><li>von Bartheld, C. S., Cunningham, D. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+tracing+with+DiI:+decalcification,+cryosectioning,+and+photoconversion+for+light+and+electron+microscopic+analysis.">Neuronal tracing with DiI: decalcification, cryosectioning, and photoconversion for light and electron microscopic analysis.</a> J. Histochem. Cytochem. 38, 725-733 (1990).</li><li>Fuller, B. C., Sumner, A. D., Kutzler, M. A., Ruiz-Velasco, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+employing+ultrasound+guidance+for+percutaneous+cardiac+muscle+injection+to+retrograde+label+rat+stellate+ganglion+neurons.">A novel approach employing ultrasound guidance for percutaneous cardiac muscle injection to retrograde label rat stellate ganglion neurons.</a> Neurosci. Lett. 363, 252-256 (2004).</li><li>Takahashi, Y., Nakajima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermatomes+in+the+rat+limbs+as+determined+by+antidromic+stimulation+of+sensory+C-fibers+in+spinal+nerves.">Dermatomes in the rat limbs as determined by antidromic stimulation of sensory C-fibers in spinal nerves.</a> Pain. 67, 197-202 (1996).</li><li>Zhou, W., Jones, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+charge+and+calcium+channel+saturation+in+bullfrog+sympathetic+neurons.">Surface charge and calcium channel saturation in bullfrog sympathetic neurons.</a> J Gen Physiol. 105, 441-462 (1995).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pflugers Arch. 391, 85-100 (1981).</li><li>Margas, W., Mahmoud, S., Ruiz-Velasco, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscarinic+Acetylcholine+Receptor+Modulation+of+Mu+({micro})+Opioid+Receptors+in+Adult+Rat+Sphenopalatine+Ganglion+Neurons.">Muscarinic Acetylcholine Receptor Modulation of Mu ({micro}) Opioid Receptors in Adult Rat Sphenopalatine Ganglion Neurons.</a> J. Neurophysiol. 103, 172-182 (2010).</li></ol>

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 membrane3,14. Using this method as a guide, one can inject the dye into any target...

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.&#160;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 term retention in neurons, and the lack of transfer between neurons3. The afferent neurons labeled with DiI can be easily visualized using a fluorescent microscope for detailed study. However, one limitation is that markers such as DiI can be lost from cells during cell permeabilization needed for immunocytochemistry with intracellular epitopes4. Here we describe a method that uses DiI injected into skeletal muscle to identify muscle afferent neurons in the DRG. We also describe a cell permeabilization method that retains DiI in the neuron while permitting antibody access to intracellular epitopes. We demonstrate use of both patch clamp electrophysiology and immunocytochemistry to study these identified neurons, as well as describe the distinct advantages of each technique in studying ion channels that control neuronal excitability. Finally, we describe potential problems that can hinder interpretation of electrophysiological data. We have used these methods to identify muscle and cutaneous afferent neurons and determine the voltage-gated sodium (NaV) channels expressed in these identified neurons8. However, these methods can be adapted to a variety of preparations for studying identified subsets of neurons innervating a particular target tissue or brain nucleus.

<table border="0" cellpadding="0" cellspacing="0" width="480">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">NaV 1.8</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:136px;">Ab93616</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:195px;">NaV 1.1 / 1.6/ 1.7 and 1.9</td>
			<td style="width:71px;">Alamone Labs, Jerusalem, Israel</td>
			<td style="width:136px;">ASC- 001/ 009 /008 /017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Alexa Fluor secondary antibodies</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:136px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Normal goat serum</td>
			<td style="width:71px;"></td>
			<td align="right" style="width:136px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:195px;">Fluoro-Gel</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:136px;">17985</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

identification of specific sensory neuron populations for study of expressed ion channels

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&#39;-dioctadecyl-3,3,3&#39;,3&#39;-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.

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...

Watch this Scientific Journal Video about Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels at JoVE.com

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

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.

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 ...

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...