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 of the anesthetics by testing pain reflexes.</li>
	<li>DiI Solution: Weigh DiI powder and dissolve in sterile 100% DMSO to achieve a 1.5% solution (15 mg/ml). Store the solution at room temperature in a dry atmosphere until use.</li>
	<li>DiI Injections: shave the hair over the calf area on both legs of the anesthetized rat using an electric razor.
	<ol>
		<li>Fill a 1 ml sterile syringe (1&#188; in&#160;27 G needle) with 200 &#181;l of 1.5% DiI solution. Slowly insert the needle parallel to the long-axis of each gastrocnemius muscle, and inject 100 &#181;l DiI into the muscle while slowly backing the needle out of the muscle (~10 sec) without withdrawing from the muscle.</li>
		<li>Prior to withdrawing the needle from the muscle, pull back on the syringe plunger to ensure the dye no longer flows from the tip, wait 10 sec, and then withdraw the needle from the muscle. This step prevents the leakage of DiI from the needle onto the skin, which could label cutaneous afferents.</li>
		<li>Repeat for other leg.</li>
		<li>Allow 4-5 days for the DiI to be transported from muscle to the DRG.</li>
	</ol>
	</li>
</ol>
2. Dissection and Neuronal Isolation
<ol>
	<li>Solutions:
	<ol>
		<li>Prepare Hank&#8217;s Balanced Salt Solution (HBSS) and Earle&#8217;s Balanced Salt Solution (EBSS) according to manufacturer&#8217;s instructions, filter sterilize and store at 4 &#176;C.</li>
		<li>Culture media (MEM Plus): Mix 89 ml minimal essential media (MEM), 10 ml fetal bovine serum (FBS), 1 ml penicillin-streptomycin solution, filter sterilize and store at 4 &#176;C.</li>
		<li>Polylysine solution: dissolve poly-L-lysine hydrobromide powder (10 mg/100 ml) in 0.1 M sodium borate solution, pH 8.4, filter sterilize and store aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Coating coverslips
	<ol>
		<li>Clean the glass coverslips (12 mm round) in 70% ethanol for 3-4 hr and air dry in sterile culture hood.</li>
		<li>Place the dried coverslips in the polylysine solution O/N.</li>
		<li>Remove from polylysine solution and wash in sterile distilled water 2x for 5 min each to remove any excess polylysine.</li>
		<li>Separate the coverslips, dry in culture hood, and store at 4 oC.</li>
	</ol>
	</li>
	<li>Setting up for dissection
	<ol>
		<li>Place on ice a beaker with 10 ml HBSS, a 35 mm Petri dish and a 100 mm Petri dish.</li>
		<li>Weigh out enzymes: 7 mg collagenase, 1 mg of DNAse and 10 mg of trypsin.</li>
		<li>Preheat a shaking water bath to 37 oC and place into the bath a 15 ml centrifuge tube containing 10 ml of EBSS to warm up.</li>
	</ol>
	</li>
	<li>Dissection:
	<ol>
		<li>Sacrifice rat according to an IACUC approved protocol.&#160;Carbon dioxide was used to euthanize the rat.</li>
		<li>Quickly remove the back by cutting open the skin on the back, remove the connective and muscle tissue along the spinal column for better exposure and remove the lumbar section of the vertebral column (end of the thoracic region to the iliac bone; Figure 1).</li>
		<li>Place the isolated vertebral column into the 100 mm Petri dish, and using a sterile Pasteur pipette apply chilled HBSS solution to keep the preparation moist and cold.</li>
		<li>Cut the muscles from the sides of the vertebral column. Perform a dorsal laminectomy, using rongeurs or scissors, and gently remove the bone enclosing the recesses containing the DRG.</li>
		<li>Repeatedly apply ice cold HBSS (~1 ml) during this procedure.</li>
		<li>Isolate the DRG by cutting the peripheral and central nerves (leaving 1-2 mm of nerve to facilitate handling of the ganglia)</li>
		<li>Place the isolated DRG into the 35 mm Petri dish containing ice-cold HBSS buffer.</li>
		<li>Repeat until all L-4 and L-5 DRG have been isolated. Thoracic DRG can be isolated and processed separately as a negative control for DiI labeling.</li>
		<li>Place the dish with isolated DRG under a dissection microscope and remove the excess nerves and blood vessels using micro scissors and forceps.</li>
		<li>Cut open the DRG for better exposure to the enzymes, but do not desheath the DRG.</li>
	</ol>
	</li>
	<li>DRG neuron dissociation:
	<ol>
		<li>Add the enzymes into the warm EBSS media (kept in the water bath). Shake briefly to dissolve, and filter sterilize.</li>
		<li>Using a sterile Pasteur pipette, place all DRG into the enzyme solution. Minimize the volume of HBSS that is added with DRG since the HBSS will dilute the enzyme mixture.&#160; Gas the flask with 95% O2 and 5% CO2 for 60 sec before tightly capping the flask.</li>
		<li>Incubate the flask in the shaking water bath set at 37 &#176;C for 60 min at 240 rpm.</li>
		<li>At the end of incubation, vigorously shake the flask 50x to dissociate the DRG5.</li>
		<li>Under sterile conditions, add 5 ml of MEM Plus to the flask to stop the enzymes.</li>
		<li>Transfer flask contents to a sterile 15 ml centrifuge tube and centrifuge at 16 x g for 6 min.</li>
		<li>Remove and discard the supernatant, add 5 ml of MEM Plus to the sample. Triturate gently with pipette to break up the pellet.</li>
		<li>Repeat centrifugation step, remove and discard the supernatant and add 500 &#181;l of MEM Plus. Gently triturate with a 1 ml pipette tip on a pipette to make a cell suspension.</li>
		<li>Place one poly-lysine coated glass coverslip into an empty sterile 35 mm dish. Add a drop (~50 &#181;l) of the cell suspension to the center of the coverslip.</li>
		<li>Repeat until cell solution is exhausted.</li>
		<li>Place the 35 mm dishes into a 37 &#176;C and 5% CO2 incubator for 1 hr to allow the neurons to adhere to the coverslips.</li>
		<li>Add an additional 2 ml of media into each dish and return to the incubator.</li>
	</ol>
	</li>
</ol>
3. Patch Clamp Recording
<ol>
	<li>Solutions for recording: these solutions are for recording NaV currents. Examples of solutions with differing composition can be found in these references5-7.
	<ol>
		<li>External solution: (in mM) 45 NaCl, 100 N-methyl-d-glucamine (NMG)&#8226;Cl, 4 MnCl2, 10 Na&#8226;HEPES and 10 glucose, with pH = 7.4 and osmolarity = 320 mOsm.</li>
		<li>Internal solution: (in mM) 104 NMG&#8226;Cl, 14 Creatine&#8226;PO4, 6 MgCl2, 10 NMG&#8226;HEPES, 5 Tris&#8226;ATP, 10 NMG2&#8226;EGTA, and 0.3 Tris2&#8226;GTP with pH 7.4 and osmolarity = 300 mOsm.</li>
	</ol>
	</li>
	<li>Measurement of ionic currents:
	<ol>
		<li>Pull recording electrodes from glass capillaries on an electrode puller and heat-polish the tips using a microforge. The resistance of the electrodes is typically 2-3 M&#937;</li>
		<li>Identify the DiI labeled DRG neurons using a microscope with epifluorescence and a TRITC cube. DiI has an excitation wavelength of 550 nm and emission wavelength of 570 nm.</li>
		<li>Fill the glass electrode with internal solution (only filing ~ 1 cm of the electrode tip) and gently tap to remove any trapped bubbles. Place in electrode holder and move to cell using a micromanipulator.</li>
		<li>Once the electrode touches the cell, apply gentle suction via the suction tube, until a tight seal is formed ( determined by the increase in electrode resistance).</li>
		<li>Clamp the membrane voltage at the desired holding potential (typically -60 to -120 mV).</li>
		<li>Obtain the whole-cell patch clamp configuration by applying stronger suction to remove the membrane patch at the tip of the electrode. The transition to whole-cell configuration is observed electrically as an increase in capacitative current8-10.</li>
		<li>Compensate the series resistance using the compensation circuitry of the patch clamp amplifier (use at least 80% compensation)8,10.</li>
	</ol>
	</li>
</ol>
4. Immunocytochemistry
<ol>
	<li>Fix the neurons on the coverslips with 4% paraformaldehyde for 1 hr and then permeabilize with 2% Tween 20 in phosphate buffered saline (PBS) for 10 min.</li>
	<li>Incubate the fixed cells O/N in a blocking solution (normal serum or Bovine Serum Albumin) plus the primary antibody, which is an antibody developed to bind to a specific antigen of interest.</li>
	<li>Incubate a control set of neurons O/N in blocking solution that lacks the primary antibodies.</li>
	<li>Wash the coverslips with PBS buffer 3x for 5 min followed by 2 washes of 5 min each with 1% Tween 20 to remove unbound primary antibody.</li>
	<li>Incubate test and control coverslips with the appropriate fluorescent tagged secondary antibody, which binds to the primary antibody to permit visualization.</li>
	<li>Wash the coverslips with PBS 3x for 5 min to remove excess secondary antibody.</li>
	<li>Mount the coverslips on a clean glass slide using mounting media.</li>
	<li>Apply clear nail polish to edge of the coverslip to fix it place. Once dry, visualize the neurons and capture images using the software accompanying the fluorescence microscope. 
	Note: In this work, a Nikon Microscope was used with NIS-elements BR 3.2 software. The exposure time was optimized and kept the same for control and test slides.</li>
	<li>Measure fluorescence intensity and cell size from individual neurons using imaging software such as ImageJ (rsbweb.nih.gov/ij/index.html).</li>
</ol>

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The authors declare no conflicts of interest.

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 organ of interest to identify the innervating afferent neurons. A literature search will provide the information needed to locate the best injection site and the DRGs to be isolated 13,15,16.
The injection of DiI needs to be done with care to inject only the intended tissue. It is also important to ensure that the dye is well distributed within the tissue. We optimized our dye distribution in the muscle by injecting along the longitudinal muscle axis. The needle is inserted into the muscle and the injection is done while withdrawing the needle along the muscle. Leakage is prevented by gently pulling back the syringe plunger prior to removing the needle from the muscle, which avoids dye contact with skin. However, in cases of accidental skin labeling, we exclude those rats from our recordings. As a further safeguard, the cutaneous dermatomes covering our injection area are primarily L6 and S117, so there is a low probability of unnoticed DiI leaks labeling cutaneous afferents in L4 and L5.
We have also used DiI to label cutaneous afferents by injecting the DiI just under the skin within the L4-L5 dermatomes11,17. DiI labeling can also be used to label neuronal tracts either in vivo or in post-mortem tissue14.
Speed is essential to obtain viable neurons following isolation. Thus, the dissection of the DRG from the animal should be done very quickly, but gently. There is trade off in that one can damage the neurons if the dissection is done too quickly. We generally complete the dissection within 20 min. Another key to maintaining good cell viability is to keep the tissue cold and moist during the dissection. We apply chilled HBSS solution about every 60-90 sec during the dissection procedure. Speed is less important once the DRG are placed in ice cold HBSS (35 mm dish) for debridement.
For electrophysiology recordings, the recording solutions are critical in determining which types of ionic currents can be observed. The solutions that we describe here are the ones we use to record NaV currents. Examples of solutions with differing composition can be found in these references5-7. One problem for recording NaV currents is the large size of the currents. We addressed this by lowering the external Na+ concentration. A normal mammalian extracellular Na+ concentration is 145 mM, but we use a Na+ concentration of 50 mM NaCl to reduce the current amplitude and greatly improve voltage clamp control. Other Na+ concentrations can be used. The goal is to reduce the current size sufficiently to maintain proper control of membrane voltage, while maintaining sufficient current amplitude to clearly resolve the NaV current from background (i.e. signal to noise). In our solutions, the Na+ and K+ are replaced with NMG, which is a monovalent cation that does not permeate active ion channels18. The Ca2+ in the solution is replaced with Mn2+, which maintains membrane surface charge screening 18 while not readily permeating calcium channels11.&#160; Removal of K+ and Ca2+ from the solution prevents contamination of our NaV current by KV and CaV currents.
Another problem for voltage clamp recordings is the resistance of the pipette, which produces a problem for patch clamp amplifiers in controlling membrane voltage. The problem is that the pipette tip resistance is in series with the membrane resistance so that part of the voltage drop occurs at the pipette tip instead of the complete voltage drop occurring at the membrane. The larger the pipette resistance, the larger the voltage drop at the pipette tip, instead of the cell membrane. This problem can be controlled for by the series resistance compensation circuitry of the patch clamp amplifier 8-10. However, this circuitry can induce oscillations when the series resistance is too high, which will prevent proper compensation9,10. There is no absolute resistance that will trigger oscillations, but we generally encounter problems with series resistances &#62;8 M&#937;.
Electrophysiology is often used to examine functional ion channels in neurons19, but one limitation is that this technique generally allows one to record from only one cell at a time, which can make it difficult to gauge expression of ion channels in a population of neurons. Immunocytochemistry has the advantage of labeling all neurons in a population that express a particular channel type. Thus, electrophysiology and immunocytochemistry are synergistic techniques that facilitate the identification of ion channels in a diverse population of neurons, such as those found in the DRG. The immunocytochemistry labeling method described here can be used to label any channel of interest.
The labeling method described here can be used to identify any afferent neurons of interest to facilitate their study16. The neuronal isolation protocol can be adapted to isolate DRG neurons from other regions and neurons from other ganglia16,20 . The combination of patch clamp electrophysiology and immunocytochemistry are complimentary to enhance our knowledge and understanding of the expressed ion channels11,12. The study of neuronal subtypes is critical for better understanding of the differences in receptor and ion channel expression that govern excitability, which could lead to the identification of targets that selectively impact the excitability of these neurons to treat disease.

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><tbody><tr><td>NaV 1.8</td><td>Abcam</td><td>Ab93616</td><td></td></tr><tr><td>NaV 1.1 / 1.6/ 1.7 and 1.9</td><td>Alamone Labs, Jerusalem, Israel</td><td>ASC- 001/ 009 /008 /017</td><td></td></tr><tr><td>Alexa Fluor secondary antibodies</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Normal goat serum</td><td></td><td></td><td></td></tr><tr><td>Fluoro-Gel</td><td>Electron Microscopy Sciences</td><td>17985</td><td></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 positively labeled neurons. The threshold was determined by fitting a Gaussian distribution to the large peak of unlabeled neurons, which provided mean intensity and standard deviation (SD) (Figure 1D). The threshold level was set to the mean intensity plus 2x&#160;SD so that any neurons with intensity &#62; threshold are marked as labeled11,12 .
Electrophysiology:
For patch clamp recordings, we selected brightly labeled, isolated neurons with no obvious neurites (neuronal outgrowths). Neurites cause space clamp problems that make the neurons difficult or impossible to properly voltage clamp since the ion channels in neurites are several space constants away from the current passing electrode. Thus, the decay of the injected current is too large to control the membrane voltage at these distant locations. Other problems that can erode voltage clamp performance are large membrane capacitance (large cells), high series resistance, and large currents. Figure 2 shows an example of sodium currents recorded from two neurons. The current in the first neuron (Figure 2A) is smaller and membrane voltage control is good as seen from the correspondence of peak currents over the three voltages (-20 to 0 mV) and steady increase in current from -30 to +10 mV in the current&#8211;voltage (I-V) relationship. The membrane voltage control in the second neuron (Figure 2B) is very poor as seen from the sigmoidal activation and late peak of the current at -20 mV (bold trace). The I-V also shows a threshold-like behavior between -20 and -10 mV where the current jumps from a low level to peak in 10 mV. Note also the very slow deactivation of current at -40 mV in Figure 2B (compare with Figure 2A). All of these are signs of very poor voltage control and the neuron in Figure 2B is not suitable for study8-10.
Immunocytochemistry: 
The ion channels that we studied had intracellular facing antigenic sites. Thus, the neurons needed to be permeabilized so that the antibodies could gain access to these sites. However, this is a problem since DiI can be lost from neurons upon permeabilization4. By permeabilizing the DRG neurons with Tween 20, we were able to get good antibody labeling and retain DiI for identification of muscle afferent neurons11,12.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50782/50782fig1highres.jpg" src="/files/ftp_upload/50782/50782fig1.jpg" width="600px" /> 
Figure 1.&#160;DiI Injection and cell labeling. (A) A representation of DiI injection into the gastrocnemius muscle and retrograde transportation from muscle to DRG. The L-4 and L-5 DRG are labeled. (B, C) Afferent neurons are shown in the bright field image (B) and a fluorescent image shows the one DiI labeled muscle afferent neuron (C). The white bar indicates 50 &#181;m. (D) A histogram of the DiI fluorescent intensity of sensory neurons. The large peak is from unlabeled neurons and is fit with a Gaussian equation to determine mean intensity and the standard deviation (SD). The dashed vertical line represents the threshold for DiI labeling, which was the mean Gaussian peak plus 2 x SD. The histogram comprises data from 1047 neurons with 167 being labeled with DiI (Intensity &#62; threshold). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50782/50782fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50782/50782fig2highres.jpg" src="/files/ftp_upload/50782/50782fig2.jpg" width="600px" /> 
Figure 2.&#160;Voltage clamp in a cell. (A) A representative muscle afferent neuron with good control of membrane voltage. The NaV currents at -20, -10, and 0 mV are shown on the left and the current-voltage relationship (I-V) is shown on the right. The I-V relationship was generated by measuring peak NaV current at each indicated voltage. (B) Currents are shown (left) from a neuron with large NaV currents and very poor voltage control, which is characterized by the late peak of the NaV current at -20 mV (bold trace) and the increase from small to maximum current of the I-V (right) between -20 mV to -10 mV. Compare that with the relatively even incremental increases in current between -30 and +10 mV for the I-V shown in panel A. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50782/50782fig2highres.jpg" target="_blank">Click here to view larger image</a>.

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.

Identifikation von spezifischen sensorischen Neuronenpopulationen für das Studium der Ausgedrückt Ionenkanäle

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 ...

identification-specific-sensory-neuron-populations-for-study

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.7K Aufrufe.  AT Still University of Health Sciences. 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.

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

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

Profiling Voltage-gated Potassium Channel-mRNA Expression in nigralen Neuronen mit Einzel-Zell RT-PCR-Techniken

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Forschung

Neurowissenschaften

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Ein Leitfaden zur<em&gt; In vivo</em&gt; Single-Unit-Aufnahme von Optogenetically Identifizierte kortikalen inhibitorischen Inter

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforated-Patch-Clamp-Aufzeichnung der Maus Riechzellen in Intact Neuroepithel: Funktionelle Analyse von Neuronen, die eine bestimmte Geruchsrezeptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Recapitulation of an Ion Channel IV Curve Using Frequency Components

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.
METHODS: Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages.
RESULTS AND CONCLUSIONS: In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|&lt;0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.

There are technical obstacles to measuring current flux through multiple ion channels simultaneously, and later discerning what portion of the transmembrane current is due to each channel type. To address this need, this method presents a way to generate the IV curve of individual channel types using specific frequency components.

Rekapitulation eines Ionenkanals IV Curve mit Frequenzkomponenten

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into ...

Biologie

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Eine Methode zum Ziel und Isolieren Airway-innervating sensorischen Neuronen bei Mäusen

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In eingehenden Physiologische Analyse definierter Zellpopulationen bei der akuten Gewebeschnitten der Maus Vomeronasalorgans

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

Einzelne Zelle Multiplex reversen Transkription Polymerase-Kettenreaktion nach Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Reaktionen auf Geschmacksreize

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ein-Kanal-Cell-Attached-Patch-Clamp-Aufnahme

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Identifikation von spezifischen sensorischen Neuronenpopulationen für das Studium der Ausgedrückt Ionenkanäle

Zusammenfassung

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Ein-Kanal-Cell-Attached-Patch-Clamp-Aufnahme