We thank Ivan Rodriguez and Benoit von der Weid for generating the FPR-rs3-i-venus mouse line, their constructive criticism and fruitful discussions. This work was funded by grants of the Volkswagen Foundation (I/83533), the Deutsche Forschungsgemeinschaft (SP724/6-1) and by the Excellence Initiative of the German federal and state governments. MS is a Lichtenberg Professor of the Volkswagen Foundation.

All animal procedures were in compliance with local and European Union legislation on the protection of animals used for experimental purposes (Directive 86/609/EEC) and with recommendations put forward by the Federation of European Laboratory Animal Science Associations (FELASA). Both C57BL/6 mice and Fpr-rs3-i-Venus mice were housed in groups of both sexes at room temperature on a 12 hr light/dark cycle with food and water available ad libitum. For experiments young adults (6-20 weeks) of either sex were used. No obvious gender-dependent differences were observed.
1. Solution Preparation
<ol>
	<li>Prepare extracellular solution S1: 4-(2-Hydroxy-ethyl)piperazine-1-ethanesulfonic acid (HEPES) buffered extracellular solution containing (in mM) 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES; pH = 7.3 (adjusted with NaOH); osmolarity = 300 mOsm (adjusted with glucose).</li>
	<li>Prepare extracellular solution S2: Carbogen-oxygenated (95% O2, 5% CO2) extracellular solution containing (in mM) 125 NaCl, 25 NaHCO3, 5 KCl, 1 CaCl2, 1 MgSO4, 5 BES; pH = 7.3; osmolarity = 300 mOsm (adjusted with glucose).</li>
	<li>Prepare 4% low-gelling temperature agarose solution (S3) for tissue embedding: 4% low-gelling temperature agarose. Dissolve 2 g low-gelling agarose in 50 ml of S1 in a 100 ml glass laboratory bottle together with a magnetic stirrer. To melt the agarose, put the bottle in a water bath (with lid not tightly closed) at 70 &#176;C for approximately 20 min or until solution becomes transparent while stirring. Cool down and keep melted agarose in a second water bath at 42 &#176;C until further use.</li>
	<li>Prepare intracellular solution S4: Pipette solution containing (in mM) 143 KCl, 2 KOH, 1 EGTA, 0.3 CaCl2 (free Ca2+ = 110 nM), 10 HEPES, 2 MgATP, 1 NaGTP; pH = 7.1 (adjusted with KOH); osmolarity = 290 mOsm.</li>
	<li>Modify/adjust composition of S1, S2 and S4 according to individual experimental design (e.g., pharmacological blockers to isolate certain types of voltage-activated currents).</li>
</ol>
2. Workspace Preparation
<ol>
	<li>Fill oxygenating slice storing chamber with S2 at least 30 min prior to dissection, place on ice for temperature and pH adjustment and oxygenate solution continuously.</li>
	<li>Fill reservoir for slice superfusion at recording setup with S2 and oxygenate continuously.</li>
	<li>Prior to dissection, fill vibratome chamber with S2 and arrange crushed ice around the chamber. Alternatively, transfer spare ice cold oxygenated solution from oxygenating chamber into vibratome chamber and keep oxygenating continuously.</li>
	<li>Arrange surgical tools and consumables.</li>
	<li>Place ice gel pack under dissecting microscope, cover with a paper towel to prevent VNO tissue from freezing to bottom of the dish.</li>
	<li>Clean razor blade by briefly rinsing in 70% ethanol and distilled water and mount to vibratome. Replace for every slicing session.</li>
</ol>
3. VNO Dissection and Embedding
<ol>
	<li>Sacrifice animal by brief exposure to CO2 and decapitate using sharp surgical scissors. Note: As time from sacrificing the animal to putting the VNO capsule on ice is critical, minimize the time to less than 2 min. To maintain tissue viability, embed both fully dissected VNOs in agarose in less than 30 min.</li>
	<li>Remove the lower jaw with large surgical scissors. Enter through the mouth cavity and cut the mandible bones and muscle of each side separately.</li>
	<li>Place the remaining part of the head upside down in the large Petri dish.</li>
	<li>Pull away the skin of the upper jaw and around the tip of the nose with medium forceps to gain better access to the incisors.</li>
	<li>Use bone scissors to cut away the largest part of the incisors at a ~45&#176; angle in the rostral direction (Figure 1A). This will ease removal of the VNO capsule from the nasal cavity. 
	Note: Do not cut to the root of the tooth to prevent damage to the tip of the VNO capsule.</li>
	<li>Grab the rigid upper palate at its rostral part with medium forceps and carefully peel back in one piece at a flat angle (Figure 1B). 
	Note: Repeatedly rinse with ice-cold S2.</li>
	<li>Use micro spring scissors to cut the bony fusion between the tip of the vomer bone and the jaw. Insert the scissor tips with the curved part of the tip pointing outward away from the VNO and carefully cut the bone in small steps on both the left and right side lateral to the VNO capsule.</li>
	<li>To remove the VNO capsule, use micro spring scissors to cut through the vomer bone at the caudal part and carefully lift the vomer bone out of the nasal cavity using medium forceps. Immediately transfer the VNO to a small petri dish under a stereo microscope on an ice gel pack where the remaining steps of the dissection will be performed.</li>
	<li>Rinse the VNO in a small amount of ice-cold S2 to prevent the tissue from drying out.</li>
	<li>Separate the cartilaginous capsules that contain the VNO soft tissues by grabbing the back of the vomer bone with medium forceps. Position the capsule for a dorsal view so that a split between both VNOs becomes visible (Figure 1Di).</li>
	<li>Use the tip of fine forceps to separate both cartilage VNO capsules from the central bone while keeping the vomer bone pinned down at the rear part. 
	Note: Use forceps only at the rim of the cartilage capsule and be very careful not to pierce through the cartilage as the delicate sensory epithelium is easily damaged.</li>
	<li>Once the two VNOs are separated, start removing the cartilage encapsulating the first VNO.</li>
	<li>Grab the top rim of the capsule with one fine forceps and split away the cartilage wall that was previously attached to the vomer bone (medial side).</li>
	<li>To remove remaining cartilage turn the VNO with its curved lateral side to the bottom of the dish and securely pin down the cartilage on one side using forceps. Carefully move the second fine forceps from the back side at a very flat angle between cartilage and VNO to loosen the connection between tissue and cartilage.</li>
	<li>Slowly peel the VNO away from the cartilage by holding it at its caudal tip, to avoid damaging the sensory epithelium.</li>
	<li>Once the VNO is levered from the capsule, make sure to remove all remaining small cartilage parts as any remaining pieces of cartilage will detach the tissue from surrounding agarose during the slicing process.</li>
	<li>Place a small drop of ice-cold S2 on the first dissected VNO to prevent tissue damage. A large blood vessel on the lateral side will become visible indicating that the organ is still intact and was not grossly damaged during the dissection (Figure 2Aii). In case the blood vessel got damaged, it will still be worthwhile to carry on with slicing the VNO as long as the overall morphology was not clearly impaired.</li>
	<li>Immediately dissect the second VNO.</li>
	<li>To embed the VNOs, fill both small petri dishes to the rim with melted S3 (stored in water bath at 42 &#176;C; see 1.3).</li>
	<li>Hold the VNO on the broader caudal end with fine forceps to avoid damage to the sensory epithelium.</li>
	<li>Immerse the VNO in the agarose and move it horizontally back and forth several times to remove the film of extracellular solution as well as any air bubbles from its surface.</li>
	<li>Position the VNO vertically with the caudal tip facing the bottom of the dish. Instead of pinching the VNO with the forceps directly, adjust orientation by moving the forceps tip in close proximity to the VNO. 
	Note: Orientation during embedding is crucial as it determines slice plane and accessibility to sensory neurons during the experiment.</li>
	<li>Place dishes on gel ice pack and wait until agarose has solidified. 
	Note: Do not change VNO orientation once the agarose has started solidifying as this will detach the tissue from the surrounding agarose.</li>
</ol>
4. Coronal VNO Tissue Slicing
<ol>
	<li>Use small spatula to remove agarose block from the small dish into the lid of a large Petri dish, flip the agarose upside down leaving the caudal tip of the VNO facing upwards.</li>
	<li>Cut the block into a pyramidal shape using a surgical scalpel (3-4 mm at the tip, 8-10 mm at the bottom). Take care not to damage the embedded tissue.</li>
	<li>Use super glue to fix the pyramid-shaped block to the center of the vibratome specimen plate and wait ~1 min for the glue to dry completely.</li>
	<li>Transfer the specimen plate to the slicing chamber and prepare the second VNO, accordingly. Keep plate with the second specimen at 4 &#176;C until use.</li>
	<li>Use a vibrating blade microtome with the following settings: thickness: 150-200 &#181;m; speed: 3.5 a.u. = 0.15 mm/sec; frequency: 7.5 a.u. = 75 Hz. Transfer slices to oxygenating chamber until use after briefly inspecting slice morphology under the dissecting microscope. Slices can be kept for several hr.</li>
</ol>
5. Single-cell Electrophysiological Recordings
<ol>
	<li>For recordings, use an upright fixed-stage microscope equipped with water immersion objectives, Dodt or infrared differential interfering contrast (IR-DIC), and epi-fluorescence as well as a cooled CCD-camera. For data acquisition, use a patch-clamp amplifier, head stage, AD/DA interface board and a PC (including recording software).</li>
	<li>Prepare a stock of 10-20 patch pipettes (4-7 M&#8486;). Pull pipettes from borosilicate glass capillaries (1.50 mm OD/0.86 mm ID) using a micropipette puller and fire-polish with a microforge. 
	Note: Fire polishing the pipette tips is crucial when patching the rather small VSNs. This will help to prevent rupturing the cell membrane when applying negative pressure in order to obtain a high resistance seal. After polishing the pipette opening should be approximately 1 &#181;m.</li>
	<li>Keep pipettes in a pipette storage jar to prevent damage and dust accumulation until use.</li>
	<li>Prepare perfusion system by filling solution reservoirs and tubing according to experimental design. 
	Note: Remove air bubbles from tubing completely as they will strongly interfere with electrophysiological recordings.</li>
	<li>Adjust pressure to achieve ~3 ml/min flow. 
	Note: High pressures will cause movement of the tissue slice as well as termination of electrophysiological recordings.</li>
	<li>Transfer VNO slice to imaging chamber and fix the slice position using stainless steel anchor wired with 0.1 mm thick synthetic fibers (Figure 2B-C). 
	Note: Do not cover the tissue slice with one of the synthetic fiber threads but rather the agarose surrounding the slice.</li>
	<li>Transfer imaging chamber to recording setup and continuously superfuse slice with oxygenated S2 at room temperature via a bath application.</li>
	<li>Adjust the suction capillary to the surface of the solution to create a slow suction for constant exchange of bath solution. Adjust the 8-in-1 multi-barrel &#34;perfusion pencil&#34; above and close to the non-sensory part of the VNO slice that contains the blood vessel (Figure 2C, 3A)50. It will be beneficial to arrange perfusion pencil and recording pipette to be facing the slice from opposite directions.</li>
	<li>Connect reference electrode and bath solution using an L-shaped agar bridge (filled with 150 mM KCl).</li>
	<li>Fill patch pipette with pipette solution S4.</li>
	<li>Mount the pipette over the silver chloride-coated patch electrode connected to the head stage without scraping off the coating and attach firmly.</li>
	<li>Apply slight positive pressure (approximately 1 ml on a 10 ml plastic syringe) to the patch pipette before entering the bath.</li>
	<li>Lower the pipette into the bath using micro manipulators far enough to be able to submerge the objective without hitting it (Figure 2D).</li>
	<li>Monitor pipette resistance (Rpip, between 4-7 M&#8486;) using electrophysiology software connected to the head stage. 
	Note: If Rpip is &#60;4 M&#8486; the glass tip is broken. If R pip is &#62;10 M&#8486;, the tip is most likely clogged and the pipette must be replaced.</li>
	<li>Visualize the VNO slice with a CCD-camera using infrared-optimized differential interference contrast (DIC) and identify FPR-rs3-i-Venus expressing cells (or similarly labeled neurons) using fluorescence illumination and an appropriate filter cube.</li>
	<li>Focus and target fluorescent or non-fluorescent cells depending on experimental design.</li>
	<li>To approach the cell body, use hand wheels for maximum sensitivity. Due to positive pressure, a small dent in the cell soma membrane becomes visible once the pipette tip is in close proximity.</li>
	<li>Release positive pressure and apply slight negative pressure to suck in the cell membrane in order to gain a high resistance seal (1-20 G&#8486;). Apply short and gentle suction to disrupt the cell membrane and establish the whole-cell configuration.</li>
	<li>Monitor access resistance constantly during experiment. 
	Note: Only include neurons exhibiting small and stable access resistance (&#8804;3% of Rinput; change &#60;20% over the course of the experiment) into analysis.</li>
</ol>

The authors have nothing to disclose.

The VNO is a chemosensory structure that detects semiochemicals. To date, the majority of vomeronasal receptors remains to be deorphanized as only few receptor-ligand pairs have been identified. Among those, V1rb2 was described to be specifically activated by the male urinary pheromone 2-heptanone30, V2rp5 to be activated by the male specific pheromone ESP157 as well as V2r1b and V2rf2 to be activated by the MHC peptides SYFPEITHI48 and SEIDLILGY58, respectively. A prerequisite...

Most animals rely heavily on their chemical senses to interact with their surroundings. The sense of smell plays an essential role for finding and evaluating food, avoiding predators and locating suitable mating partners. In most mammals, the olfactory system consists of at least four anatomically and functionally distinct peripheral subsystems: the main olfactory epithelium1,2, the Grueneberg ganglion3,4, the septal organ of Masera5,6 and the vomeronasal organ. The VNO comprises the peripheral sensory structure of the accessory olfactory system (AOS), which plays a major role in detecting chemical cues that convey information about identity, gender, social rank and sexual state7-10. The VNO is located at the base of the nasal septum right above the palate. In mice, it is a bilateral blind-ending tube enclosed in a cartilaginous capsule11-13. The organ consists of both a crescent-shaped medial sensory epithelium that harbors the VSNs and of a non-sensory part on the lateral side. Between both epithelia lies a mucus-filled lumen which is connected to the nasal cavity via the narrow vomeronasal duct14. A large lateral blood vessel in the non-sensory tissue provides a vascular pumping mechanism to facilitate entry of relatively large, mostly non-volatile molecules such as peptides or small proteins into the VNO lumen through negative pressure15,16. The structural components of the VNO are present at birth and the organ reaches adult size shortly before puberty17. However, whether the rodent AOS is already functional in juveniles is still subject to debate18-20.
VSNs are distinguished by both their epithelial location and the type of receptor they express. VSNs show a bipolar morphology with an unmyelinated axon and a single apical dendrite that protrudes towards the lumen and ends in a microvillous dendritic knob. VSN axons fasciculate to form the vomeronasal nerve that leaves the cartilaginous capsule at the dorso-caudal end, ascends along the septum, passes the cribriform plate and projects to the accessory olfactory bulb (AOB)21,22. The vomeronasal sensory epithelium consists of two layers: the apical layer is located closer to the luminal side and harbors both V1R- and all but one type of FPR-rs-expressing neurons. These neurons coexpress the G-protein &#945;-subunit G&#945;i2 and project to the anterior part of the AOB23-25. Sensory neurons located in the more basal layer express V2Rs or FPR-rs1 alongside G&#945;o and send their axons to the posterior region of the AOB26-28.
Vomeronasal neurons are likely activated by rather small semiochemicals29-33 (V1Rs) or proteinaceous compounds34-38 (V2Rs) that are secreted into various bodily fluids such as urine, saliva and tear fluid37,39-41. In situ experiments have shown that VSNs are also activated by formylated peptides and various antimicrobial/inflammation-linked compounds25,42. Moreover, heterologously expressed FPR-rs proteins share agonist spectra with FPRs expressed in the immune system, indicating a potential role as detectors for sickness in conspecifics or spoiled food sources25 (see reference43).
Fundamental to understanding receptor-ligand relationships and downstream signaling cascades in specific VSN populations is a detailed evaluation of their basic biophysical characteristics in a native environment. In the past, the analysis of cellular signaling has greatly benefitted from genetically modified animals that mark a defined population of neurons by coexpressing a fluorescent marker protein30,44-49. In this protocol, a transgenic mouse line that expresses FPR-rs3 together with a fluorescent marker (Fpr-rs3-i-Venus) is used. This approach exemplifies how to employ such a genetically modified mouse strain to perform electrophysiological analysis of an optically identifiable cell population using single neuron patch-clamp recordings in acute coronal VNO tissue slices. An air pressure-driven multi-barrel perfusion system for sensory stimuli and pharmacological agents allows quick, reversible and focal neuronal stimulation or inhibition during recordings. Whole-cell recordings in slice preparations allow for a detailed analysis of intrinsic properties, voltage-activated conductances, as well as action potential discharge patterns in the cell&#39;s native environment.

<table border="0" cellpadding="0" cellspacing="0" width="846">
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			<td height="21" style="height:21px;width:323px;">Large petri dish, 90 mm</td>
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			<td style="width:275px;">removing skin and palate</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Micromanipulator Device&#160;</td>
			<td style="width:133px;">Luigs &#38; Neumann</td>
			<td style="width:116px;">SM-5</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Micropipette puller, vertical two-step</td>
			<td style="width:133px;">Narishige</td>
			<td style="width:116px;">PC-10&#160;</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Microscope</td>
			<td style="width:133px;">Leica Microsystems</td>
			<td style="width:116px;">CSM DM 6000 SP5</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Noise eliminator 50/60 Hz (HumBug)</td>
			<td style="width:133px;">Quest Scientific</td>
			<td style="width:116px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Objective&#160;</td>
			<td style="width:133px;">Leica Microsystems</td>
			<td style="width:116px;">HCX APO L20x/1.00 W</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Oscilloscope</td>
			<td style="width:133px;">Tektronik</td>
			<td style="width:116px;">TDS 1001B</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Osmometer&#160;</td>
			<td style="width:133px;">Gonotec</td>
			<td style="width:116px;">Osmomat 030</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Perfusion system 8-in-1</td>
			<td style="width:133px;">AutoMate Scientific</td>
			<td style="width:116px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">pH Meter five easy</td>
			<td style="width:133px;">Mettler Toledo</td>
			<td style="width:116px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Pipette storage jar</td>
			<td style="width:133px;">World Precision Instruments</td>
			<td style="width:116px;">e212</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Recording chamber&#160;</td>
			<td style="width:133px;">Luigs &#38; Neumann</td>
			<td style="width:116px;">Slice mini chamber</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Razor blades</td>
			<td style="width:133px;">Wilkinson Sword GmbH</td>
			<td style="width:116px;">Wilkinson Sword Classic</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:323px;">Oxygenating slice storage chamber; alternative commercial chambers are e.g. BSK1 Brain Slice Keeper (Digitimer) or the Pre-chamber (BSC-PC; Warner Instruments)</td>
			<td style="width:133px;">custom-made</td>
			<td style="width:116px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Stereo microscope</td>
			<td style="width:133px;">Leica Microsystems</td>
			<td style="width:116px;">S4E</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Trigger interface&#160;</td>
			<td style="width:133px;">HEKA Elektronik</td>
			<td style="width:116px;">TIB-14 S</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Vibratome&#160;</td>
			<td style="width:133px;">Leica Microsystems</td>
			<td style="width:116px;">VT 1000 S</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Water bath&#160;</td>
			<td style="width:133px;">Memmert</td>
			<td style="width:116px;">WNB 45</td>
			<td style="width:275px;"></td>
		</tr>
	</tbody>
</table>

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.

To gain insight into the biophysical and physiological properties of defined cell populations, we perform acute coronal tissue slices of the mouse VNO (Figure 1-2). After dissection, slices can be kept in ice-cold oxygenated extracellular solution (S2) for several hr. At the recording setup, a constant exchange with fresh oxygenated solution (Figure 2D) ensures tissue viability throughout the experiment. We here employ a transg...

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

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

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Research

JoVE Journal

Neuroscience

7.5K Views.  RWTH Aachen University. 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. 

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

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

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.

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

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

Biology

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 ...

Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from Vomeronasal Sensory Neurons

Source: Ackels, T., et al. <a href="https://app.jove.com/v/54517">In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ.</a> J. Vis. Exp. (2016)
The protocol video describes establishing a whole-cell patch clamp for electrophysiological recording from the sensory neurons of the vomeronasal organ (VNO) in mice. A coronal VNO slice with fluorescently labeled sensory neurons is placed in a recording setup. A labeled neuron is patched with a recording pipette in a whole-cell configuration for electrophysiological recording in response to chemical stimuli.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Solution Preparation

	Prepare ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Preparation of Mouse Vomeronasal Organ for Vibratome Sectioning

Source: Ackels, T., et al. <a href="https://app.jove.com/v/54517">In-depth physiological analysis of defined cell populations in acute tissue slices of the mouse vomeronasal organ.</a> J. Vis. Exp. (2016)
This video outlines the isolation of a mouse vomeronasal organ (VNO) and tissue preparation for vibratome sectioning. Sections are used to characterize the biophysical and physiological attributes of a specific group of sensory neurons.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
Both ...

Neuronal Culture Techniques

Primary Neuronal Culture

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

For this protocol, have mice available between 2 and 4 weeks old. In this presentation, mice expressing OR/IRES/tauGFP are used.
By using OR/IRES/tauGFP mice, all neurons expressing the OR of interest will be labeled with GFP. This method can be used for OR expressed in all zones. However, dissections and recordings will be easier for OR expressed in the dorsal zone.
Begin by anesthetizing and then decapitating a mouse.
The outcome of this protocol depends on the quality of the dissection. These dissection steps must be as short as possible and be precise.
To begin the surgery, use dissecting scissors to make a longitudinal cut along the dorsum. Pull skin apart to remove it. Then, cut along the lower jaw and make a coronal cut parallel to the teeth. Thus, remove the upper incisor teeth. Now, make a coronal cut around the head, behind the eyes. Discard the posterior head, and transfer the anterior portion to ice-cold Ringer&#39;s.
Now, under a dissection microscope, isolate the septum within 5 to 10 minutes. Start with a longitudinal cut along the ventral side. Then, cut the dorsal bones longitudinally along the dorsal lateral side of the nasal cavity.
Next, remove most of the bones and the palate. Transfer the isolated septum and attached epithelium to a container of oxygenated Ringer&#39;s at room temperature. Within five minutes, make the final preparations before recording.
The solution must not be too warm, or the preparation will look damaged. To access the neuroepithelium for recording, first, peel the epithelium from the underlying septum with forceps. Then, using micro-Vannas scissors, cut the anterior end of the septum to release the epithelium.
Now, carefully remove the vomeronasal organ. Cut it off where it is attached dorsally. The isolated epithelium can now be moved to the recording chamber, mucus side up. Once in place, use a harp to flatten the tissue. Then, transfer the chamber to an upright microscope. Visualize the neuroepithelium under a 40x water immersion objective while perfusing it continually with fresh Ringer&#39;s at room temperature.
To begin, search for the cells of interest, which, in this case, express EGFP. So 480-nanometers light is used to scan for cells that emit light in the 530 to 550-nanometer range. The targeted dendritic knobs and OSNs, when reviewed at 80 to 160x under bright field, should be clearly distinguishable from the supporting cells.
Now, load the electrode with the nystatin solution. Tap out any bubbles that form, and attach the electrode to the holder. Now, apply some positive pressure. The expected resistance is between 15 and 20 megaohms. Then, bring the electrode to the cell while maintaining the positive pressure.
When the resistance increases to about 40 megaohms, release the pressure and apply a slight negative pressure. If a gigaseal is made, the membrane potential should be clamped at about negative 75 millivolts.
Now that the cell is open, proceed with the experiment. Apply stimulation protocols and perfuse with pharmacological treatments as needed. To test with a single odorant, record 200 to 500 milliseconds of spontaneous activity. Apply the odorant for 500 milliseconds, and then, measure responses for up to 10 seconds.