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....

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.

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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...

Watch this Scientific Journal Video about In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ at JoVE.com

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 ...

The overall goal of this procedure is to perform single cell electro-physiological analysis of a defined population of sensory neurons in acute tissue slices from the mouse vomeronasal organ. This method can help elucidate key questions in the field of chemosensation, such as the mechanisms of see-no chemical detection. The main advantage of this technique is that by recording from sensory neurons in acute tissue slices, these cells can be investigated in their native environment.To begin this procedure, after sacrificing the animal, remove its lower jaw, by inserting a pair of large surgical scissors through the mouth cavity, and cut the mandible bones and muscle of each side separately. Next, remove the skin of the upper jaw and around the tip of the nose with medium forceps, to gain better access to the incisors. Then use the bone scissors to cut away the largest part of the incisors at a 45 degree angle in the rostral direction to ease the VNO capsule removal from the nasal cavity.Do not cut the root of the tooth to prevent damage to the tip of the VNO capsule. After that, grab the rigid upper palate at its rostral part with medium forceps, and carefully peel back in one piece at a flat angle. Repeatedly rinse the naval cavity with ice cold solution S2.Then use micro spring scissors to cut the bone effusion between the tip of the vomer bone and the jaw.Insert the scissor tips with the curved part pointing away from the VNO. And carefully cut the bone in small steps on both the left and right side, lateral to the VNO capsule. To remove the VNO capsule, 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 should be performed. Next, rinse the VNO in a small amount of ice cold S2, to prevent the tissue from drying out. Then, separate the cartilaginous capsules that contain the VNO soft tissues by grabbing the back of the vomer bone with medium forceps.For that, position the capsule for a dorsal view so that a split between both VNOs becomes visible. 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. Make sure the medial cartilage wall that was previously attached to the vomer bone is removed.When removing cartilage around the VNO use forceps only at the rim of the cartilage capsule. And be very careful not to damage the delicate sensory epithelium. 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.Then 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. To avoid damaging the sensory epithelium slowly peel the VNO away from the cartilage by holding it at its caudal tip. Once the VNO is levered from the capsule, remove all the remaining small cartilage parts as any remaining pieces of cartilage will detach the tissue from the surrounding agarose during the slicing process.Next, place a small drop of ice cold S2 on the first dissected VNO to prevent tissue damage. To embed the VNOs, fill both small petri dishes to the rim with melted S3.After that, immerse each VNO in 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. Position the VNO vertically, with the rostral tip facing the bottom of the dish.Subsequently, place both the dishes on a gel ice pack, and wait until agarose has solidified. Do not change VNO orientation once the agarose has started solidifying, as this will detach the tissue from the surrounding agarose. In this procedure, use a small spatula to remove the agarose block from the small dish into the lid of a large petri dish, and flip the agarose upside down, leaving the rostral tip of the VNO facing upwards.Next, cut the block into a pyramidal shape using a surgical scalpel, and take care not to damage the embedded tissue. Then, use super glue to fix the pyramid shaped block to the center of the vibratome specimen plate. And wait about one minute for the glue to dry completely.Slice the specimen into 150 to 200 micrometer thick sections. Briefly inspect the slice morphology under the dissecting microscope. Then transfer all the slices to the oxygenating chamber until use.In this step, transfer a VNO slice to the imaging chamber and fix the slice position using stainless steel anchor wired with point one milometer thick synthetic fibers. Do not cover the tissue slice with any of the synthetic fiber threads. Next, transfer the imaging chamber to the recording setup and continuously superfuse the slice with oxygenated S2 at room temperature.Adjust the suction capillary to the surface of the solution to create a constant exchange of bath solution. Then adjust the eight in one multi barrel profusion pencil above and close to the non sensory part of the VNO slice that contains the blood vessel. Afterward connect the reference electrode and bath solution using the L shaped agar bridge filled with 150 millimolar potassium chloride.Subsequently fill the patch pipette with pipette solution S4.Mount the pipette over the silver chloride coated electrode connected to the head stage without scraping off the coating and attach firmly. Then apply slight positive pressure to the patch pipette before entering the bath. Monitor the pipette and make sure that it is between four megaohms and 10 megaohms.Visualize the VNO slice with a CCD camera using infrared optimized DIC, and identify FPR-rs3 i-Venus expressing cells or similarly labeled neurons using florescence illumination and an appropriate filter cube. Approach the cell body using hand wheels for maximum sensitivity. Due to the positive pressure a small dent on the cell soma membrane becomes visible once the pipette tip is close proximity.Then release the positive pressure and apply slight negative pressure to suck in the cell membrane in order to gain a high resistance seal of one to 20 gigaohms. Subsequently apply short and gentle suction to disrupt the cell membrane and establish the whole cell configuration. In this figure, a confocal image of a coronal VNO tissue slice shows the distribution of fluorescent FPR-rs3 Tau Venus positive neurons in the vomeronasal sensory epithelium.The FPR-rs3 Tau Venus positive neurons exhibit a single apical dendrite ending in a knob like structure at the luminal border. And shown here are the whole cell patch clamp recordings performed from the VSN soma. Here are the representative traces from wholesale patch clamp recordings of a TTX sensitive fast activating sodium current, and FPR-rs3 positive VSNs.Voltage step recordings under control conditions reveal a voltage dependent fast and transient inward current. TTX treatment at one micromolar strongly diminishes the current. And the digitally subtracted trace shown here reveals the TTX sensitive voltage gated sodium current.The representative current clamp traces show depolarization and hyper-polarization and trains of action potentials generated upon step-wise positive and negative current injection. Once mastered, the whole dissection and tissue slicing process can be done in approximately one hour for both VNOs. Following this procedure, other methods such as extra-cellular loose patch recordings or calcium imaging can be performed in order to increase throughput when analyzing larger populations of neurons.After watching this video, you should have a good understanding of how to make acute tissue slices from the mouth vomeronasal organ, and how to perform single cell electro-physiological recordings from vomeronasal sensory neurons.

in-depth-physiological-analysis-defined-cell-populations-acute-tissue

Research

JoVE Journal

Neuroscience

7.5K visualizações.  RWTH Aachen University. The overall goal of this procedure is to perform single cell electro-physiological analysis of a defined population of sensory neurons in acute tissue slices from the mouse vomeronasal organ. This method can help elucidate key questions in the field of chemosensation, such as the mechanisms of see-no chemical detection. The main advantage of this technique is that by recording from sensory neurons in acute tissue slices, these cells can be investigated in their native environment.To be...