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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 epithelium^1,2, the Grueneberg ganglion^3,4, the septal organ of Masera^5,6 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 state^7-10. 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 capsule^11-13. 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 pressure^15,16. 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 debate^18-20.

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 α-subunit G_αi2 and project to the anterior part of the AOB^23-25. Sensory neurons located in the more basal layer express V2Rs or FPR-rs1 alongside G_αo and send their axons to the posterior region of the AOB^26-28.

Vomeronasal neurons are likely activated by rather small semiochemicals^29-33 (V1Rs) or proteinaceous compounds^34-38 (V2Rs) that are secreted into various bodily fluids such as urine, saliva and tear fluid^37,39-41. In situ experiments have shown that VSNs are also activated by formylated peptides and various antimicrobial/inflammation-linked compounds^25,42. 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 protein^30,44-49. 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's native environment.

Protokół

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

1. 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).
2. 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).
3. 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 °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 °C until further use.
4. 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.
5. 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).

2. Workspace Preparation

1. 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.
2. Fill reservoir for slice superfusion at recording setup with S₂ and oxygenate continuously.
3. 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.
4. Arrange surgical tools and consumables.
5. Place ice gel pack under dissecting microscope, cover with a paper towel to prevent VNO tissue from freezing to bottom of the dish.
6. Clean razor blade by briefly rinsing in 70% ethanol and distilled water and mount to vibratome. Replace for every slicing session.

3. VNO Dissection and Embedding

1. 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.
2. Remove the lower jaw with large surgical scissors. Enter through the mouth cavity and cut the mandible bones and muscle of each side separately.
3. Place the remaining part of the head upside down in the large Petri dish.
4. 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.
5. Use bone scissors to cut away the largest part of the incisors at a ~45° 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.
6. 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.
7. 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.
8. 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.
9. Rinse the VNO in a small amount of ice-cold S₂ to prevent the tissue from drying out.
10. 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 1D_i).
11. 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.
12. Once the two VNOs are separated, start removing the cartilage encapsulating the first VNO.
13. 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).
14. 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.
15. Slowly peel the VNO away from the cartilage by holding it at its caudal tip, to avoid damaging the sensory epithelium.
16. 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.
17. 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 2A_ii). 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.
18. Immediately dissect the second VNO.
19. To embed the VNOs, fill both small petri dishes to the rim with melted S₃ (stored in water bath at 42 °C; see 1.3).
20. Hold the VNO on the broader caudal end with fine forceps to avoid damage to the sensory epithelium.
21. 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.
22. 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.
23. 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.

4. Coronal VNO Tissue Slicing

1. 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.
2. 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.
3. 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.
4. Transfer the specimen plate to the slicing chamber and prepare the second VNO, accordingly. Keep plate with the second specimen at 4 °C until use.
5. Use a vibrating blade microtome with the following settings: thickness: 150-200 µm; 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.

5. Single-cell Electrophysiological Recordings

1. 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).
2. Prepare a stock of 10-20 patch pipettes (4-7 MΩ). 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 µm.
3. Keep pipettes in a pipette storage jar to prevent damage and dust accumulation until use.
4. 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.
5. 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.
6. 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.
7. Transfer imaging chamber to recording setup and continuously superfuse slice with oxygenated S₂ at room temperature via a bath application.
8. 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 "perfusion pencil" 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.
9. Connect reference electrode and bath solution using an L-shaped agar bridge (filled with 150 mM KCl).
10. Fill patch pipette with pipette solution S₄.
11. Mount the pipette over the silver chloride-coated patch electrode connected to the head stage without scraping off the coating and attach firmly.
12. Apply slight positive pressure (approximately 1 ml on a 10 ml plastic syringe) to the patch pipette before entering the bath.
13. Lower the pipette into the bath using micro manipulators far enough to be able to submerge the objective without hitting it (Figure 2D).
14. Monitor pipette resistance (R_pip, between 4-7 MΩ) using electrophysiology software connected to the head stage.
    Note: If R_pip is <4 MΩ the glass tip is broken. If R _pip is >10 MΩ, the tip is most likely clogged and the pipette must be replaced.
15. 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.
16. Focus and target fluorescent or non-fluorescent cells depending on experimental design.
17. 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.
18. Release positive pressure and apply slight negative pressure to suck in the cell membrane in order to gain a high resistance seal (1-20 GΩ). Apply short and gentle suction to disrupt the cell membrane and establish the whole-cell configuration.
19. Monitor access resistance constantly during experiment.
    Note: Only include neurons exhibiting small and stable access resistance (≤3% of R_input; change <20% over the course of the experiment) into analysis.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Chemicals
Agarose (low-gelling temperature)	PeqLab	35-2030
ATP (Mg-ATP)	Sigma-Aldrich	A9187
Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)	Sigma-Aldrich	B9879
Calcium chloride	Sigma-Aldrich	C1016
Ethylene glycol tetraacetic acid (EGTA)	Sigma-Aldrich	E3889
Glucose	Sigma-Aldrich	G8270
GTP (Na-GTP)	Sigma-Aldrich	51120
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Sigma-Aldrich	H3375
Magnesium chloride	Sigma-Aldrich	M8266
Potassium chloride	Sigma-Aldrich	P9333
Potassium hydroxide	Sigma-Aldrich	03564
Sodium chloride	Sigma-Aldrich	S7653
Sodium hydrogen carbonate	Sigma-Aldrich	S5761
Sodium hydroxide	Sigma-Aldrich	S8045
Name	Company	Catalog Number	Comments
Surgical tools and consumables
Large Petri dish, 90 mm	VWR		decapitation, dissection of VNO capsule
Small Petri dish, 35 mm	VWR		lid for VNO dissection, dish for embedding in agarose
Sharp large surgical scissor	Fine Science Tools		decapitation, removal of lower jaw
Strong bone scissors	Fine Science Tools		cutting incisors
Medium forceps, Dumont tweezers #2	Fine Science Tools		removing skin and palate
Micro spring scissors, 8.5 cm, curved, 7 mm blades	Fine Science Tools		cutting out VNO
Two pairs of fine forceps, Dumont tweezers #5	Fine Science Tools		dissecting VNO out of cartilaginous capsule
Small stainless steel spatula	Fine Science Tools		handling agarose block and tissue slices
Surgical scalpel			cutting agarose block into pyramidal shape
Name	Company	Catalog Number	Comments
Equipment
Amplifier	HEKA Elektronik	EPC-10
Borosilicate glass capillaries (1.50 mm OD/0.86 mm ID)	Science Products
CCD-camera	Leica Microsystems	DFC360FX
Filter cube, excitation: BP 450-490, suppression: LP 515	Leica Microsystems	I3
Fluorescence lamp	Leica Microsystems	EL6000
Hot plate magnetic stirrer	Snijders	34532
Microforge	Narishige	MF-830
Micromanipulator Device	Luigs & Neumann	SM-5
Micropipette puller, vertical two-step	Narishige	PC-10
Microscope	Leica Microsystems	CSM DM 6000 SP5
Noise eliminator 50/60 Hz (HumBug)	Quest Scientific
Objective	Leica Microsystems	HCX APO L20x/1.00 W
Oscilloscope	Tektronik	TDS 1001B
Osmometer	Gonotec	Osmomat 030
Perfusion system 8-in-1	AutoMate Scientific
pH Meter five easy	Mettler Toledo
Pipette storage jar	World Precision Instruments	e212
Recording chamber	Luigs & Neumann	Slice mini chamber
Razor blades	Wilkinson Sword GmbH	Wilkinson Sword Classic
Oxygenating slice storage chamber; alternative commercial chambers are: e.g., BSK1 Brain Slice Keeper (Digitimer) or the Pre-chamber (BSC-PC; Warner Instruments)	custom-made
Stereo microscope	Leica Microsystems	S4E
Trigger interface	HEKA Elektronik	TIB-14 S
Vibratome	Leica Microsystems	VT 1000 S
Water bath	Memmert	WNB 45