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

Published: September 10th, 2016



1Department of Chemosensation, Institute for Biology II, RWTH Aachen University, 2Mill Hill Laboratory, The Francis Crick Institute

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 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.

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 id....

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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........

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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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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.......

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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.


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Name Company Catalog Number Comments
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
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
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

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