We want to thank particularly Monique Nenniger Tosato for her excellent technical support as well as Jean-Yves Chatton for his scientific advices and the imaging CIF platform of the UNIL for the microscope equipment. Funding was provided by the Swiss National Science Foundation.

1. Dissection of the mouse VNO
<ol>
	<li>Use adult mice. As pheromone sensing is implicated in multimodalities, careful distinction between strains, genotypes, sexes and ages is important and will influence your results. Here, we choose adult VG mice30 previously used for the identification of a pheromone-receptor pair (2-heptanone-V1rb2)31 (Fig. 1A).</li>
	<li>Before starting the dissection, prepare fresh cold artificial cerebro-spinal fluid (ACSF; NaCl 118 mM, NaHCO3 25 mM, D-Glucose 10 mM, KCl 2 mM, MgCl2 2 mM, NaH2PO4 1.2 mM, CaCl2 2 mM; pH 7.4) saturated with oxycarbon (95% O2 : 5% CO2). For that, mix all the ACSF components except CaCl2. Saturate the solution by directly bubbling it with oxycarbon. In the end, add the CaCl2 and adjust with ddH20 to the desired volume. Keep the ACSF solution on ice until use.</li>
	<li>Euthanasia of the mouse should be preferentially done by cervical dislocation or by CO2 inhalation just before the experiment.</li>
	<li>Cut the mouse head. Place it under a dissecting microscope in cold ACSF continuously oxycarbonated.</li>
	<li>Cut the lower jaw and position the head in order to visualize the palate (Fig. 1B).</li>
	<li>Make an incision horizontally in the upper part of the palate (Fig. 1B) with micro scissors and remove the palate membrane with micro dissecting forceps. Hydrate and clean the exposed cavity with ACSF (Fig. 1C).</li>
	<li>Cut the upper and the lower part of the nasal septum (Fig. 1C). Delicately extract the nasal septum containing the VNO and place it directly in ACSF on ice (Fig. 1D).</li>
	<li>Separate vertically the two parts of the VNO using micro dissecting forceps and delicately remove the cartilaginous capsule of the VNO (Fig. 1E). Make sure that all the cartilaginous pieces are removed.</li>
</ol>
2. VNO tissue slice preparation
The VNO tissue slice preparation described in this section 2) is mainly for calcium imaging investigations. VNO slices can also be used for immunohistostainings on floating slices to verify the structural integrity of the tissue (please see section 3) of the protocol).
<ol>
	<li>Prepare low-melting agar (3% in standard PBS) and your working place. You will need ACSF, a microtome and supports, micro dissecting forceps, embedding molds (22 x 22 x 20 mm), precision wipes, brushes, tissue culture plates, cyanacrylat glue, ice and a thermometer.</li>
	<li>Fill the embedding mold with 3% low-melting agar. Make sure that the temperature is not higher than 41&deg;C before placing vertically the shortly wiped VNO (Fig. 2A) to be able to obtain coronal slices.</li>
	<li>Place the embedding mold on ice for 1 min until solidification and then separate it from the agar block. Cut the agar block in a pyramidal shape and place it with glue on the microtome support (Fig. 2B).</li>
	<li>Cut coronal VNO slices with the microtome, with a speed of 0.5 mm/s, an amplitude of 0.7 mm and a thickness of 100 &mu;m in cold ACSF and collect the tissue slices with a brush before placing them in a tissue culture plate filled with cold ACSF.</li>
	<li>If your slices contain endogenous fluorescence like GFP (in gene-targeted neurons) you can select them under a fluorescent stereomicroscope (Fig. 2C).</li>
</ol>
3. Immunohistochemistry on VNO floating slices
The goal of this procedure is to verify the integrity of the VNO tissue slices obtained in section 2). Acetylated-tubulin is a microtubule / cytoskeleton marker expressed in dendrites and microvilli of VNO sensory neurons. For calcium imaging experiments, go directly to section 4) of the protocol. The immunohistostaining method on floating VNO slices can be adapted to the evaluation of the expression of any protein of interest. For this purpose, we recommend that you fix the VNO for 1h at 4&deg;C in a fixative solution (paraformaldehyde 4% in PBS, pH 7.6) after the point 1.7) of the protocol and use from this point PBS at pH 7.6 rather than ACSF solution.
<ol>
	<li>In the tissue culture plate, fix your slices of interest in a fixative solution (paraformaldehyde 4% in PBS, pH 7.6) 1h at 4&deg;C.</li>
	<li>Block your tissue slices overnight at 4&deg;C in a PBS solution containing NGS (normal goat serum) 10% and triton X-100 at 0.5%.</li>
	<li>Incubate slices with the primary antibody (anti-acetylated-tubulin, Mouse, 1:2000) for 16h at room temperature (RT) in a PBS solution containing NGS 5% and Triton X-100 at 0.25%.</li>
	<li>Wash 3 times for 5 minutes with a PBS solution containing NGS 2%.</li>
	<li>Incubate in the dark with the secondary antibody (Cy5-conjugated Goat anti-Mouse, 1:200) in a PBS solution containing NGS 2% for 1h at RT.</li>
	<li>Wash 3 times for 5 minutes with PBS containing NGS 2%, PBS NGS 1% and PBS only.</li>
	<li>Mount your slices in a fluorescent mounting media (containing or not DAPI) by placing them in the centre of the hydrophobic delimited zones (use a hydrophobic pen) and cover the slices with coverslips.</li>
	<li>Make observations and acquisitions with confocal microscopy and a software allowing 3D reconstructions (Fig. 2D-F).</li>
</ol>
4. Calcium imaging on VNO slices
<ol>
	<li>The following procedures will take place in the dark. Incubate VNO slices for 1h at 37&deg;C in a loading solution composed of cold ACSF with Fura-2AM (7 &mu;M; stock solution of 1 mM in DMSO) and pluronic 0.1% (w/v; stock solution at 20 % in ddH2O).</li>
	<li>Keep the loaded slices on ice with oxycarbonation until use. Loaded slices can be kept for 3-4h.</li>
	<li>Place the loaded slices with a brush in a perfusion chamber containing ACSF and maintain the slices with a tissue slice anchor (Fig. 3A).</li>
	<li>Place the perfusion chamber on the stage of a calcium imaging microscope (Fig. 3B) and continuously perfuse with oxycarbonated ACSF at RT. Temperature can be adapted with the use of a bipolar temperature controller.</li>
	<li>Observations of the loaded VNO slices (Fig. 3C-E) are performed with an inverted fluorescence microscope, with a 25x or 63x objective and a CooL SNAP-HQ camera. Illumination is done sequentially (340/380 nm) with a polychromator instrument and recorded at a rate of 5Hz. Filtered emission is done at 510 nm. Intracellular calcium ratio changes (&Delta;F = 340 nm/380 nm) are monitored with an appropriate software.</li>
	<li>Chemostimulation should be selected according to your own experimental purpose. Here, perfusions of ACSF containing a pheromone mix (isobutylamine, 2-heptanone, 4-heptanone, 2-heptanol, pentylacetate, dimethylpyrazine, &alpha;/&beta;-farnesene, 10-6 M), 2-heptanone (10-6 M) or freshly collected male and female (1:1) mouse urine (1:100) are performed32. Perfusion of ATP (125 &mu;M) is used as a viability test at the end of the experiments (Fig. 3F) and should indicate a proportion of approximately 80% viable neurons. Under these conditions, slices can be used up to 2 hours.</li>
	<li>GFP-tagged subpopulations of sensory neurons are visualised by specific illumination (here the V1rb2 expressing neurons) and can be analysed precisely (Fig. 3G-I).</li>
</ol>
5. Representative Results:
To establish a functional assay to investigate the multi transduction pathways taking place in the mouse VNO, or to identify new pheromone-receptor pairs, we take advantage of the VG mouse line (Fig. 1A). In this line, one out of 240 V1Rs receptors, the V1Rb2 receptor is GFP-tagged allowing the easy visualization of a particular subpopulation of vomeronasal sensory neurons. After extraction and dissection of the VNO (Fig. 1E), we prepare acute VNO slices (Fig. 2C). We fix a fraction of them, in order to check and validate the tissular integrity of the preparation by performing immunohistostainings against the acetylated-tubulin protein (Fig. 2D-F). The other fraction of the slices is used for calcium imaging experiments. For that, slices are loaded with Fura-2AM (Fig. 3C-E) and the intracellular calcium level of each neuron is monitored as a &Delta;F ratio (Fig. 3F). Viability of the neurons is evaluated by perfusion of ATP (125 &mu;M), after which rapid and transient increase of the &Delta;F ratio is expected (Fig. 3F). Urine being a major source of pheromones, perfusion of freshly collected mouse urine (1:100) is used as an endogenous control. It triggers calcium transients in approximately 50% of the recorded neurons (cell 1 and 2, Fig. 3F). Under GFP illumination, V1Rb2 positive neurons are observable, and perfusion of its known pheromonal ligand, 2-heptanone, initiates cellular activation (cell 1, Fig. 3I). Interestingly, neuronal activations are also observable in a fraction of neurons that do not express the V1Rb2 receptor (GFP negative neurons) (cell 2, Fig. 3I), demonstrating the complexity of pheromone coding in the VNO.
<img alt="Figure 1" src="/files/ftp_upload/3311/3311fig1.jpg" /> 
Figure 1. Dissection of the mouse vomeronasal organ. (A) A mouse from the VG line. (B) After euthanasia of the mouse, the full head is placed under a binocular microscope and the lower jaw is removed in order to visualize the palate. A small horizontal incision is done (black dashed line). (C) After the removal of the palate, the nasal septum lined with the VNO is cut in the upper and the lower part (white dashed lines) to facilitate VNO extraction. (D) The VNO is positioned in ACSF solution and separated (insert : higher power view of the two parts). (E) The cartilaginous capsule of the VNO is delicately removed, and the VNO without its cartilaginous capsule is used for the rest of the experiments. Scale bars are: B-E, 1 mm.
<img alt="Figure 2" src="/files/ftp_upload/3311/3311fig2.jpg" /> 
Figure 2. Mouse vomeronasal acute slice preparation and tissular integrity. (A) The mouse VNO is vertically embedded in an agar 3% solution. The temperature of the agar must be lower than 41&deg;C. (B) The agar block is cut in a pyramidal shape and 100 &mu;m VNO sections (black dashed line) are generated in ACSF. (C) VNO slices can be selected under a fluorescent stereomicroscope in order to visualize a specific population of tagged sensory neurons. (D-F) Tissular integrity can be evaluated by immunohistostainings against the acetylated-tubulin protein, a higher power view of the V1rb2 gene-targeted neurons demonstrate the perfect conservation of the preparation. Neurons with their long dendrites reaching the surface of the lumen (E) as well as the expression of the structural protein in the dendritic knobs and microvilli can be observed (F). Scale bars are: B, 5 mm; C, 60 &mu;m; D, 40 &mu;m; E-F, 20 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3311/3311fig3.jpg" /> 
Figure 3. Calcium imaging and pheromone detection in vomeronasal acute tissue slices. (A) After a Fura-2AM loading phase, VNO slices are placed in a perfusion chamber and are maintained with an adapted anchor (high power view). (B) Experiments are performed in the dark and VNO slices are continuously perfused with ACSF at RT with a vacuum system. The temperature can be chosen using a temperature controller system composed by a bipolar Pelletier element and a thermal probe. This particular experiment was performed at RT. (C-E) VNO slices are observable under Hoffman phase contrast (C, Hv) or Fura 380 nm illumination before (D) and during neuronal activation (E, here with ATP). (F) Neuronal viability is evaluated with perfusion of ATP (125 &mu;M). Here, chemostimulation is done with urine (Urine, 1:100) or with a mix of mouse pheromones (mix ph., 10-6 M) at a constant flow rate of 165 ml/h. Intracellular calcium levels (&Delta;F) can be recorded individually for each cell (Cell 1 to 3). (G) A V1Rb2 neuron is visualized under GFP illumination (1). (H) The loading of this neuron (1) as well as a non-V1rb2 expressing neuron (2) is shown. (I) The V1rb2 neuron is able to respond to 2-heptanone with an intracellular calcium increase. This particular non-V1rb2 expressing neuron also responds to 2-heptanone (cell 2). Scale bars are: C-E, 20 &mu;m; G-H, 15 &mu;m; &Delta;F = 340 nm / 380 nm. (F and I) Duration of stimulus application is indicated by bars under each trace. The arbitrary color scale represents the fluorescence intensity.

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No conflicts of interest declared.

The calcium imaging method presented here enables to record, in an acute slice preparation, the pheromonal responses of mouse VNO neurons. With this approach, dissection from the animal to the vomeronasal neurons is, with some practice, easily achievable and provides a long-living tissue preparation. It allows time-consuming experiments like pharmacological investigations or the study of pheromonal coding. With this physiological technique, large populations as well as precise neuronal subpopulations can be analysed specifically. In addition, this method can be easily adapted to immunohistochemical approaches or experiments based on other calcium dyes. The use of these combined methodologies will surely allow the identification of new pheromonal ligands for the many orphan chemoreceptors expressed in the mouse vomeronasal organ.

<table><tbody><tr><td>Adenosine 5&amp;rsquo;-triphosphate disodium salt solution (ATP)</td><td>Sigma-Aldrich</td><td>A6559-25UMO</td><td></td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A7002-100G</td><td>Preferentially for immunohistochemistry</td></tr><tr><td>Agar (low-melting)</td><td>Sigma-Aldrich</td><td>A0701-25G</td><td>Preferentially for calcium imaging</td></tr><tr><td>CL-100 Bipolar Temperature Controller</td><td>Warner Instruments</td><td>W64-0352</td><td></td></tr><tr><td>Confocal Microscope</td><td>Leica Microsystems</td><td>SP5 AOBS</td><td></td></tr><tr><td>Cy5-AffiniPure Goat Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>115-175-071</td><td>Protect from light</td></tr><tr><td>DMSO (Dimethyl Sulfoxide)</td><td>Merck &amp;amp; Co., Inc.</td><td>317275-100ML</td><td></td></tr><tr><td>Embedding molds, Peel-A-Way</td><td>Polysciences, Inc.</td><td>18646A-1</td><td> 22 x 22 x 20 mm</td></tr><tr><td>Fluorescent mounting media (Vectashield with DAPI, 10 ml)</td><td>Rectolab</td><td>H-1200</td><td></td></tr><tr><td>FURA-2AM</td><td>Teflabs</td><td>0103</td><td>Protect from light</td></tr><tr><td>Glisseal N (vacuum grease, 60 g)</td><td>Borer Chemie</td><td>Glisseal N</td><td></td></tr><tr><td>Large bath recording chamber (RC-26G)</td><td>Warner Instruments</td><td>64-0235</td><td></td></tr><tr><td>MetaFluor (software for calcium imaging; v.7.6.5.0)</td><td>Visitron Systems</td><td></td><td></td></tr><tr><td>Monoclonal Anti-Tubulin, acetylated Mouse antibody</td><td>Sigma-Aldrich</td><td>T6793-.2ML</td><td></td></tr><tr><td>Normal goat serum (NGS, 10 ml)</td><td>Interchim</td><td>UP379030</td><td></td></tr><tr><td>Platform for chambers (P-1)</td><td>Warner Instruments</td><td>64-0277</td><td></td></tr><tr><td>Pluronic F-127, 2 g</td><td>Invitrogen</td><td>P-6867</td><td></td></tr><tr><td>Roti Coll (Dosing bottle, 10 g)</td><td>Carl Roth GmbH</td><td>0258.1</td><td></td></tr><tr><td>SC-20 Dual In-line Solution heater/Cooler</td><td>Warner Instruments</td><td>W64-0353</td><td></td></tr><tr><td>Slice anchor (SHD-26GKIT)</td><td>Warner Instruments</td><td>64-0266</td><td></td></tr><tr><td>Stereofluorescence microscope </td><td>Leica Microsystems</td><td>MZ16FA</td><td></td></tr><tr><td>Super PAP PEN (hydrophobic pen)</td><td>Pelco International</td><td>22309</td><td></td></tr><tr><td>Triton X-100</td><td>Fluka</td><td>93420-250ML</td><td></td></tr><tr><td>Vibrating blade microtome VT 1200S</td><td>Leica Microsystems</td><td>14048142066</td><td></td></tr><tr><td>VisiChrome high speed polychromator system</td><td>Visitron Systems</td><td></td><td></td></tr><tr><td>3D Data Visualization (Imaris; v.6.3.1)</td><td>Bitplane Scientific Software</td><td></td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation at JoVE.com

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

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-pheromone-sensing-mouse-vomeronasal-acute-tissue-slice

Universidad de Cartagena UDEC

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Neuroscience

16.2K Views.  University of Lausanne. 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.

Video: Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

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 ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

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 ...

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

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

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

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

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://appjove.unicartagenaproxy.elogim.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://appjove.unicartagenaproxy.elogim.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.

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Summary

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Chapters in this video

Multielectrode Array Recordings of the Vomeronasal Epithelium

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

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

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

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

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

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

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

Preparation of Mouse Vomeronasal Organ for Vibratome Sectioning

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice