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 border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Adenosine 5&#x2019;-triphosphate disodium salt solution (ATP)</td>
			<td>Sigma-Aldrich</td>
			<td>A6559-25UMO</td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica Microsystems</td>
			<td>SP5 AOBS</td>
			<td>&nbsp;</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</td>
			<td>317275-100ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Embedding molds, Peel-A-Way</td>
			<td>Polysciences</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>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>Large bath recording chamber (RC-26G)</td>
			<td>Warner Instruments</td>
			<td>64-0235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MetaFluor (software for calcium imaging; v.7.6.5.0)</td>
			<td>Visitron Systems</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Tubulin, acetylated Mouse antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T6793-.2ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Normal goat serum (NGS, 10 ml)</td>
			<td>Interchim</td>
			<td>UP379030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Platform for chambers (P-1)</td>
			<td>Warner Instruments</td>
			<td>64-0277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pluronic F-127, 2 g</td>
			<td>Invitrogen</td>
			<td>P-6867</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Roti Coll (Dosing bottle, 10 g)</td>
			<td>Carl Roth</td>
			<td>0258.1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SC-20 Dual In-line Solution heater/Cooler</td>
			<td>Warner Instruments</td>
			<td>W64-0353</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Slice anchor (SHD-26GKIT)</td>
			<td>Warner Instruments</td>
			<td>64-0266</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereofluorescence microscope </td>
			<td>Leica Microsystems</td>
			<td>MZ16FA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Super PAP PEN (hydrophobic pen)</td>
			<td>Pelco International</td>
			<td>22309</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fluka</td>
			<td>93420-250ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vibrating blade microtome VT 1200S</td>
			<td>Leica Microsystems</td>
			<td>14048142066</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VisiChrome high speed polychromator system</td>
			<td>Visitron Systems</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3D Data Visualization (Imaris; v.6.3.1)</td>
			<td>Bitplane Scientific Software</td>
			<td>&nbsp;</td>
			<td>&nbsp;</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

Research

JoVE Journal

Neuroscience

16.1K 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 ...

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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 ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

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 ...