We thank Andrea Moran together with members of Lab Animal Service Facility (LASF) at Stowers Institute for their excellent support on animal husbandry and technical services. This work is supported by funding from Stowers Institute and the NIH (NIDCD 008003) to CRY. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Deafness and Other Communication Disorders or the National Institutes of Health. U.S. patent pending for the tetO-G-CaMP2 mice for Stowers Institute, CRY and LM.

1. Solution preparation
<ol>
 <li>Prepare 10X R1, 10X R2 and 10X R3 solutions according to the table. 
 R1 
 <table border="1">
 <tbody>
 <tr>
 <td>Chemicals</td>
 <td>MW(g/mol)</td>
 <td>mM (1X)</td>
 <td>10X stock (g/L)</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>58.44</td>
 <td>125</td>
 <td>73.05</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>74.55</td>
 <td>2.5</td>
 <td>1.86</td>
 </tr>
 <tr>
 <td>MgCl2</td>
 <td>1 M stock</td>
 <td>1</td>
 <td>10 ml</td>
 </tr>
 <tr>
 <td>CaCl2&middot;2H2O</td>
 <td>147.02</td>
 <td>2</td>
 <td>2.94</td>
 </tr>
 <tr>
 <td>NaH2PO4&middot;H2O</td>
 <td>137.99</td>
 <td>1.25</td>
 <td>1.72</td>
 </tr>
 </tbody>
 </table>
 
 R2 
 <table border="1">
 <tbody>
 <tr>
 <td>Chemical</td>
 <td>MW(g/mol)</td>
 <td>mM (1X)</td>
 <td>10X stock (g/L)</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>84.01</td>
 <td>25</td>
 <td>21</td>
 </tr>
 </tbody>
 </table>
 
 R3 
 <table border="1">
 <tbody>
 <tr>
 <td>Chemicals</td>
 <td>MW(g/mol)</td>
 <td>mM (1X)</td>
 <td>10X stock (g/L)</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>58.44</td>
 <td>125</td>
 <td>73.05</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>74.55</td>
 <td>2.5</td>
 <td>1.86</td>
 </tr>
 <tr>
 <td>MgCl2</td>
 <td>1 M stock</td>
 <td>2</td>
 <td>20 ml</td>
 </tr>
 <tr>
 <td>CaCl2&middot;2H2O</td>
 <td>147.02</td>
 <td>2</td>
 <td>2.94</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>1 M stock</td>
 <td>5</td>
 <td>50 ml</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>Make 1 liter of mouse artificial cerebro-spinal fluid (mACSF) on the day of the experiment by mixing 100 ml of 10X R1 and 100 ml of 10X R2 in double distilled water (DDW) and add 1.8 g of dextrose (final 10 mM). This preparation method prevents calcium carbonate precipitation at high pH. The osmolarity of mACSF is around 310-315 mOsm/L. Adjust the osmolarity with dextrose or water if necessary. Aerate the solution with Carboxygen gas (95% oxygen and 5% carbon dioxide) for at least 30 minutes before adjusting the pH of the solution to 7.2-7.4.</li>
 <li>Make 1 liter of Ringer's solution on the day of the experiment by mixing 100 ml of 10X R2 and 100 ml of 10X R3 in DDW and add 1.8 g of dextrose (10 mM). The osmolarity and pH of Ringer's solution should be similar to those of the mACSF. Aerate the solution with Carboxygen gas.</li>
 <li>Prepare 4% low melting agarose (LMA) in either mACSF or Ringer's solution. Aliquot the melted agarose in eppendorf tubes and store at 4&deg;C till use. LMA, which is composed of purified polysaccharide, remains fluid at 37&deg;C and solidifies quickly at temperature below 25&deg;C. These properties make it ideal for embedding VNO and live tissue physiology. Other impure materials, such as agar, should not be the choice for live tissue experiment as uncharacterized components may interfere with neuronal response.</li>
 <li>Prepare pheromone stimuli in Ringer's solution. For mouse urine, 1:100 dilution give rise to robust responses.</li>
</ol>
2. Preparation of VNO slice
<ol>
 <li>Before sacrificing the animal, put two tubes of LMA on a heat block and set the temperature to &gt;60&deg;C to melt the agarose. Once the gel liquefies, transfer the tubes to a 37&deg;C heat block.</li>
 <li>Decapitate a G-CaMP2 mouse following CO2 euthanasia. Cut the mandible bones with scissors to remove the lower jaw. Peel off the ridged upper palate tissue to expose the nasal cavity (Figure 1A). Separate the jawbones by inserting a surgical blade between the two front incisors. Carefully remove the jawbones to expose VNO. At this point, the whole VNO can be lifted up from the nasal cavity by holding on the tail bone (Figure 1B). Transfer the VNO to cold oxygenated mACSF solution immediately.</li>
 <li>Under dissection scope, separate the two VNOs by using the tip of #5 forceps gently slide along the wall of septal bone (Figure 1C). Peel away the vomer bone that encases the VNO tissue. Gently lift the entire VNO tissue from the bone cavity. Extreme care should be taken in this step not to damage the neuroepithelium. Small bone fragments left on the tissue surface must be removed completely before embedding. Fragments left on the tissue may be caught by the cutting blade to pull the tissue out of the agarose block.</li>
 <li>Use the forceps to hold the posterior end of the VNO tissue and gently submerge it into the melted agarose (Figure 1D). Quickly cool the tube on ice to solidify the agarose. The embedding and cooling process should take less than 2 minutes.</li>
 <li>VF300 tissue slicer (Precisionary Instruments, Greenville, NC) is used to make sections. Cut the agarose block containing the VNO in shape and glue it to the tissue holder (Figure 1E and F). Insert the tissue holder into the metal cylinder and fill the remaining chamber with additional LMA. Once the LMA solidifies on ice, proceed to sectioning immediately. Supply cold oxygenated mACSF into the sectioning chamber and start cutting at 180-200 &mu;m thickness per slice. Adjust the advancing speed and vibration frequency so that the tissue is not compressed during sectioning and VNO does not fall off from the agarose. Collect and transfer the sectioned slices to mACSF incubation chamber (Figure 2A). The slices are viable for 6-8 hours in oxygenated mACSF at room temperature. Figure 2B and C show DIC and 2-photon images of a G-CaMP2 VNO slice, respectively.</li>
</ol>
3. Imaging chamber set up
<ol>
 <li>Place the VNO slice in the middle of the perfusion chamber (Siskiyou, Grants Pass, OR) and hold the slice down with a slice anchor (Warner Instruments, Hamden, CT) (Figure 3A). The threads of the anchor should only press against the LMA part of the slice but not the VNO tissue. Oxygenated mACSF is delivered to the perfusion chamber through inlet port at &sim;100 &mu;l/sec and the liquid is drained through a suction needle via the outlet port to provide a continuous flow of fresh mACSF (Figure 3A and B).</li>
 <li>Fill a 30 ml syringe with Ringer's solution and clamp it to the syringe pump (New Era Pump Systems, Farmingdale, NY). Set the pump speed to 300-600 &mu;l/min to provide a continuous flow of Ringer's solution over the slice (Figure 3C).</li>
 <li>Connect the outlet of the Ringer's to a HPLC injection loop (Chromtech, Apple Valley, MN) (Figure 3D). Different loop size can be selected for precise control of the volume being delivered. We use a 20 &mu;l loop in our experiments. The injection loop control has two flow routes (Figure 3E). At the "load" position, samples can be loaded into the sample loop with a Hamilton precision syringe. Excess sample exits the sample loop through waste outlet. Ringer's solution injected by the syringe pump bypasses the sample loop and goes directly to the outlet, which is connected to the perfusion tip (Figure 3A and E). At "injection" position, the pump solution flows through the sample loop and push the stimuli into the outlet (Figure 3E). Prior to the imaging experiment, air bubbles should be chased out to ensure smooth flow of the perfusion fluid. Other commercial or custom made injection systems can also be implemented for stimulus delivery.</li>
 <li>Adjust the perfusion tip under a 5X or 10X lens so that the tip is about 1 mm away from the VNO slice.</li>
 <li>When a new perfusion system is setup, it is advisable to measure the sample delay time and duration with fluorescent dye. Load 0.1% rhodamine 6G dye into the sample loop and switch the valve to inject position at 5 seconds after the start of image acquisition (Figure 3F). The fluorescent signal is detected between 10-30 seconds. The smooth curve of fluorescent signal also indicates that there is little turbulence generated under the dipping lens and that adequate perfusion of oxygenated mACSF reaches the slice.</li>
</ol>
4. Time lapse imaging
<ol>
 <li>We use the Zeiss AxioSkope FS2 microscope with a 10X or 20X water-dipping lens for time lapse imaging. Standard GFP bandpass filter (450-490nm) is used for G-CaMP2 signals. The epifluorescent images are acquired by a CCD camera (Zeiss HRM) with 1X1 or 2x2 binning depending on the expression levels of G-CaMP2.</li>
 <li>Set the acquisition speed to 1 frame per second. Adjust the intensity of the light to minimize bleaching of the G-CaMP2 signals and photo damage to the cells. For each experiment, we normally acquire a 60-frame image stack (&sim;60-70 seconds). Switch the valve to injection position at a specific time point (eg, 5 second in Figure 3F) for one set of experiment to obtain consistent time delay in all trials.</li>
 <li>Perform a test run using Ringer's solution as the stimulus. Re-adjust the perfusion setup if movement artifact is introduced during sample injection.</li>
 <li>Perform a positive control run with mouse urine diluted at 1:100 in Ringer's solution. The typical maximal &Delta;F/F value of G-CaMP2 response to mouse urine is around 20-40%. If needed, one can confirm the viability of the slice by delivering 10 mM KCl in Ringer's to stimulate the slices at the end of the experiments.</li>
 <li>Wash the Hamilton syringe in Ringer's solution at least three times after loading one stimulus. Wash the sample loop with Ringer's solution at least three times between different stimuli. These steps prevent cross contamination among different samples.</li>
 <li>Wait for 4-10 min for the VSNs to recover before applying the next stimulus.</li>
</ol>
5. Data analysis
<ol>
 <li>Perform image registration of all the images acquired from one slice. We use a custom-written VBA script in AxioVision (Carl Zeiss, North America) to automate this process. All image frames within the same experiment are registered against a common chosen reference frame with elastic registration (Figure 4A). Elastic registration, also known as nonlinear registration, is a category of image registration technique emphasizing the transformation of a target image non-rigidly to a reference image. Here we used the implementation from AxioVision. 
 
 This reference frame is chosen arbitrarily from the image stacks.</li>
 <li>Perform image subtraction to identify the responding cells. We use custom written macros in ImageJ v1.42 (<a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a>, NIH, Bethesda, MD) to automate this process. A minimal projection image is generated for each stack. Responding cells emerge after the minimal projection is subtracted from the raw stacks (Figure 4B).</li>
 <li>Identify region of interest (ROI) from the subtracted stacks and obtain the ROI coordinates using Multi-Measure plugin from ImageJ. Process all stacks for one experiment and save all ROI coordinates in a ROI master list (Figure 4C).</li>
 <li>Use the ROI master list to measure cell responses from raw image stacks with custom-written macro and Multi-Measure plugin from ImageJ (Figure 4D). Plot response curves and heatmap in Matlab (Mathworks Inc., Natick, MA).</li>
</ol>
6. Representative Results
An example of the VNO imaging experiment using urine samples collected from four individual mice is shown in Figure 5. The slice responds to urine stimulation with diverse patterns of activation. About 80 cells are identified showing response to at least one of the urine stimuli and their response &Delta;F/F values are plotted in the heatmap (Figure 5A). The response traces of cells 1, 2 and 3 are plotted in Figure 5B showing the time course activation by urine stimuli. Cell 1 displays response to both female urine samples but not the male samples, whereas cell 2 shows the opposite patterns of activation and responds to both male samples only. Cell 3 is activated by both individual male and female samples. Cells 1 and 2 show characteristic response to sex specific cues in the urine 15.
<img alt="Figure 1" src="/files/ftp_upload/3404/3404fig1.jpg" /> 
Figure 1 Schematic illustration of VNO dissection process. A. The anatomical location of VNO in the mouse head. The head of a G-CaMP2 mouse has been dissected and is laid upside down with the soft tissue removed from the palate to expose the VNO. Inlet shows a fluorescent image of the same picture. The neuroepithelia of the VNO are bright green. B. A side view of the isolated VNO that is enclosed in the vomer bone (top) and the fluorescent image of the same VNO (bottom). C. A coronal view of the VNO and dissection process. One VNO is separated from the septum and the vomer bone can then be removed to extricate the neuroepithelium. D. VNO is embedded into LMA. E. The embedded block is glued to the tissue holder for sectioning. The tissue holder is advanced at 180-200 &mu;m per slice pushing the agarose block out of the metal barrel for sectioning. The cutting blade is positioned closely to the metal barrel. F. Side view of the VF300 tissue slicer system. B.V., blood vessel. N.E., neuroepithelium.
<img alt="Figure 2" src="/files/ftp_upload/3404/3404fig2.jpg" /> 
Figure 2 VNO slices. A. VNO slices are maintained in oxygenated mACSF at room temperature. B. A DIC picture of the VNO slice. C. A 2-photon image of the VNO from G-CaMP2 mouse. Scale bar, 50 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3404/3404fig3.jpg" /> 
Figure 3 Illustration of perfusion system setup. A. A typical perfusion chamber with inlet and outlet. VNO slice is positioned in the center of the chamber and pressed down with a tissue anchor. The middle threads are removed from the tissue anchor so that the VNO tissue is not pressed. B. The perfusion chamber is placed on microscope stage under the dipping objective. The mACSF inlet, suction outlet and perfusion tip are indicated. C. The single barrel syringe pump provides continuous flow of Ringer's through the perfusion tip. D. The HPLC injection loop system is adopted for convenient stimulus sample loading and injection. E. Schematic illustration of the flow directions at loading and injection positions. Ringer's, green. Stimulus sample, magenta. Arrows indicate direction of flow. F. Delay and wash off time measurement of the perfusion system. Rhodamine 6G fluorescent dye is loaded to the sample loop and the valve is switched to the injection position at 5th second, which is indicated with an arrow. The fluorescent signal change is detected between 10-30 seconds and diminishes afterwards.
<img alt="Figure 4" src="/files/ftp_upload/3404/3404fig4.jpg" /> 
Figure 4 Image processing pipeline. A. All image frames for one slice are registered against a reference frame. B. Subtraction of minimal projection of a stack from its raw frames. Responding cells become prominent after subtraction. C. ROIs are selected and their coordinates are compiled into master list. D. Measurement of responding cells using ROI master list from raw stack.
<img alt="Figure 5" src="/files/ftp_upload/3404/3404fig5.jpg" /> 
Figure 5 A representative G-CaMP2 VNO imaging experiment. A. The &Delta;F/F response heatmap from a slice stimulated with individual male and female urine. Urine samples are collected from individual male and female mice. X-axis, responding cells identified from the slice. Y-axis, urine samples used in the experiment. Color bar indicates the &Delta;F/F value. B. Response time course of 3 representative cells marked in A. Arrows indicate the time of stimulus application. F-FVB, female FVB; F-CBA, female CBA; M-FVB, male FVB; M-BL6, male C57BL/6.
Table of specific reagents and equipment:
<table border="1">
 <tbody>
 <tr>
 <td>
 Name of the reagent</td>
 <td>
 Company</td>
 <td>
 Catalogue number</td>
 <td>
 Comments (optional)</td>
 </tr>
 <tr>
 <td>perfusion chamber</td>
 <td>Siskiyou</td>
 <td>PC-H</td>
 <td>horizontal</td>
 </tr>
 <tr>
 <td>slice hold-down</td>
 <td>Warner Instruments</td>
 <td>SHD-22CL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>perfusion port holder</td>
 <td>ALA scientific, Inc.</td>
 <td>MPIOH-S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>syringe pump</td>
 <td>New Era Pump Systems, Inc.</td>
 <td>NE-300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>low pressure injection valve</td>
 <td>Chromtech</td>
 <td>V-451</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PEEK tubing</td>
 <td>Chromtech</td>
 <td>1531</td>
 <td>1/16" OD, 0.25mm ID</td>
 </tr>
 <tr>
 <td>PEEK sample loop</td>
 <td>Chromtech</td>
 <td>1803</td>
 <td>20 &mu;L</td>
 </tr>
 <tr>
 <td>Hamilton syringe</td>
 <td>Chromtech</td>
 <td>80630</td>
 <td>100 &mu;L</td>
 </tr>
 </tbody>
</table>

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No conflicts of interest declared.

The majority of the vomeronasal receptors (VRs) remain as orphan receptors since their discovery by Dulac and Axel 5. The pheromone ligands for these chemosensory receptors and their roles in mediating animal behaviors are not well understood. Until now, only one pair of ligand/receptor, the ESP1 peptide and its cognate receptor, Vmn2r116 (V2Rp5), has been identified and shown to convey specific social information 19,23. Another receptor, V1rb2, has been shown to respond to 2-heptanone, which presum...

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.

Watch this Scientific Journal Video about Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor at JoVE.com

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

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 ...

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10.7K vistas.  Stowers Institute for Medical Research. 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.

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

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

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.

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. ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

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 ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.