This project was funded by the DFG Excellence Cluster 171, the Center for Nanoscale Microscopy and Molecular Physiology of the Brain, the Bernstein Center for Computational Neuroscience and the ENC-Network, an Erasmus Mundus Joint Doctoral Program. The authors thank Stephan Junek, Mihai Alevra and Guobin Bao for providing MATLAB codes and custom-written programs for image evaluation and data analysis.

All experiments with Xenopus laevis tadpoles were performed according to the guidelines approved by the G&#246;ttingen University Committee of Ethics in Animal Experimentation.
1. Electroporation
<ol>
	<li>Choose animals of stages 49-54 according to Nieuwkoop and Faber7.</li>
	<li>Make sure that the recording setup consists of a stereomicroscope with a large working distance and an electroporation device which can apply voltage pulses of 20 V for 20 msec at a frequency of at least 2 Hz. Apply the voltage pulses via two platinum electrodes with a rough diameter of 200 &#181;m so that they can be inserted into the tadpoles&#39; nostrils without causing damage.</li>
	<li>Prepare crystals of a dextran conjugated dye (e.g., Calcium Green 10 kDa Dextran, or Alexa Fluor 647 10 kDa Dextran) by dissolving the typically delivered amount of 5 mg in about 100 &#181;l of distilled water and letting small droplets of 2 &#181;l dry on a sheet of Parafilm. 
	Note: The crystals dry in less than a day and can be stored afterwards at -18 &#176;C in the freezer for a period of over a year.</li>
	<li>Anesthetize the animals by placing them in tap water containing 3% Tricaine methane sulfonate for 1-2 min until it reaches the surgical anesthesia state characterized by decreased heart rate, loss of all movements and lack of responses to mechanical stimuli.</li>
	<li>Immerse the anesthetized animal for 10 sec in pure tap water.</li>
	<li>Place the animal on a gel cushion and fix it by placing needles around it without harming it. 
	Note: Do not fix the animal by poking needles through its skin.</li>
	<li>Gently dry the area surrounding the nostrils with a tissue.</li>
	<li>Place the animal under the stereomicroscope and focus on the nostrils.</li>
	<li>Use forceps to pick a dye crystal of size matching the nostril hole, place it in one of the nasal cavities and wait until it dissolves completely, which takes less than 1 min. If the crystals are smaller, add 2 or 3 into each cavity until they produce a non-translucent high-concentrated solution.</li>
	<li>Place the cathode on the skin of the tadpole and the anode into one of the nostrils. 
	Note: This particular setting applies to the dyes cited in step 1.3. For dyes with a different polarity, the staining results may be improved by placing the cathode into the nostrils. If unsure about the polarity of the dye, both electrodes can be placed simultaneously in the nostrils (one per nostril), and the polarity alternated between single voltage pulses.</li>
	<li>Apply six pulses of 20 V and 20 msec with approximately 0.5 sec of stimulus interval. 
	Note: Small bubbles should appear around the electrode in the nostril during the electroporation provided that the electrodes are in contact with the skin and the solution in the nostril. If no bubbles are visible check the connecting cables and make sure the electroporating device is delivering the desired voltage pulse.</li>
	<li>Repeat the procedure (1.9-1.11) for the second nostril.</li>
	<li>Transfer the electroporated tadpole into a beaker filled with tap water at room temperature. Wait 5 to 10 min till the animal regains consciousness and resumes its swimming. Feed it and let it recover for at least 1 day during which the dye is transported along the olfactory nerve to the olfactory bulb.</li>
	<li>Use the electroporated animals within 1-7 days following the electroporation for the best imaging results. Give at least 24 hr time for dye transport and recovery before imaging.</li>
</ol>
2. Whole Mount Preparation
<ol>
	<li>Anesthetize the animal in tap water containing 3% Tricaine methane sulfonate until all movements have stopped and it no longer responds to mechanical stimulations.</li>
	<li>Transfer the tadpole to a gel cushion under a stereomicroscope and fix it tightly by poking needles through the skin on each side of the forebrain.</li>
	<li>Sacrifice the tadpole by severing its spinal cord.</li>
	<li>Use a scalpel to dissect out a block of tissue containing both nasal cavities, the olfactory nerves and olfactory bulbs (Figure 1A). Make the first incision close to left nostril-without touching it-and move the blade forward cutting alongside the left olfactory nerve and bulb until the telencephalic-diencephalic border. Do likewise on the right side. Isolate the preparation from the rest of the nervous system by making a final cut posterior to the telencephalon.</li>
	<li>Dispose of the body of the tadpole and carefully flip the tissue block upside down so that the ventral side of the brain preparation faces upwards.</li>
	<li>Pin the tissue block again by inserting two needles between the olfactory nerves.</li>
	<li>Put a drop of Frog Ringer solution (98 mM NaCl, 2 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 5 mM glucose, 5 mM Na-pyruvate, 10 mM HEPES, adjusted to pH 7.8, osmolarity of 230 mOsmol/L) on the tissue.</li>
	<li>Remove the meninges covering the axon sorting zone and the olfactory bulbs by making three incisions using fine scissors. Starting from the posterior edge of the tissue block, cut caudorostrally closely along the left olfactory bulb until the entrance point of the olfactory nerves into the bulbs (the axon sorting zone).</li>
	<li>Repeat step 2.8 for the right hemisphere.</li>
	<li>Raise the meninges with forceps and make the third cut, perpendicular to the previous ones at the axon sorting zone. 
	Note: The ventral side of the olfactory bulb is now accessible for imaging and bolus loading.</li>
	<li>To improve the image quality in the transmitted light image, repeat the procedure for the dorsal side. These extra steps can facilitate the navigation of the micropipette for bolus loading, but are not strictly necessary.</li>
	<li>Transfer the sample to a recording chamber filled with Frog Ringer for further processing and imaging. Make sure that the ventral side is facing upwards and secure the sample with a net of nylon fibers spanned over a small platinum frame.</li>
	<li>For an easier application of stimuli place the nasal cavities on top of one of the nylon fibers.</li>
</ol>
3. Bolus Loading
<ol>
	<li>Dissolve 50 &#181;g of the calcium AM dye (e.g., Fluo-8 AM) in 20 &#181;l of dimethyl sulfoxide (DMSO) containing 20% of Pluronic F-127 (w/v) to prepare a stock solution of AM dye. Freeze the stock solutions in small aliquots of 1-2 &#181;l volume. The stock solution is stable for at least half a year but avoid freeze-thaw cycles.</li>
	<li>Use a micropipette puller and pull pipettes with a resistance of 5-8 M&#937; and a tip diameter of 1-2 &#181;m.</li>
	<li>Dissolve the prepared stock solution of the dye in Frog Ringer&#39;s solution at a concentration of 250-500 &#181;M.</li>
	<li>Add MK571 to reach a concentration of 500 &#181;M to block multidrug resistance transporters.</li>
	<li>Fill the micropipette with 10 &#181;l of the solution using an elongated pipette tip and remove all air bubbles by flicking the micropipette.</li>
	<li>Mount the micropipette in the pipette holder and make sure that pressure can be applied either manually with a syringe or a pneumatic drug ejection device and monitor the applied pressure with a gauge.</li>
	<li>If possible, use a microscope setup with the capability to excite the injected dye so that the outflow from the micropipette tip can be visualized and adjusted. Ideally, use a confocal imaging setup. Use an upright microscope with an immersion objective so that the tissue can be reached with the micropipette.</li>
	<li>Place the whole mount preparation under the microscope, follow the olfactory nerve until the bulb and focus onto the area of interest in the olfactory bulb.</li>
	<li>Perfuse fresh Ringer solution through the recording chamber in order to increase the viability of the preparation and wash out the dye leaking from the micropipette tip.</li>
	<li>Lower the pipette onto the surface of the olfactory bulb, with the tip pointing into the rostral direction of the preparation.</li>
	<li>Apply a small and constant positive pressure (~25 hPa) to the micropipette to prevent clogging of the pipette and gently insert it into the tissue.</li>
	<li>Once the pipette has breached the outer tissue layers, move it in a rostro-dorsal direction into the mitral cell layer. (Note that a counter staining of olfactory receptor neurons by electroporation is helpful to adjust the micropipette&#39;s position.) Ideally, place the pipette tip at a distance of 50-100 &#181;m from the desired recording site.
	<ol>
		<li>For staining the temperature-sensitive &#947;-glomerulus, target an area about 50 &#181;m rostrally from the position of the presynaptic neuropil of the &#947;-glomerulus.</li>
	</ol>
	</li>
	<li>Apply a positive pressure in the range of 100-200 hPa. Adjust the pressure strength depending on the size of the micropipette tip. Confirm the outflow from the pipette by watching for slight tissue movements visible under brightlight illumination when pressure is applied for the first time.</li>
	<li>Maintain a constant pressure for about 10 min while the micropipette remains in the tissue. If possible, check the neuropil in the meantime for cell loading under fluorescent illumination as successful loading will result in visible cell somata staining with increasing intensity over time. 
	Note: Often the reason for unsuccessful loading is pipette clogging due to tissue entering the tip or aggregated dye clusters. It is sometimes possible to rescue the pipette by gently breaking its tip against the lower surface of the recording chamber. However, the pipette tip should not exceed a few micrometers. Compensate larger pipette openings by applying lower pressure during bolus loading.</li>
	<li>After the 10 min of loading, reduce the applied pressure to zero and check the staining. 
	Note: The size of the stained area varies significantly and depends on several parameters like amount of injected dye and location of ejection site. A good staining covers an area of about 100 &#181;m x 100 &#181;m.</li>
	<li>If the stained area is too small or does not cover the desired recording site, repeat steps 3.10-3.13. Use the same micropipette for the next injection if it has not yet clogged.</li>
	<li>Wait at least 30 min after the last injection before starting any experiments to allow dye uptake and deesterification. Continue to perfuse the recording chamber with fresh Ringer solution at all times.</li>
</ol>
4. Measurement Settings
<ol>
	<li>Make sure that the measurement setup consists of a confocal microscope with sufficient speed to record three dimensional volumes. Choose an acquisition rate of at least 1 Hz per stack. 
	Note: Suitable setups include for example line-illumination microscopes scanning the sample line-wise instead of point by point. A simple realization of such a setup has been previously described6. Other options are spinning disk microscopes. If no such setup is available, it is still possible to measure smaller areas with a normal point-scanning setup.</li>
	<li>Set the measurement parameters so that a volume large enough to fit the size of a glomerulus can be covered. 
	Note: The typical thickness of a recording volume is in the range of 20 &#181;m which should be covered by at least 5 layers.</li>
	<li>Check all presynaptic measurements for bleaching. Adjust the laser power until the average fluorescence intensity of the recorded images does not drop over the time course of the recording.</li>
	<li>Limit the measurement time and area for recordings on the postsynaptic side to avoid bleaching as much as possible, although for measurements exceeding 20-30 sec bleaching is likely to occur.</li>
</ol>
5. Odor Application and Temperature Experiments
<ol>
	<li>Pour 25 ml of fresh Ringer solution (previously stored at 4 &#176;C) in a 50 ml tube. Place the tube in an ice bucket.</li>
	<li>Monitor the temperature of the cooled Ringer by inserting the clean probe of a thermometer into the tube. Wait until the temperature drops below 1 &#176;C before starting the experiment.</li>
	<li>Prepare 50 ml of L-histidine dissolved in Ringer solution at a concentration of 10 &#181;M.</li>
	<li>Use a funnel applicator8 or similar application systems allowing stimulus delivery concomitantly to the perfusing Ringer so that the water flow in the chamber remains constant and uninterrupted during the release of the stimulus solution. Position the funnel in such a way that the distal outlet is less than 1 mm away from the olfactory epithelium.</li>
	<li>Place a NiCr-Ni temperature sensor connected to a digital thermometer close to the epithelium and the outlet of the funnel applicator. Wire the thermometer output port to a computer to record and visually display voltage changes reflecting small temperature fluctuations.</li>
	<li>Before starting the experiment, connect another thermosensor to a standard thermometer to establish the voltage-to-temperature scale factor.</li>
	<li>Survey the bath temperature in the recording chamber and ensure that it does not exceed 22 &#176;C.</li>
	<li>Start the image acquisition and sequentially apply 200-400 &#181;l of cold Ringer, L-histidine and room-temperature Ringer (20-22 &#176;C) via an electronic pipette with an interstimulus interval of 20-30 sec. For a better control of the stimulus application, release the stimulus with a trigger signal sent by the chosen imaging setup to the pipette if possible. Repeat the application protocol for reproducible results.</li>
	<li>Take sets of longer recordings with several rounds of stimulation since they are preferable for post-imaging analysis with activity correlation imaging. 
	Note: A compromise between the recording time and the slice viability has to be found. Measurements of ca. 2 min covering 6 stimulus applications provide sufficient activity for a good reconstruction of the network.</li>
</ol>
6. Image Processing Using Activity Correlation Imaging (ACI)
<ol>
	<li>Image Processing of the Pre-synaptic Recordings
	<ol>
		<li>In recordings with stimulation by cold Ringer solution and histidine, distinguish the temperature-sensitive &#947;-neuropil from the neighboring histidine-sensitive glomerulus by their different response profiles (see Kludt et al.5).</li>
		<li>Create a maximum intensity projection in the axial direction from recordings of the brain preparation in which ORNs have been electroporated with two different dyes to visualize the bilateral innervation of the &#947;-glomerulus.</li>
	</ol>
	</li>
	<li>Image Processing of the Post-synaptic Recordings Using Activity Correlation Imaging (ACI)
	<ol>
		<li>Select recordings with sufficient time points (more than 100 frames) and a decent amount of activity (at least two events). 
		Note: The activity can be either spontaneous to reveal the processes of single mitral cells or induced by several applications of stimuli during the same recording to reveal the cellular networks activated by the olfactory stimuli.</li>
		<li>Choose recordings where the measured structures move no more than 2-3 pixels over the time course of the recording. 
		Note: Recordings with too much movement can be rescued by applying a shift correction procedure described in Kludt et al.5</li>
		<li>Check whether the recordings suffer from bleaching. If the average intensity of the whole image drops over the time course of the recording, bleach correction is necessary, otherwise skip the next two steps.</li>
		<li>Calculate the linear trend for the time trace of each pixel by performing a linear regression.</li>
		<li>Subtract the result from each pixel individually to eliminate the linear component of the bleaching. 
		Note: As an alternative Legendre fitting can be used as described by Bao and Schild9.</li>
		<li>Download the ready-to-use MATLAB script together with a step-by-step guide for activity correlation imaging (ACI) as described by Junek et al.6 
		Note: Follow the steps described in Junek et al.6 as an alternative to using the provided MATLAB script.</li>
		<li>Move the files &#39;aci.m&#39;, &#39;matVis.m&#39; and &#39;aci_roiSelector.m&#39; from the downloaded container to the MATLAB path of the system used for data evaluation.</li>
		<li>Load the raw data acquired in step 5.8 as a variable to MATLAB&#39;s user workspace organized as a [x,y,z,t]-matrix with x and y referring to the lateral dimensions, z, the axial direction and t, the time course.</li>
		<li>Call &#39;aci&#39; from the MATLAB command line.</li>
		<li>In the user interface (UI) which appears, select &#39;Prepare Data&#39;, then select the variable containing the data and a directory in which the results will be saved. 
		Note: For a full description of the UI see the manual accompanying the aci script.</li>
		<li>Scroll through the measured z-layers by moving the corresponding slider in the UI to get an overview of the variance map displayed.</li>
		<li>Enter the size of the region of interest (ROI) into the UI. For a mitral cell soma adjust the ROI to span approximately 10 &#181;m in the lateral and 5 &#181;m in the axial direction. For a glomerulus, the slightly higher values of 20 &#181;m laterally and 10 &#181;m axially are appropriate. 
		Note: The ROI size has to be entered as a number of pixels, which depends on the pixel scaling chosen for the recording.</li>
		<li>Select a ROI containing the &#947;-glomerulus and additional regions for each visible soma of the surrounding mitral cells by clicking with the middle mouse button onto the center of the cell/glomerulus.</li>
		<li>Close the main UI which triggers the calculation of correlation maps for all reference traces. The result is automatically saved and displayed.</li>
	</ol>
	</li>
</ol>

<ol><li>Mamasuew, K., Breer, H., Fleischer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Grueneberg+ganglion+neurons+respond+to+cool+ambient+temperatures%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">Grueneberg ganglion neurons respond to cool ambient temperatures.</a> Eur J Neurosci. 28 (9), 1775-1785 (2008).</li>
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<li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNieuwkoop%2C+PD+%22Normal+Table+of+Xenopus+laevis%22">Normal Table of Xenopus laevis</a>. , Garland Publishing Inc. New York. (1994).</li>
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The authors have nothing to disclose.

The methods presented herein aim at recording temperature processing in the olfactory bulb of Xenopus laevis tadpoles. The protocol stains first and second order neurons in the olfactory bulb and provides a sample preparation in which the olfactory system remains mainly intact. Thus, the activation of the temperature-sensitive &#947;-glomerulus can be monitored and compared with its chemo-sensitive neighboring glomeruli. The unique bilateral innervation of this glomerulus is visualized by cell electroporation with spectrally different dyes. Furthermore, bolus loading allows for the staining of mitral cells spanning a large volume within the olfactory bulb. The neuronal network processing temperature-induced signals is revealed by taking calcium measurements with repeated stimulus applications and subsequently analyzing the data with activity correlation imaging.

The protocol highlights two sophisticated staining procedures, both of which require cautious manipulation and practice in order to achieve satisfying and reproducible results. During the electroporation any injury of the animals has to be avoided, especially when positioning the electrodes into the nostrils. Optimally, no contact with the olfactory epithelium should occur. Note that the animals are still living after the electroporation procedure and their recovery time must be taken into account. If the staining remains too weak after one round of electroporation, which can happen depending on the types of dyes used, its intensity can be enhanced by increasing dye concentration in the nostrils. Since dextran-coupled molecules are transported via several mechanisms including slow axonal transport (at speed of 1-2 mm/day10) and passive diffusion, another alternative is to wait 48 hours after electroporation before sacrificing the animals. Alternatively, the electroporation may be repeated after one day of recovery.
Bolus loading is a critical step since the amount of dye entering the mitral cells is difficult to regulate and depends on various parameters like pipette tip size and location of the application. Monitoring the procedure under a confocal fluorescence microscope proves to be useful for adjusting the duration of dye application and thus generating similar staining results across preparations. Furthermore, previously electroporated tadpoles should be used to determine the best position for the dye application by identifying the position of the small cluster (comprising the &#947;-glomerulus). The most critical step during measurements is to avoid both shift and bleaching of the sample. The shift can be avoided by carefully positioning the Ringer flow under the microscope. As to limit the bleaching of the area of interest, the measurement time should be reduced to the essential.
Bolus loading staining with calcium-sensitive dyes only provides very limited contrast since healthy cells generally have low calcium levels and thus show weak basal fluorescence. Applying activity correlation imaging circumvents this limitation by generating contrast based on activity and highlights structures with similar calcium signals. This post-acquisition analysis method computes the correlation factor between the calcium signal of a selected region of interest (reference trace) and that of each individual pixel in the 3D volume. Therefore, the results obtained strongly depend on the activity pattern selected as reference trace. If the main focus is to visualize mitral cell innervation patterns, a reference signal derived from spontaneous neuronal activity is preferred, and choosing the most active mitral cells will produce the best results. For revealing the chemo- or thermo-sensitive networks of mitral cells, reference traces only containing responses to either histidine or cold Ringer should be selected. The selection of an entire glomerulus or mitral cell soma as region of interest may not always provide a clear reference trace, especially if the structures responding to the two different stimuli are lying on top of each other. In such a case, it is often useful to select a smaller area of the glomerulus or cell body as region of interest.
In the last decades, electroporation has been described as an efficient method to stain single or multiple cells11,12. Here it is used to specifically label olfactory receptor neurons. Dextran-conjugated molecules give the highest efficiency, and for non-calcium sensitive dyes, the range of selection is broad and covers the complete spectrum typically used in fluorescence microscopy13. However, calcium-sensitive dyes which are successfully electroporated into olfactory receptor neurons are at the moment limited to calcium-green dextran, and Fluo-4 dextran if still commercially available. Besides, the recordings primarily target superficial layers on the ventral surface of the olfactory bulb only, since the penetration depth of fast measurement techniques is limited. Two-photon imaging can partly overcome this limitation but often lacks speed and restricts the amount of selectable calcium-sensitive dyes further.
We described here a protocol for measuring temperature-induced activity in the olfactory bulb. The brain neuropil is scanned as a three dimensional volume to visualize the complex cellular networks involved in the olfactory processing of temperature. Measuring temperature-induced activity in the olfactory bulb has been very recently reported5 and requires a specifically customized procedure combining different techniques. A major asset of the techniques presented above is that hundreds of cells are imaged in three dimensions in a preparation where most of the olfactory system remains intact. These advantages put high demands on the staining techniques as well as the brain preparation and imaging. For example, cell electroporation and bolus loading hit large amounts of cells in the olfactory epithelium and bulb, and thus enable the visualization of complete cellular networks. Furthermore, the delivery of chemical indicators via bolus loading instead of genetically encoded fluorophores enables measurements in a potentially larger set of species. Other alternatives like bath incubation with AM dyes primarily work in slices which damage the olfactory bulb severely, leaving only a few hundred micrometers of intact tissue. In comparison, the whole mount preparation used in our protocol ensures for example that the bilateral innervation of the &#947;-glomerulus remains intact and the recordings are thus taken in a still operative system. Finally, the imaging itself is done by line-illumination microscopy allowing the acquisition of 3D volumes. Line-illumination microscopy is one of the confocal techniques providing the highest possible acquisition rates6 which are necessary to cover a large fraction of the olfactory bulb. Slower acquisition systems may be used but have the disadvantage that the size of the recorded volume must be reduced. In recent years, other methods for fast image acquisition have been developed and can be used as alternatives14,15. Nevertheless, line-illumination microscopy remains one of the easiest methods to gain both sufficient speed and resolution. Here follows some information as guidelines for selecting suitable imaging setups. Since calcium imaging is done within from thick brain preparations, the setup should provide decent confocality and the objectives should have numerical apertures of 1.0 or higher. For a reference point, the recordings taken with the line illumination microscope correspond to images taken with a standard laser scanning microscope with a pinhole size of 0.5-1 airy units. Fast acquisition speed is desirable. A volume with a thickness of 20 &#181;m covered by at least 5 layers, a lateral field of view of 100 &#181;m x 100 &#181;m and a pixel size of 0.5 &#181;m or smaller should be scanned at a minimum speed of 1 Hz per stack. Reducing the confocality can increase the amount of photons counted and thus allows for faster acquisitions if necessary, but has the drawback of recording more out-of-focus light. However, since such an approach increases the thickness of the optical slices, it can actually facilitate the tracing of dendrites through different z-planes after application of ACI6.
The necessary tools to extensively study temperature processing in olfactory bulb networks are presented herein. Temperature-induced activity is recorded in the first and second order neurons via calcium-sensitive dyes and signals both arriving and departing from the &#947;-glomerulus. Furthermore, the extent to which individual mitral cells process both chemical and temperature information can be assessed. Since the preparation leaves the olfactory bulb intact, the role of the bilateral innervation in the olfactory processing can be further studied. The procedure is also useful for revealing whether and how thermo- and chemoinformation is encoded in overlapping olfactory networks5. Finally, the techniques mentioned above are not limited to the study of the temperature responses in the olfactory bulb but can be applied for a more general evaluation of the olfactory system, especially the cellular processing networks in large three dimensional volumes. Bolus loading and activity correlation imaging are powerful tools to observe and compare the activity of dozens of neurons, making them applicable to different brain networks16.

Over the last years, temperature sensitivity has no longer been described as a somesthetic sense only, but also as a physiological function relevant for the olfactory system. In rodents, the main olfactory bulb receives input from the Grueneberg ganglion (GG), an organ in the nasal cavity, consisting of thermosensitive neurons. GG neurons respond to cool temperatures1 as well as to chemical stimuli, and their chemosensitivity is modulated by temperature fluctuations2. These observations suggest that the olfactory bulb may integrate chemical and temperature information collected at the nose. In order to explore this hypothesis, we present here a set of experiments enabling the detection of temperature responses in the olfactory bulb of non-transgenic animals, using the Xenopus laevis larva as a model. The organization of the olfactory system in these animals closely resembles that of mammals. The olfactory receptor neurons of premetamorphic X. laevis terminate in tufts, and make synaptic contacts with the dendrites of second-order neurons, the mitral cells. Pre- and postsynaptic fibers intermingle and form skein-like neuropil structures called glomeruli3. The abundant synapses of the glomerular layer represent the first processing center of olfactory information. Mitral cells further integrate the sensory input and convey it to higher olfactory areas.
We have developed a protocol combining electroporation of olfactory receptor neurons (ORNs) with calcium-sensitive and non-sensitive dyes followed by bolus loading of the postsynaptic network of glomeruli and mitral cells. The staining by electroporation of two spectrally distinct dyes loaded in the nasal cavities serves to single out the &#947;-glomerulus3 through its bilateral innervation by ORNs from both olfactory epithelia. Thus, the location of the &#947;-glomerulus is identified prior to further measurements. Subsequently, bolus loading4 with Fluo-8 acetoxymethyl (Fluo-8 AM) is carried out in a volume comprising the &#947;-glomerulus. Imaging calcium changes with fast confocal microscopy allows the visualization of temperature responses in the 3D neuropil surrounding the &#947;-glomerulus, a unique temperature-sensitive glomerulus in this system5. Mitral cells innervating this specific structure can also be identified by their Ca2+ signals responsive to induced temperature drops. Next, activity correlation imaging6 uses the specific Ca2+ traces of these cells to reveal the dendritic morphology of thermosensitive mitral cells. Alternating repeated applications of cold Ringer solution and chemical odorants in one measurement can be used to visualize the mitral cell networks for odor and temperature processing surrounding the &#947;-glomerulus and identify potential overlaps. To unambiguously assign the responses to either the chemical or the temperature stimulus, we constantly monitor temperature at the olfactory epithelium.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>1064040500</td><td></td></tr><tr><td>Potassium chloride</td><td>Merck Millipore</td><td>1049360250</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Merck Millipore</td><td>1023820250</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Merck Millipore</td><td>1058330250</td><td></td></tr><tr><td>D(+)-Glucose</td><td>Merck Millipore</td><td>1083371000</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td></td></tr><tr><td>HEPES</td><td>Merck Millipore</td><td>1101100250</td><td></td></tr><tr><td>Calcium Green 10 kDa Dextran</td><td>Thermo Fisher Scientific</td><td>C-3713</td><td></td></tr><tr><td>Dextran, Alexa Fluor 647</td><td>Thermo Fisher Scientific</td><td>D-22914</td><td></td></tr><tr><td>Dextran, Alexa Fluor 546</td><td>Thermo Fisher Scientific</td><td>D-22911</td><td></td></tr><tr><td>Fluo-8 AM</td><td>TEFlabs</td><td>203</td><td></td></tr><tr><td>MK571</td><td>Alexis Biochemicals</td><td>340-021-M005</td><td></td></tr><tr><td>MS-222</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>Pluronic acid F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td>powder</td></tr><tr><td>L-Histidine monohydrochloride monohydrate</td><td>Sigma-Aldrich</td><td>53370</td><td></td></tr><tr><td>DMSO</td><td>Merck Millipore</td><td>1029522500</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Electronic pipette</td><td>BrandTech</td><td>HandyStep Electronic Repeating Pipette</td><td></td></tr><tr><td>NiCr-Ni thermocouple</td><td>Greisinger Elektronik</td><td>GTF 300</td><td></td></tr><tr><td>Micropipette puller</td><td>Narishige</td><td>Model PC-10</td><td>two-step puller</td></tr><tr><td>Funnel applicator</td><td>(Custom-made)</td><td></td><td></td></tr><tr><td>Line-illumination microscope</td><td>(Custom-made)</td><td></td><td>otherwise, a commercially available spinning disk microscope</td></tr><tr><td>Objective W Plan-Apochromat 63X/1.0&amp;nbsp;</td><td>Zeiss</td><td>441470-9900-000</td><td></td></tr><tr><td>Objective W Plan-Apochromat 40X/1.0 DIC</td><td>Zeiss</td><td>441452-9900-000</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MATLAB</td><td>The MathWorks</td><td></td><td>from R2010b upwards</td></tr></tbody></table>

recording temperature-induced neuronal activity through monitoring calcium changes in the olfactory bulb of xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

The electroporation of olfactory receptor neurons (ORNs) was achieved with Alexa Fluor dyes or calcium indicators conjugated with Dextran molecules for anterograde labelling via active axonal transport. While the former dyes provide bright staining of the sensory neurons and their axon terminals branching in the glomerular layer of the bulb, the latter allow the measurement of neuronal activity in these cells (Figures 1 and 2). First, the position of the thermosensitive &#947;-glomerulus and its innervation pattern was visualized by electroporating Alexa Fluor 647 Dextran and Alexa Fluor 546 Dextran in the left and right olfactory epithelia, respectively (Figure 1A). Twenty-four hr after the procedure, the ORNs in the nostrils, the two olfactory nerves and the glomeruli in both hemispheres were visible under fluorescent microscopy. The different glomerular clusters were identifiable by their respective positions, notably the small cluster comprising the &#947;-glomerulus (Figure 1B). A small number of contralateral olfactory fibers ran through the contralateral olfactory bulb, crossed the anterior commissure and terminated in the ipsilateral &#947;-glomerulus (Figure 2A).
In order to record calcium responses of the presynaptic fibers of the &#947;-glomerulus, Calcium Green Dextran was electroporated in ORNs, according to the same procedure. Negative temperature jumps were induced at the nostril via the controlled release of ice-cold Ringer solution (0-1 &#176;C). A 3D volume comprising the &#947;-glomerulus was imaged under a fast line-illumination confocal microscope. &#916;Ts of -1 &#176;C were sufficient to trigger cold responses in the &#947;-glomerulus and its afferents, recognizable as reversible peaks in the &#916;F/F Ca2+ traces (Figure 2B, C).
Further experimental steps were carried out to measure cold-induced activity in the mitral cells connected to the &#947;-glomerulus via their ramifying dendritic endings. These postsynaptic fibers and the surrounding neuropil were effectively stained by bolus loading of the calcium-sensitive dye Fluo-8 AM, performed a few days after Alexa 647 Dextran had been electroporated into ORNs (Figure 3A, B). Mitral cells were filled with Fluo-8 AM, and the olfactory epithelium was stimulated twice according to the following paradigm: cold Ringer, histidine (10 &#181;M) and room-temperature Ringer, applied subsequently. Two reference traces were taken from regions of interest in the recorded volume, one responding exclusively to temperature drops, the other one, to histidine only. Activity correlation imaging (ACI)6 was computed based on the selected reference traces to visualize with high contrast the dendritic morphology of the postsynaptic networks corresponding either to the temperature or the histidine-responsive Ca2+ signal (Figure 3C, D). Finally, thermosensitive and chemosensitive maps were color-coded and overlaid on top of each other, showing how temperature and chemical information is conveyed from individual glomeruli to the olfactory second-order neurons (Figure 3E). For a description of the integration and processing of both types of information in shared olfactory networks, see Kludt et al.5
<img alt="Figure 1" src="/files/ftp_upload/54108/54108fig1.jpg" /> 
Figure 1: Overview of the ORN electroporation and bolus loading. (A) Olfactory receptor neurons in both nasal cavities of X. laevis larvae were electroporated with Alexa dyes or calcium-sensitive dyes coupled to Dextran molecules. The fluorescent indicators were transported anterogradely until the terminal axonal arborization. 24 hr after electroporation, the glomerular layer in both hemispheres showed fluorescent staining. (B) Schematic of the cellular organization of the olfactory bulb in one hemisphere. The glomerular layer spans the bulb in clusters: the medial, small, intermediate and lateral clusters. Olfactory information is transferred from the receptor neurons to mitral cells via excitatory synapses in glomeruli. Periglomerular cells and granule cells are inhibitory neurons modulating olfactory processing and encoding. The &#947;-glomerulus (cyan) was easily identified as the small neuropil where ipsilateral (red) and contralateral (orange) olfactory fibers merged. (C) Bolus loading was achieved in the vicinity of the &#947;-glomerulus to stain the post-synaptic neuropil consisting primarily of mitral cells and their dendritic trees branching considerably in the glomerular layer. <a href="https://www.jove.com/files/ftp_upload/54108/54108fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54108/54108fig2.jpg" /> 
Figure 2: Electroporation of ORNs reveals structural and functional connectivity. (A) Bilateral innervation of the &#947;-glomerulus by ipsilateral (green) and contralateral (red) olfactory receptor neurons (ORNs). Scale bar = 50 &#181;m. (B) The olfactory bulb after electroporation with the calcium-sensitive dye Calcium Green Dextran. The intermedial (IMC), medial (MC) and the small cluster (SC) are visible. The image is a maximum projection of 100 &#181;m-thick measurement volume. Scale bar = 50 &#181;m. (C) Close-up view of the small cluster. The recorded volume (12 &#181;m) is represented in a maximal projection. The image depicts the basal fluorescent level in gray and the color-coded &#916;F/F map as an overlay. The maximum response to stimulation with cold Ringer solution is plotted. The &#947;-glomerulus reacted strongly while the two neighboring glomeruli remain silent. The inset shows the &#916;F/F trace for the &#947;-glomerulus corresponding to the indicated region of interest. The blue bar represents the stimulus application. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54108/54108fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54108/54108fig3.jpg" /> 
Figure 3: Bolus loading and ACI dissociate thermosensitive and chemosensitive networks. (A) Axons of ORNs terminating in the small cluster were stained by electroporation with the non-calcium sensitive dye Alexa 647 Dextran. The dotted line outlines the &#947;-glomerulus. (B) Image of the same region as in (A) in the second measurement channel after bolus loading with the calcium-sensitive dye Fluo-8 AM. Some mitral cell somata were visible but the contrast was limited. Following the arrows, two response traces were plotted, which were used for activity correlation imaging (ACI). Blue, red and black bars below the traces depict the onset of application of cold Ringer, histidine (10 &#181;M) and room-temperature Ringer as negative control, respectively. The two Ca2+ traces were taken from different regions of interest of the measured volume. (C) The ACI result of the trace in (B) highlighting areas responding predominantly to cold Ringer. (D) The ACI result of the trace in (B) highlighting areas responding predominantly to histidine. (E) Overlay of the two ACI maps. Mitral cells responding to histidine and the innervated glomerulus (red) were easily distinguishable from thermosensitive mitral cells and the &#947;-glomerulus (cyan). All images of this figure are maximum intensity projections of a 28 &#181;m-thick volume. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54108/54108fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis at JoVE.com

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

recording-temperature-induced-neuronal-activity-through-monitoring

Research

JoVE Journal

Neuroscience

7.2K Views.  Georg-August-Universität Göttingen. Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose. 

Video: Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

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 ...

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Biology

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Developmental Biology

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Recording Neuronal Activity During Odor Plume Navigation with Miniscope Imaging

Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to monitor behavior and record Ca2+ transients in dorsal CA1 stratum pyramidale neurons in the hippocampus (dCA1) in mice navigating an odor plume in a 50 cm x 50 cm x 25 cm odor arena. An epifluorescence miniscope focused through a gradient-index (GRIN) lens imaged Ca2+ transients in dCA1 neurons expressing the calcium sensor GCaMP6f in Thy1-GCaMP6f mice. The paper describes the behavioral protocol to train the mice to perform this odor plume navigation task in an automated odor arena. The methods include a step-by-step procedure for the surgery for GRIN lens implantation and baseplate placement for imaging GCaMP6f in CA1. The article provides information on real-time tracking of the mouse position to automate the start of the trials and delivery of a water reward. In addition, the protocol includes information on using an interface board to synchronize metadata describing the automation of the odor navigation task and frame times for the miniscope and a digital camera tracking mouse position. Moreover, the methods delineate the pipeline used to process GCaMP6f fluorescence movies by motion correction using the NorMCorre algorithm followed by identification of regions of interest with EXTRACT. Finally, the paper describes an artificial neural network approach to decode spatial paths from CA1 neural ensemble activity to predict mouse navigation of the odor plume.

Author Spotlight: Understanding Processing of Olfactory and Spatial Information by Brain with Real-Time Behavioral Analysis

This protocol investigates the brain-behavior relationship in hippocampal CA1 in mice navigating an odor plume. We provide a step-by-step protocol, including surgery to access imaging of the hippocampus, behavioral training, miniscope GCaMP6f recording and processing of the brain, and behavioral data to decode the mouse position from ROI neural activity.

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

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