This work is supported by FEDER Funds through the Algarve Regional Operational Program - CRESC Algarve 2020 and by National Funds through FCT - Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia under the ALG-01-0145-FEDER-30262 project, and FCT projects PTDC/BIA-BMA/30262/2017, UID/Multi/04326/2019 and DL57/2016/CP1361/CT0041 (awarded to ZV).

Animal maintenance and experimentation was carried out in certified experimental facilities and followed Portuguese national legislation (DL 113/2013) under a &#39;group-1&#39; license by the Veterinary General Directorate, Ministry of Agriculture, Rural Development and Fisheries of Portugal. As this protocol involves animal handling, it has to be approved by the local and/or national body that regulates welfare of animals used in scientific experiments, in addition researchers need to have the appropriate training and licenses to carry out such procedures.
1. Stimulus preparation
NOTE: Most fish have highly sensitive olfactory system, thus great care must be taken when preparing the olfactory stimuli to be used in the experiment. The glassware used to make up the stimuli should be washed in 5% bleach (sodium hypochlorite), rinsed thoroughly with tap water and dried. Immediately prior to use rinse the glassware thoroughly with seawater (the same water used to make the stimulus dilutions). Take care that none of this water comes into contact with bare skin.
<ol>
	<li>Make 100 mL of 10-2 M L-glutamine, L-leucine and L-serine; store 1 mL aliquots of each at -20 &#176;C until use.</li>
	<li>On the day of the experiment, prepare from these aliquots, 10-3 M to 10-7 M solutions (in steps of x10 dilution) using both control and high CO2 seawater. 
	NOTE: L-serine (10-3 M) will be used as a positive control, or standard. Water used to make up the dilutions of the stimuli, and treated in exactly the same way as the stimuli but without addition of any odorant, will be used as the negative control or blank.</li>
</ol>
2. Preparation of control and high CO2&#160;water

<ol>
	<li>Prepare control water by collecting 1 L of charcoal-filtered seawater.
	<ol>
		<li>Using a pH probe, check the pH; it should be around 8.2. If not, bubble with atmospheric air until this pH is reached.</li>
		<li>Using an alkalinity titrator measure the alkalinity of the water.</li>
		<li>Measure the water temperature and salinity.</li>
	</ol>
	</li>
	<li>Prepare high CO2 water by filtering 1 L of sea water, then bubble CO2 until the desired pH is reached.
	<ol>
		<li>Using a pH probe, check the pH; it should be around 7.7.</li>
		<li>Using an alkalinity titrator, measure the alkalinity of the water.</li>
		<li>Measure the water temperature and salinity.</li>
	</ol>
	</li>
	<li>Determine CO2 pressure in both control and high CO2 water using a software designed to calculate CO2 parameters in water (e.g., CO2Calc software13).
	<ol>
		<li>In the input window add the values of water pH, temperature, salinity and total alkalinity (Figure 1).</li>
		<li>Select the constants, units and scales (see the recommended values in Figure 2).</li>
		<li>Press the button process to determine CO2 pressure. 
		NOTE: Figure 3 shows an example of a result sheet.</li>
	</ol>
	</li>
</ol>
3. Preparation of the fish
NOTE: A seabream of 200&#8722;400 g is used in this protocol.
<ol>
	<li>Anesthetize the fish byimmersion in aerated natural seawater containing MS222 (ethyl-3-aminobenzoate methanesulfonate salt). When response to a tail pinch has stopped, inject into the flank muscle the neuromuscular blocker gallamine triethiodide (10 mg&#183;kg-1 in physiological saline). 
	NOTE: The concentration of anesthetic used varies among species; for a seabream of 200&#8722;400 g, use 200 mg&#183;L-1 buffered with 400 mg&#183;L-1 NaHCO3.</li>
	<li>Place the anesthetized fish on a cushioned support. The exact shape and size depend on the model species; for seabream (200&#8722;400 g), use a padded V-shaped support, made in house.</li>
	<li>Place a silicon tube (diameter = 10 mm) in the mouth, connect the tube to a submersible pump in a reservoir of anesthetic-containing, aerated seawater, and pump water over the gills at ~100 mL&#183;min-1&#183;kg-1. 
	NOTE: The size of the silicon tube used will depend on the size of the fish.</li>
	<li>Insert the earthing pin into the flank muscle and connect it to the head-stage of the amplifier).</li>
	<li>Cover the fish with damp cloth (or paper towel) with only the head exposed, ensuring that the covering does not impede the exit of water from the gills. 
	NOTE: The eyes can be covered with pieces of damp paper/cloth or black plastic.</li>
	<li>Position the tube of the stimulus-delivery system, i.e., the glass tube connected to a supply of background seawater, into the nostril. 
	NOTE: Micro-hematocrit tubes can be used (length = 75 mm, ID = 1.15 mm, OD = 1.55 mm); these can be pulled to a finer point on an electrode puller for use with smaller fish. It is important to ensure that the olfactory epithelium is kept wet during the surgery (described below).</li>
	<li>Expose the olfactory nerves by removing the skin and bone of the skull between the eyes (the olfactory nerves usually run together between the eyes) with the aid of a dental (ideally) or hobby (e.g., Dremel) drill or jeweler&#39;s polisher (with dental drill-bits) under a dissecting microscope (inside a Faraday cage).
	<ol>
		<li>In the seabream, remove the part of the skull immediately above the eyes, with a circular saw, from just anterior to the eyes to just posterior to them. Then, using a drill bit, remove the bone between the eyes; the olfactory nerves lie between the eyes.</li>
	</ol>
	</li>
	<li>Once sufficient bone has been cleared, remove the fat and connective tissue overlying the nerves using fine forceps; take care to not damage the nerves or puncture any blood vessels. 
	NOTE: Experience will help to refine the dissection; the smaller the dissection, the more stable the preparation will be. However, sufficient tissue must be cleared; for the unexperienced, when the olfactory bulbs are just visible, clear slightly more anteriorly to expose the part of the nerves as they join the bulbs to allow the correct positioning of the electrodes.</li>
	<li>Clean the electrodes prior to use by connecting them to negative pole of a 3V DC source (e.g., two AA batteries in series) and placing the tip in physiological saline (or seawater diluted 1:3 in freshwater) for 20&#8722;30 s; a steady stream of small bubbles should be seen coming from the tip.</li>
	<li>Once the olfactory nerves are exposed, insert the recording electrodes (held on micro-manipulators) in a position that gives a maximal response to the standard (e.g., 10-3 M L-serine), and a minimal response to the blank. Use parylene-coated tungsten electrodes (Table of Materials) connected to the head-stage of an alternating current (AC) preamplifier. 
	NOTE: In the seabream, the strongest responses to amino acids are usually seen with the electrodes placed in the lateral side of the nerve, close to where it joins the olfactory bulb. This may hold true for other species, as the glomerular organization of the bulb is broadly similar among species. However, experience is always the best teacher.</li>
</ol>
4. Electrophysiological recording
NOTE: As with most electrophysiology, multi-unit recording needs to take place within a Faraday cage. However, extracellular recording does not usually require an anti-vibration table; most movement will come from the fish. Nevertheless, a strong, stable table is required with a metal surface to secure the magnetic bases of the micro-manipulator stands.
<ol>
	<li>Set up a stimulus delivery system to allow the rapid switching from clean background water to stimulus-containing water, e.g., by using a solenoid-operated three-way valve. Connect the common outlet to the tube carrying water to the olfactory rosette, and place one line in a seawater reservoir and the other in the test solution. 
	NOTE: When the valve is switched (by passing DC current), the water flow switches from background water to that containing the odorant. The stimulus should be given for long enough to see a clear peak in the integrated response, followed by a period of accommodation; the time used in the current protocol is 4 s, but longer time may be necessary depending on the species.</li>
	<li>Connect the valve driver to the trigger of an analogue-digital converter (e.g., Digidata); when the valve is switched from background to stimulus-containing line, this will start the recording of the data. Configure the software to start recording at the trigger event and continue for a predetermined period (e.g., 10 s). 
	NOTE: Ten seconds should be enough, but this can be shortened or lengthened, depending on the experimental question.</li>
	<li>Check the stability of the preparation by testing (recording and measuring the amplitude of the integrated response) repeatedly with the standard, 10-3 M L-serine in this case, and allowing 1 min to elapse between successive stimuli. 
	NOTE: Depending to some extent on species and odorant, the responses should have an amplitude within 10% of each other (as a rule of thumb), and should have a rapid onset, rise to maximum activity, and return to baseline after the stimulus is absent (Figure 4).</li>
	<li>Record the olfactory nerve responses to amino acids in control sea water (from the lowest to the highest concentration) and allow 1 min to elapse between successive stimuli. 
	NOTE: It is possible that, for some species and/or some odorants, more time is necessary. But for amino acids and seabream, 1 min is sufficient.</li>
	<li>Record the response to 10-3 M serine and a control water blank solution.</li>
	<li>Change the background water from control seawater to high CO2 seawater, by placing the background line into the bottle with high CO2 seawater. 
	NOTE: It is advisable to insert another hematocrit tube (or equivalent) into the end of the stimulus and background lines to avoid touching the water and ensure the end of the tube remains in the water.</li>
	<li>Prior to testing the response of the olfactory nerve to amino acids in high CO2 seawater, condition the olfactory epithelium with high CO2 water by following the high CO2 water over the olfactory epithelium for a few minutes. 
	NOTE: Experience has shown that, for seabream, 5 min is sufficient.</li>
	<li>Record the olfactory nerve responses to amino acids in high CO2 seawater (from the lowest to the highest concentration).</li>
	<li>Record the response to a high CO2 water blank solution.</li>
	<li>Record the response to 10-3 M serine and a control water blank solution. 
	NOTE: The raw signal (nerve activity) should be filtered (low pass around 2,000&#8722;5,000 Hz, high pass 50&#8722;300 Hz) and passed to an analogue-digital converter (Table of Materials). For easier quantification of nerve activity, the raw signal can also be integrated using a leaky integrator (Table of Materials) and passed to the analogue-digital converter, and from there, both raw and integrated signals to a computer running the appropriate software (e.g., Axoscope).</li>
</ol>
5. Data analysis
<ol>
	<li>Subtract the amplitude of the integrated response to the blank (in mV) from the amplitude of the integrated responses to all stimuli.</li>
	<li>Normalize responses to stimuli by dividing the amplitude of the previous response to the standard (10-3 M serine); this reduces inter- and intra-fish variability.</li>
	<li>Calculate the thresholds of detection by linear regression of the concentration-response curves (on a semi-logarithmic plot), according to the formula log(N + 1.5) = alog C + B, where C is the molar concentration, N is the normalized response amplitude, and a and b are constants7,14. 
	NOTE: The threshold of detection is then the value for x where y = 0.1761 (i.e., log 1.5; N = 0); the concentration above which a response will be seen (i.e., the fish can smell it). Some odorants evoke sigmoidal concentration-response curves when plotted semi-logarithmically (e.g., calcium15,16; in this case, the normalized data can be fitted to a three-parameter Hill plot which will give the maximum response amplitude and the EC50 (i.e., [odorant] that gives half-maximum response; also a measure of sensitivity).</li>
	<li>Compare the thresholds of detection and/or the maximum response amplitude and the EC50 of stimuli tested in control water and those tested in high CO2 water.</li>
</ol>

The authors have nothing to disclose.

The current study describes the use of multi-unit (extracellular) recording from the olfactory nerve of the seabream (S. aurata), a marine sparid of great importance in aquaculture. However, this experimental approach can be broadly applied to other fish; the surgery and exact placement of electrodes will clearly depend on the anatomy of the olfactory system, and the choice and concentration of anesthetic may depend on the species under study. For example, the olfactory nerve of the goldfish (Carassius auratus) is short; in this case, recording the EEG from the olfactory bulb would be easier. The choice of odorant may also depend, to some extent, on the species. The current study used amino acids. As far as the authors are aware, all fish species investigated to date have olfactory sensitivity to amino acids1,18. This sensitivity has been implicated is diverse processes such as food location, chemical communication and recognition of natal waters19,20,21,22,23. However, the sensitivities of different species are, broadly speaking, rather similar and do not depend on lifestyle or habitat. They are also well-defined molecules and are cheaply and easily available. These reasons make them ideal test stimuli for studies on olfaction in fish, especially those investigating the effects of anthropogenic disturbances (e.g., acidification or pollution), where results can be easily compared across species24.
Depending on the species in question, preparations for multi-unit recording can remain stable for several hours; the amplitude of response to the internal standard (10-3 M L-serine in the current study) should not vary by more than 10% between successive tests. Any significant deviation from this rule of thumb could be due to: (i) movement of the fish, and therefore displacement of the electrodes and/or nose-tube; (ii) contamination of the water, for example, by coming into contact with the experimenter&#39;s hands (especially if lower concentrations of a given odorant give larger responses than higher concentrations); or (iii) deterioration of the health of the preparation). In case (i), the fish should be checked for having moved; if so, reposition it, and add more anesthetic to the water and/or give another dose of gallamine triethiodide. Allow 5 min and retest the standard. If the response is still smaller, then reposition the electrodes and/or nose-tube until a sufficiently large response is recorded. In case (ii), simply remake a fresh dilution series of the odorant, using clean glassware and water. In case (iii), check that the flow of water over the fish&#39;s gills is adequate, that the water is flowing over the gills (i.e., exiting via the opercula, rather than the mouth), and the water is well-aerated. Different fish species have widely different temperature preferences; ensure that the laboratory) temperature (and that of the water in contact with the fish) is as close to the temperature that the fish are kept at as possible. Ensure, too, that the fish are not stressed, and avoid moving them (even from one tank to another) for at least a week prior to recording. Electrical noise is, of course, the bane of an electrophysiologist&#39;s life; however, the current article is not the appropriate medium to discuss how to overcome/reduce this. Nevertheless, &#39;The Axon Guide&#39; (available freely as a pdf for download from the manufacturer&#39;s website) is a source of practical advice on noise minimization. Once a large, stable response is evoked by the standard stimulus, and a concentration series gives a concentration-dependent increase in amplitude, with minimal response to the blank, recording responses to test stimuli can begin. Some authors give the same stimulus three times, and calculate the arithmetic mean for subsequent data analysis. However, these are technical replicates, and this approach will increase the time a recording session takes by three-fold. The current authors prefer to test a given odorant once, but always part of a concentration-response curve. This not only allows the calculation of the threshold of detection or EC50 (as described), but also ensures that concentrations close to those that the fish would experience in its natural environment are tested (this is not always known). Furthermore, any outlier responses, due to contamination for example, are easier to spot; these can then be repeated using a freshly made sample if necessary.
Multi-unit recording from the olfactory nerve may be invasive, but it is more sensitive than the EOG when recorded in seawater7, as it is independent of external salinity. It can therefore be used to assess olfactory sensitivity to odorants, such as calcium and sodium, changes in the concentrations of which would also affect conductivity and consequently voltages recorded15. As an estimate of the number of ORNs responding to a given odor (i.e., action potentials travelling along ORN axons from the olfactory epithelium to the bulb), it represents a raw, unprocessed signal (initial processing of olfactory input begins in the bulbs). Therefore, it is a better parameter to assess the direct effects of pollutants, such as heavy metals, and environmental changes, such as pH, on the olfactory system than the EOG or EEG24,25. Recording from the olfactory bulb in seawater with high PCO2 (and therefore low pH) may be affected by central effects of pH on neural processing; the &#39;GABAA receptor theory&#39; of ocean acidification26, whereby reduction in water pH causes a redistribution of Cl- and HCO3- ion in the CSF and a consequent shift of GABAergic activation from inhibitory (hyperpolarizing) to excitatory (depolarizing). Furthermore, in such studies, it is important to assess the effects of acidification or pollutants using odorant concentrations similar to those the fish is likely to encounter in its natural environment. For amino acids, this is in the nano to micromolar range27,28,29; close to the threshold of detection of these compounds in fish1,18. Estimation of the threshold of detection for a given odorant can give some idea of the importance and/or biological role of the olfactory sensitivity. For example, the sea lamprey (Petromyzon marinus) has high olfactory sensitivity to specific bile acids released by larvae down to a threshold of 10-13 M30; this sensitivity allows adults to locate and identify suitable spawning grounds, and therefore act as a migratory pheromone over long distance. Similarly, ripe female sea lamprey have high olfactory sensitivity to spermine (threshold 10-14 M), a polyamine released in the milt by males, which then attracts them to the nests of spermiating males31. Other fish also have olfactory sensitivity to polyamines32,33,34,35, but not with sufficiently low thresholds of detection to support a similar pheromonal role; instead, avoidance of decaying fish is suggested. Nevertheless, with such high olfactory sensitivities, it is possible to imagine that a slight reduction in sensitivity (i.e., increase in threshold), even when the response amplitude is not dramatically reduced, could cause severe problems for fish24.
When plotted semi-logarithmically, concentration-response curves to odorants can be exponential, linear or sigmoidal18. In the case of amino acids, such semi-logarithmic concentration-response curves are either linear (i.e., logarithmic), sigmoidal or power functions7. That no saturation of the response is seen (i.e., no plateau in the concentration-response curve, even at supra-environmental concentrations) is probably due to several receptors binding to individual amino acids, depending on their concentration, rather than each amino acid binding to a specific receptor; as the concentration of a given amino acid increases, more receptors are able to bind it and therefore respond. Nevertheless, fish can distinguish between mixtures of amino acids36,37,38,39; this is likely due to combinatorial patterns of activity evoked in the olfactory bulbs12,40; the axons of all ORNs expressing the same receptor protein terminate at the same glomeruli in the olfactory bulbs41,42, and one amino acid can activate more than one glomerulus.
However, highly specific odorants, such as pheromones, may evoke sigmoidal or quasi-sigmoidal concentration-response curves43,44. The inference, although not empirically tested, is that these olfactory responses are due to highly specific receptors which bind the pheromone molecule and little else. Therefore, above a given concentration, all receptors are occupied, and further increases will evoke no further responses in other ORNs. Therefore, these data can be fitted to a three-parameter Hill plot, and the maximal response, EC50 and Hill co-efficient can be calculated15,45,46. This can give valuable information, such as apparent affinity and apparent receptor number, that linear or exponential concentration-response curves cannot give.

Fish rely heavily on olfaction for many aspects of their lives including finding food, avoiding predators, assessing potential mates and migration, among others1,2,3. Therefore, assessing olfactory sensitivity in fish (What do they smell? How sensitive are they to these compounds?) is vital to fully understand these processes. Furthermore, anthropogenic effects on the environment, such as ocean acidification and pollution, may have profound effects on the olfactory system, even at sublethal levels, because it is necessarily in intimate contact with the surrounding water4. In vivo electrophysiology is the experimental approach of choice to assess olfactory sensitivity in fish. Three main techniques are available: the electro-olfactogram (EOG), the electro-encephalogram (EEG) recorded from the olfactory bulb, and multi-unit recording from the olfactory nerve5.
The EOG is the most widely used of these three6. It is a direct current (DC) field potential recorded above the olfactory epithelium and is believed to be the summed generator potentials of those olfactory receptor neurons (ORNs) responding to a given odorant. However, as it is recorded in the water, rather than inside the fish, the amplitude of response is not only dependent on the signal generated by the fish, but also on the conductivity of the surrounding water; the higher the conductivity (or the lower the resistance), the lower will be the amplitude. This may mean that the EOG is a less sensitive method in seawater than freshwater7.
The EEG recorded from the olfactory bulb is also widely used in investigation of olfaction in fish. However, the olfactory bulb is the first-order processing center for olfactory sensory input8; it is highly organized into glomeruli, and consequently the response recorded depends heavily on the position of the recording electrodes. For example, the input from ORNs detecting amino acids is processed by glomeruli in the lateral region of the olfactory bulbs, whereas that from conspecific-derived chemicals is directed to the medial region9,10,11,12. Pheromonal input may be directed to highly localized glomeruli within the olfactory bulb. Depending also on the anatomy of the species in question, the ideal recording position for a given odorant may not be easily accessible.
Multi-unit recording from the olfactory nerve circumvents the main problems with the EOG and EEG outlined above. As it records actions potentials passing down the axons of the ORNs from the epithelium to the bulb, it is a primary sensory signal. And as it is recorded inside the fish, the amplitude of response is independent of external salinity. Nevertheless, of course it has some disadvantages. Firstly, depending on the anatomy of the species, more extensive surgery is required to expose the olfactory nerve than for the EOG. Secondly, because the signal is smaller than the EOG, it requires slightly more sophisticated, and therefore expensive, equipment. A general description of other experimental approaches is given by John Caprio5. The aim of this article is to outline how to record extracellular multi-unit responses from the olfactory nerve of the seabream (Sparus aurata) in vivo to amino acid odorants as an example of this technique, and how to identify, and overcome, some of the more common problems encountered in such an experiment.

<table><tbody><tr><td>AC pre-amplifier</td><td>Digitimer Ltd (Welwyn Garden City, UK)</td><td>NL104</td><td>Neurolog pre-amplifier specifically designed for this type of recording.</td></tr><tr><td>Digidata</td><td>Molecular Devices, LLC. (San Jose, CA, USA)</td><td>1440A</td><td>Analogue-digital converter.</td></tr><tr><td>EMG Integrator</td><td>Digitimer Ltd (Welwyn Garden City, UK)</td><td>NL703</td><td>Leaky&#039; electrical integrator to integrate raw activity of the nerve.</td></tr><tr><td>Faraday cage</td><td>Made in-house</td><td></td><td>To reduce electrical noise.</td></tr><tr><td>Filter</td><td>Digitimer Ltd (Welwyn Garden City, UK)</td><td>NL125/6</td><td>Filter module for electrophysiological recording.</td></tr><tr><td>Gallamine triethiodide</td><td>Sigma-Aldrich (Portugal)</td><td>G8134</td><td>Neuromuscular blocker</td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich (Portugal)</td><td>G3126</td><td>Amino acid used as odorant</td></tr><tr><td>L-leucine</td><td>Sigma-Aldrich (Portugal)</td><td>L80000</td><td>Amino acid used as odorant</td></tr><tr><td>L-serine</td><td>Sigma-Aldrich (Portugal)</td><td>S4500</td><td>Amino acid used as odorant</td></tr><tr><td>Metalic base-plate</td><td>Any</td><td></td><td>Provides base for micro-manipulators.</td></tr><tr><td>Micro-hematocrit tubes</td><td>Any</td><td></td><td>To position water supply to the olfactory epithelium</td></tr><tr><td>Micro-manipulators</td><td>Narishige International Ltd (London, UK)</td><td>M-152</td><td>Position electrodes</td></tr><tr><td>MS222 (ethyl-3-aminobenzoate methanesulfonate salt)</td><td>Sigma-Aldrich (Portugal)</td><td>E10505</td><td>Anesthetic</td></tr><tr><td>pH probe</td><td>Hanna instruments (P&amp;oacute;voa de Varzim, Portugal)</td><td>HI12302</td><td>Probe to measure pH of water.</td></tr><tr><td>Refractometer</td><td>Hanna instruments (P&amp;oacute;voa de Varzim, Portugal)</td><td>HI96822</td><td>Refractometer to measure water salinity</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich (Portugal)</td><td>746398</td><td>For saline solution</td></tr><tr><td>Solenoid valves</td><td>The Lee Co. (Essex, CT, USA)</td><td>LFAA1201618H</td><td>For switching between background water and stimuus solutions (no longer available)</td></tr><tr><td>Stereo-microscope</td><td>Zeiss, Leica, Olympus</td><td>Any suitable model.</td><td>For dissection and placement of electrodes.</td></tr><tr><td>Titrator</td><td>Hanna instruments (P&amp;oacute;voa de Varzim, Portugal)</td><td>HI84531</td><td>Titrator to measure water alkalinity, pH and temperature.</td></tr><tr><td>Tungsten micro-electrodes 0.1 M&amp;Omega;</td><td>World Precision Instruments (Hitchin, UK)</td><td>TM31A10</td><td>Extracellular electrodes.</td></tr><tr><td>Valve Driver</td><td>Made in-house</td><td></td><td>12 V DC source for operating solenoid valves.</td></tr><tr><td>Water pump (submersible)</td><td>Any</td><td></td><td>To supply anesthetic-containing water to the gills of the fish.</td></tr></tbody></table>

extracellular multi-unit recording from the olfactory nerve of teleosts

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory sensitivity in high PCO2/low pH water. To assess the effects of ocean acidification, or olfactory sensitivity in marine fish in general, we propose that extracellular multi-unit recording from the olfactory nerve is the method of choice. Although invasive, it is sensitive, robust, reproducible and independent of external salinity (unlike the electro-olfactogram [EOG], for example). Furthermore, it records a primary sensory input into the CNS, prior to any central processing. We show that this method can show a reduction in olfactory sensitivity that is both temporary and odorant-dependent, using a range of amino acids to construct concentration-response curves and calculate the thresholds of detection.

A typical response to the positive control (10-3 M L-serine; Figure 4A) and negative control (blank; Figure 4B) recorded from the olfactory nerve of a seabream is shown in Figure 4. In the presence of the stimulus (black horizontal bar; in the olfactory cavity, in contact with the olfactory epithelium), note the rapid increase in activity (reflected in the upward deflection of the integrated signal) to a peak within about one second of stimulus onset, followed by a period of accommodation (while the stimulus is still present), and a return to baseline activity once the stimulus has ended. The absolute amplitude of the response is highly dependent on electrode position; if a low amplitude response is recorded, try changing the electrode positions. A slower rise to peak activity may be due to the tube carrying the stimulus-containing water to the olfactory epithelium being placed too far away from the epithelium; try moving the nose-tube closer to (but not touching) the epithelium. Note that, in contrast, the blank evokes little or no response. A significant positive response (i.e., increase in activity) to the blank may indicate contamination of the water used to make the dilutions of the stimuli; making fresh dilutions with clean water (and glassware) should resolve this. If not, a more thorough cleaning of the water system (including activated charcoal filters) may be necessary. A negative response (i.e., decrease in activity) may indicate a slight change in flow rate when the valve is switched due to, for example, a blockage in the valve.
A typical concentration-response curve (plotted semi-logarithmically), in this case to L-leucine (10-7 M to 10-3 M), is shown in Figure 5A. Note that increasing concentrations of the odorant evoke increasingly large increases in activity, and therefore, in the amplitude of the integrated responses. A plot of the normalized data, and their corresponding linear regression, is shown in Figure 5B. The estimated threshold of detection can be calculated from the value of x when y = 0.1761 (i.e., log1.5; where N = 0). In this case, this value is -7.48; that is, the calculated threshold for L-leucine in this fish is 10-7.48 M. The exponent &#945; can similarly be estimated from linear regression of the normalized data on a log-log plot; logN = &#945;log[odorant] + constant. The factor &#947; then gives the increase in odorant concentration required to increase the response amplitude by one log unit; that is, it is an estimate of the steepness of the concentration-response curve17. In this example, &#945; = 0.277 and &#947; = 3.61; therefore, to increase the response amplitude ten-fold (i.e., one log unit; log10 = 1), the stimulus concentration needs to be increased by 103.61-fold (4,074-fold).
A typical sigmoidal concentration-response curve (Figure 6A) when plotted semi-logarithmically, in this case to L-glutamine, is shown in Figure 6B. A similar concentration-dependent increase in response amplitude is seen; however, at the higher concentrations, this increase becomes less so that the response amplitude reaches a maximum (Nmax). This allows the data to be fitted to a three parameter Hill equation: 
<img alt="Equation 1" src="/files/ftp_upload/60962/60962eq1.jpg" />
This way, the EC50 (the odorant concentration at which a 50% maximal response is evoked) and the Hill co-efficient (a measure of the steepness of the slope of the linear part of the sigmoidal curve) can be calculated.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60962/60962fig1.jpg" /> 
Figure 1: Software screenshot showing the input window from the program CO2Calc. Highlighted (red boxes) are the fields required for carbonate parameter calculation. <a href="https://www.jove.com/files/ftp_upload/60962/60962fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60962/60962fig2.jpg" /> 
Figure 2: Software screenshot showing the input window for the appropriate constants, units and scales. Values shown are recommended for conditions under which the described experiments were carried out; they may change. <a href="https://www.jove.com/files/ftp_upload/60962/60962fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60962/60962fig3.jpg" /> 
Figure 3: Software screenshot showing the results window. <a href="https://www.jove.com/files/ftp_upload/60962/60962fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60962/60962fig4.jpg" /> 
Figure 4: Typical multi-unit responses recorded extracellularly from the olfactory nerve of seabream in vivo in response to 10-3 M L-serine (A) and blank (B). Upper traces show the integrated responses and lower traces show the raw (nerve) signal. Stimuli were applied to the olfactory epithelium (horizontal bars). Note the rapid increase in activity during the 1 s of exposure, a peak in activity, followed by a period of accommodation (while the odorant was still delivered to the epithelium) and a return to baseline levels once the odorant delivery has ceased. Little or no increase in activity is seen following stimulation with water treated the same way as odorant dilutions, with the exception of adding any odorant (blank). <a href="https://www.jove.com/files/ftp_upload/60962/60962fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60962/60962fig5.jpg" /> 
Figure 5: Typical concentration-response curve for L-leucine recorded extracellularly from the olfactory nerve in vivo. (A) As the concentration of L-leucine applied to the olfactory epithelium (horizontal bars) increases from 10-7 M to 10-3 M, a concomitant increase in activity is seen in the nerve. Upper traces show the integrated responses and lower traces show the raw (nerve) signal. (B) Linear regression (R2 = 0.97) of normalized data plotted semi-logarithmically to calculate threshold of detection as the value for log[L-leucine] when log(N + 1.5) = 0.1761 (i.e., where N = 0). In this example, this value is -7.48; the estimated threshold of detection for L-leucine in this fish is therefore 10-7.48 M. <a href="https://www.jove.com/files/ftp_upload/60962/60962fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60962/60962fig6.jpg" /> 
Figure 6: Typical concentration-response curve for L-glutamine recorded extracellularly from the olfactory nerve in vivo. (A) As the concentration of L-glutamine applied to the olfactory epithelium (horizontal bars) increases from 10-7 M to 10-3 M, a concomitant increase in activity is seen in the nerve. Upper traces show the integrated responses and lower traces show the raw (nerve) signal. (B) Semi-logarithmic plot of normalized data fitted to a three-parameter Hill equation (R2 = 0.99). For this example, the calculated EC50 = 3.11 &#181;M, and the Hill co-efficient = 0.565). <a href="https://www.jove.com/files/ftp_upload/60962/60962fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts at JoVE.com

Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts

Extracellular multi-unit recording from the olfactory nerve is a sensitive, robust and reproducible method for assessing olfactory sensitivity in marine fish. It records primary sensory input and is independent of external salinity.

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory ...

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6.7K Views.  Centro de Ciências do Mar. Dit is de meest robuuste en gevoelige methode voor het beoordelen van reukgevoeligheid bij zeevissen, omdat het een zuiver zintuiglijk signaal registreert voorafgaand aan een verwerking door het CNS. De belangrijkste voordelen van deze techniek zijn de gevoeligheid en dat het gemakkelijk kan worden overgebracht naar een andere soort uit een andere omgeving onafhankelijk van externe zoutgehalte. Om het controlewater voor te bereiden, verzamelt u een liter met houtskool gefilterd zeewater en ge...

Video: Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

Neuroscience

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

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

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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.

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.

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

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.

Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts

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