This work was supported by an intramural grant from NIH-NICHD to M.S. We thank G. Dold and T. Talbot of the NIH-NIMH Instrumentation Core Facility for assistance with designing the tastant delivery system.

<ol>
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The authors have nothing to disclose.

The methods described here permit in vivo recordings from a relatively simple animal, Manduca sexta, to characterize the activity of multiple, randomly selected GRNs over long durations (for more than 2 h), before, during and after tastant delivery. These methods also allow the rapid, sequential delivery of multiple tastant stimuli with precise temporal control, advantages that are useful for studying neural mechanisms underlying tastant representation. This protocol has been used to study how the responses of GRNs to tastants are transformed when they are transmitted to their postsynaptic target neurons (e.g., in the SEZ) by monitoring GRNs simultaneously with monosynaptically connected interneurons 10. Additionally, these methods can be adapted to the experimenter&#39;s needs, allowing the execution of complex paradigms to study fundamental aspects of gustatory coding.
When beginning our studies, one technical problem we sometimes had to troubleshoot was the inability to detect spiking signals from maxillary nerve with the tetrode wires. Possible causes for this are diverse, as the dissection protocol is challenging, and some practice is necessary to obtain a good preparation. First, during the moth dissection the maxillary nerves are easy to damage, especially during the removal of the sheath surrounding the nervous tissue. Second, if the sheath is not removed fully, the tetrode wires may not be able to access the nerve. In both cases, starting a new preparation is often the easiest way to resolve these issues. Third, there may be a problem with the tetrode wires. This can be checked by measuring the impedance of the wires which should be ~270 k&#937; at 1kHz. If the impedance value is above ~300 k&#937;, electroplate the wires with gold to achieve the desired impedance (see reference 30). Fourth, a piece of equipment may be misconnected or misbehaving.
Another possible problem is that spiking signals are recorded but the neuron(s) appear unresponsive to the tastants. This could be because the recorded neurons are insensitive to the set of tastants delivered. Also, is important to keep in mind that in addition to axons of GRNs, the maxillary nerve also carries mechanosensory fibers. Thus, it is possible to record from mechanosensory neurons instead of, or in addition to, GRNs. However, the tastant delivery system is designed to provide a constant mechanical input throughout the experiment making it unlikely that responses to a tastant will be confounded by responses to the mechanical component of its delivery. Neurons that respond to some but not other tastants, or in different ways to different tastants, can be classified unambiguously as GRNs. We recommend using freshly diluted tastants to avoid variations in tastant concentration or composition owing to compound degradation or evaporation of the solvent. We also recommend cleaning the system regularly to avoid tubing contamination and/or obstructions.
Another possible technical problem is a disadvantageous signal-to-noise ratio. This problem can often be solved by rechloriding or adjusting the position of the bath ground electrode. Other solutions might require shielding and minimizing the length of each electrical connection in the apparatus.
Finally, it is important to note that correct analysis of data obtained using tetrode recordings requires careful spike sorting. We found that fully-automated methods are generally inadequate. We recommend becoming familiar with the spike sorting literature before analyzing tetrode data 10,29,31,32,33.
Alternatives to our dissection protocol can be used. Here, we described a dissection through the ventral part of the moth head, providing access to the maxillary nerves and SEZ, but it is also possible to access these structures by dissecting through the dorsal side. We found the dorsal side preparation is not optimal for making recordings from these gustatory structures due to their deep location, but this preparation does offer the advantage of enabling recordings from higher order structures such as the mushroom body, an area that has been associated with multi-sensory integration, associative learning and memory processing 34. We have focused on the use of tetrode electrodes to record from the maxillary nerve, but, as we illustrated, standard intracellular sharp electrodes can also be used for this purpose. In addition, both techniques can be combined to perform simultaneous recordings from multiple brain areas 10. The neuroscience literature offers many examples of invertebrate models that have proven to be powerful tools for revealing fundamental principles of sensory processing, such as olfactory coding, that apply to both insects and vertebrates 35,36,37,38,39. We hope our methods will lead to fundamental new insights about gustatory coding.

The gustatory and olfactory systems generate internal representations of chemicals in the environment, giving rise to perceptions of tastes and odors, respectively. These chemical senses are essential for eliciting numerous behaviors critical for the survival of the organism, ranging from finding mates and meals to avoiding predators and toxins. The process begins when environmental chemicals interact with receptors located in the plasma membranes of sensory receptor cells; these cells, directly or through interactions with neurons, transduce information about the identity and concentration of chemicals into electrical signals. These signals are then transmitted to higher order neurons and to other brain structures. As these steps progress, the original signal always undergoes changes that promote the organism&#39;s ability to detect, discriminate, classify, compare and store the sensory information, and to select an appropriate action. Understanding how the brain transforms information about environmental chemicals to best perform a variety of tasks is a basic question in neuroscience.
Gustatory coding has been thought to be relatively simple: a widely-held view posits that every chemical molecule that elicits a taste (a &#34;tastant&#34;) naturally belongs to one of the approximately five or so basic taste qualities (i.e. sweet, bitter, sour, salty and umami) 1. In this &#34;basic taste&#34; view, the job of the gustatory system is to determine which of these basic tastes is present. Further, the neural mechanisms underlying basic taste representation in the nervous system are unclear, and are thought to be governed by either a &#34;labeled line&#34; 2,3,4,5,6 or an &#34;across fiber pattern&#34; 7,8 code. In a labeled line code, each sensory cell and each of its neural followers responds to a single taste quality, together forming a direct and independent channel to higher processing centers in the central nervous system dedicated to that taste. In contrast, in an across fiber pattern code, each sensory cell can respond to multiple taste qualities so that information about the tastant is represented by the overall response of the population of sensory neurons. Whether gustatory information is represented by basic tastes, through labeled lines, or through some other mechanism, is unclear and is the focus of recent investigation 3,8,9,10,11,12. Our own recent work suggests that the gustatory system uses a spatiotemporal population code to generate representations of individual tastants rather than basic taste categories 10.
Here we offer 3 new tools to assist in the study of gustatory coding. First, we suggest the use of the hawkmoth Manduca sexta as a relatively simple model organism amenable to electrophysiological study of taste and describe a dissection procedure. Second, we suggest the use of extracellular &#34;tetrodes&#34; to record the activity of individual GRNs. And third, we suggest a new apparatus for delivering and monitoring precisely timed pulses of tastant to the animal. These tools were adapted from techniques our lab and others have used to study the olfactory system.
Insects such as the fruit fly Drosophila melanogaster, the locust Schistocerca americana, as well as the moth&#160;Manduca sexta,&#160;have for decades provided powerful resources to understand basic principles about the nervous system, including sensory coding (e.g., olfaction 13). In mammals, taste receptors are specialized cells that communicate with neurons through complex second-messenger pathways 1,14. It is simpler in insects: their taste receptors are neurons. Further, mammalian taste pathways near the periphery are relatively complex, featuring multiple, parallel neural routes, and important components are challenging to access, contained within small bony structures 15. Insect taste pathways appear to be simpler. In insects, GRNs are contained in specialized structures known as sensilla, located in the antenna, mouthparts, wings and legs 16,17. The GRNs directly project to the subesophageal zone (SEZ), a structure whose role has been thought to be mainly gustatory 17, and which contains second-order gustatory neurons 10. From there the information travels to the body to drive reflexes, and to higher brain areas to be integrated, stored, and ultimately to drive behavioral choices 16.
It is necessary to characterize peripheral taste responses to understand how taste information is propagated and transformed from point to point throughout the nervous system. The most commonly used method to directly monitor the neural activity of GRNs in insects is the tip-recording technique 12,18,19,20,21,22,23. This involves placing an electrode directly onto a sensillum, many of which are relatively easy to access. The tastant is included within the electrode, allowing one to activate and extracellularly measure neuronal responses of GRNs in the sensillum. But, because the tastant is contained in the electrode, it is not possible to measure GRN activity before the tastant is delivered or after it is removed, or to exchange tastants without replacing the electrode 20. Another method, the &#34;side-wall&#34; recording technique, has also been used to record GRNs activity. Here, a recording electrode is inserted into the base of a taste sensillum 24, and tastants are delivered through a separate glass capillary on the tip of the sensillum. Both techniques restrict recording from GRNs to a particular sensillum. Here, we suggest a new technique: recording from randomly selected GRN axons from different sensilla, while separately delivering sequences of tastants to the proboscis. Axon recordings are achieved by placing either sharp glass electrodes or extracellular electrode bundles (tetrodes) into the nerve that carries axons from GRNs in the proboscis to the SEZ 10. In Manduca, these axons traverse the maxillary nerve, which is known to be purely afferent, allowing the unambiguous recording of sensory responses 25. This method of recording from axons, allows, for more than two hours, stable measurement of GRN responses before, during and after a series of tastant presentations.
Here, we describe a dissection procedure for exposing the maxillary nerves together with the SEZ, which can allow one to simultaneously record the responses of multiple GRNs and neurons in the SEZ 10. We also describe the use of extracellular recordings of GRNs using a custom-made 4-channel twisted wire tetrode which, when combined with a spike sorting method, permits the analysis of multiple (in our hands, up to six) GRNs simultaneously. We further compare recordings made with tetrodes to recordings made with sharp intracellular electrodes. Finally, we describe a new apparatus for delivering tastant stimuli. Adapted from equipment long used by many researchers to deliver odorants in olfaction studies, our new apparatus offers advantages for studying gustation: improving upon previous multichannel delivery system such as those developed by St&#252;rckow and colleagues (see references 26,27), our apparatus achieves precise control over the timing of the tastant delivery while providing a voltage readout of this timing; and it allows the rapid, sequential delivery of multiple tastant stimuli 10. The apparatus bathes the proboscis in a constant flow of clean water into which controlled pulses of tastant can be delivered. Each tastant pulse passes over the proboscis and is then washed away. Tastants contain a small quantity of tasteless food coloring, allowing a color sensor to monitor, with precise timing, the passage of tastant over the proboscis.

<table><tbody><tr><td>&lt;strong&gt;Dissection and Specimen Preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Polypropylene tube, 15 mL Falcon</td><td>Fisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>Needle, Short bevel, 19 G x 1 - 1/2&quot;&amp;nbsp;</td><td>&amp;nbsp;MONOJET</td><td>888200144</td><td>For applying air to remove the hair from the moth.</td></tr><tr><td>Modeling Clay-Van Aken Plastalina&amp;nbsp;</td><td>DickBlick</td><td>33268</td><td></td></tr><tr><td>Petri dish -100 x 15 mm&amp;nbsp;</td><td>VWR International</td><td>89000-304</td><td></td></tr><tr><td>Pipette tip (1 - 200 &amp;micro;L)</td><td>USA Scientific</td><td>1111-0806</td><td></td></tr><tr><td>Razor blade</td><td>Techni Edge</td><td>TE05-071</td><td></td></tr><tr><td>22 AWG standard hookup wire</td><td>AlphaWire</td><td>1551</td><td>For inserting the proboscis into the pippete tip.</td></tr><tr><td>Batik wax</td><td>Jacquard</td><td>7946000</td><td></td></tr><tr><td>Electric waxer</td><td>Almore International</td><td>66000</td><td></td></tr><tr><td>Stereo Myscroscope</td><td>Leica</td><td>MZ75</td><td></td></tr><tr><td>Dumont #1 forceps (coarse)&amp;nbsp;</td><td>World Precision Instruments</td><td>500335</td><td>For removing fat and non-nervous tissue.</td></tr><tr><td>Dumont #5 titanium forceps (fine)&amp;nbsp;</td><td>World Precision Instruments</td><td>14096</td><td>For removing fat and non-nervous tissue.</td></tr><tr><td>Dumont #5SF forceps (super-fine)</td><td>World Precision Instruments</td><td>500085</td><td>For desheathing the nervous tissue.</td></tr><tr><td>Vannas scissors (fine)</td><td>World Precision Instruments</td><td>500086</td><td>For removing the cuticle.</td></tr><tr><td>Collagenase/Dispase</td><td>Sigma-Aldrich</td><td>11097113001</td><td></td></tr><tr><td>Epoxy</td><td>Permatex</td><td>84101</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Saline Perfusion System&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Extension set with rate flow regulator</td><td>&amp;nbsp;B Braun Medical Inc.</td><td>V5200</td><td></td></tr><tr><td>IV administration set with Y injection site</td><td>&amp;nbsp;B Braun Medical Inc.</td><td>V1402</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tastant Delivery System&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>White Translucent Nylon Tubing OD 1/4&quot;, ID 1/8&quot;</td><td>Small Parts Inc.</td><td>B001JJT4SA</td><td>Rigid tube that connects the four main elements of the system.</td></tr><tr><td>Soldering iron</td><td>Circuit Specialists</td><td>ZD200BK</td><td></td></tr><tr><td>Rotary tool-Dremel</td><td>Dremel</td><td>4200</td><td></td></tr><tr><td>Polypropylene mesh, hole size (hole size 0.1 x 0.13 cm)&amp;nbsp;</td><td>Industrial Netting</td><td>XN5170</td><td>For ensuring that the probosises of different animals are placed in the same location.</td></tr><tr><td>Pressurized 16-Channel perfusion system&amp;nbsp;</td><td>Bioscience Tools</td><td>PS-16H</td><td>For tastant delivery. This system includes pinch valves, tubing, manifold, solution cylinders, valve controller and fitting accessories.</td></tr><tr><td>Polypropylene tubing, ID 0.034&quot;, ID 0.050&quot;</td><td>Becton, Dickinson &amp;amp; Co</td><td>427421</td><td>Output tube from the perfusion system.</td></tr><tr><td>Pneumatic PicoPump</td><td>World Precision Instruments</td><td>SYS-PV820</td><td>For controlling the output channel of the perfusion system.&amp;nbsp;</td></tr><tr><td>Data acquisition software system, LabVIEW PCI-MIO-16E-4 DAQ card</td><td>National Instruments</td><td>LabVIEW 2011</td><td>To control the pico pump for tastant delivery and to record the signals from the color sensor.</td></tr><tr><td>Compulab 3 Manostat peristaltic pump</td><td>Sigma</td><td>P1366</td><td>For pumping water.</td></tr><tr><td>Silicone tubing, ID 1/16&quot; OD 1/8&quot;</td><td>Cole-Parmer</td><td>WU-95802-02</td><td>To connect the water source to the peristaltic pump tubing, and the outlet tube of the pump to the rigid tube of the delivery system.</td></tr><tr><td>Color sensor-digital fiber optic sensor</td><td>Keyence</td><td>&amp;nbsp;FS-V31M</td><td>For monitoring tastant delivery.&amp;nbsp;</td></tr><tr><td>Color sensor-reflective fiber unit</td><td>Keyence</td><td>FU35-FZ</td><td>To connect the color sensor device.</td></tr><tr><td>Dental periphery Wax</td><td>Henry-Schein Dental</td><td>6652151</td><td>To secure the proboscis into the rigid tube.</td></tr><tr><td>Two 3.7 L containers</td><td></td><td></td><td>To provide water to the system, and to recollect the water waste.</td></tr><tr><td>Fast green FCF&amp;nbsp;</td><td>Sigma</td><td>F7258</td><td></td></tr><tr><td>Dressing forceps 25.5 cm</td><td>WPI</td><td>500364</td><td>To introduce moth proboscis into the proboscis hole in the rigid tube.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Electrophysiology Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D.C. amplifier</td><td>Brown-Lee</td><td>440</td><td></td></tr><tr><td>Lamp</td><td>Schott</td><td>Schott Fostec Light Source DCR 2</td><td></td></tr><tr><td>Manual micromanipulator</td><td>Leica</td><td>micromanipulator</td><td>To precisely insert the tetrodes into the animal&#039;s brain. The manipulator has to allow fine and coarse movements in x, y and z axis.</td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ75</td><td></td></tr><tr><td>Vibration-isolation table (MICRO-g lab table)</td><td>TMC</td><td>63-541</td><td></td></tr><tr><td>Oscilloscope</td><td>Tektronix&amp;nbsp;</td><td>TDS2014</td><td></td></tr><tr><td>16-channle pre-amplifier and amplifier</td><td>16 Channel MA-800 Amplifier System&amp;nbsp;</td><td>B.E.S 2013</td><td></td></tr><tr><td>Computer</td><td>Dell</td><td>optiplex 780</td><td>The following are the minimum recommended requirements. RAM: 3.32 GHz, 3GB. Processor: Intel Core&amp;nbsp;2 Duo. Graphic card: integrated Intel GMA X4500.&amp;nbsp;</td></tr><tr><td>Data acquisition software system, LabVIEW PCI-MIO-16E-4 DAQ card</td><td>National Instruments</td><td>LabVIEW 2011</td><td>To control the pico pump for tastant delivery and to record the signals from the color sensor and electrode.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tastants&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>KAc</td><td>Sigma-Aldrich</td><td>P5708</td><td></td></tr><tr><td>LiCl</td><td>Sigma-Aldrich</td><td>L9650</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>73575</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td></td></tr></tbody></table>

new methods to study gustatory coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

The activity of GRNs can be recorded using an extracellular tetrode electrode before, during and after tastant delivery (Figure 6A and 6C). Figure 6A (middle and bottom panels) shows filtered (300-6,000 Hz) voltage traces recorded by each of the four wires placed in the maxillary nerve, where signals of different amplitudes reflecting action potentials (arrows) can be observed. A 1 s pulse of tastant (1 M sucrose) was delivered 2s after the beginning of the experiment; the onset and offset of the stimulus was monitored by the color sensor (Figure 6A, upper panel). The tastant induced spikes that can be observed in each of the four channels (Figure 6A, middle panel). GRNs can be identified and distinguished from mechanosensors (none shown here) when they respond to some tastants and not others 10.
To identify and isolate single neuron responses from the tetrode recordings, we performed off-line spike sorting using a set of custom functions based on Pouzat&#39;s 31 and Kleinfeld&#39;s 32,33 methods (these methods are described in these citations: 10,29). Figure 6B shows an example of spike sorting applied to the data shown in Figure 6A, in which three well isolated units were found.
Raster plots from Figure 6C depict responses of the three isolated units in Figure 6B to six different tastants (1 M sucrose, maltose and NaCl; 100 mM caffeine and 10 mM berberine and lobeline) delivered in sequence (4 trials/tastant are shown). As shown in Figure 6C, the recorded GRNs have different levels of baseline activity, ranging from silent (GRNs 1) to low or moderate (GRN 2 and 3). After tastant onset, GRNs show diverse activity patterns and exhibit different selectivity to the tastants. For example, GRN 1 responded only to sucrose, whereas GRN 2 responded to maltose and NaCl with burst of spikes and to lobeline with spiking only at the onset of the stimulus. In addition, some responses are locked to the timing of the stimulus (e.g. GRN 1 response to sucrose), whereas other responses outlast the duration of the stimulus (e.g. GRN 3 response to berberine) or contain both excitatory and inhibitory components (e.g. Figure 7 GRN 2 responses to NaCl and sucrose). For more information about the diverse sensitivities and activity patterns of GRNs see reference 10.
To validate the use of the tetrode and spike-sorting techniques to record GRNs from the maxillary nerve, we performed intracellular recordings from GRN axons in other animals by using conventional sharp glass electrodes (resistance of 80-120 M&#937;). We found that the activity patterns obtained by using intracellular recordings were similar to responses observed by using the tetrode technique (Figure 7). The voltage traces in the green box in Panel A were recorded intracellularly from GRN 1 during 5 consecutive trials of sucrose presentation, and the same responses are shown as raster plots in panel B. (Note that this type of response matches that obtained with tetrodes and shown in Figure 6C.) GRN 2, recorded with sharp intracellular electrodes, shows a broader response pattern.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55868/55868fig6v2.jpg" /> 
Figure 6: Representative Results from Maxillary Nerve Recordings with Extracellular Tetrodes. (A) Filtered (300-6,000 Hz) voltage traces recorded by each of the four wires at the maxillary nerve are shown (middle panel). A 1 s pulse of tastant was applied during the time period indicated by red shading in the middle panel. The onset and offset of the stimulus was monitored by the color sensor as indicated by the red voltage trace on the upper panel, and is denoted on the middle panel by the red-shaded area. The dotted horizontal lines denote +50 (top), 0 (middle) and -50 (bottom) &#181;V. An enlargement of the raw voltage traces corresponding to the area inside the vertical dotted lines is shown (bottom panel). Examples of spikes are indicated by the arrows. (B) An example of spike sorting applied to the data shown in panel A. The waveforms recorded at each of the four extracellular wires (1-4) are identified with three different GRNs (units 1-3) contributing to the recorded signals. Individual events (colored thin lines) and the mean (thick black line) are shown for the three units. A number of statistical criteria have to be considered to reliably identify independent units using the spike-sorting method (see references 10,29). (C) Raster plots representing responses of the three isolated units to a sequence of six different tastants (4 trials/tastant are shown). The time period of tastant delivery (1 s) is indicated by the red-shaded area. The concentrations of tastants were either 1 M (sucrose, maltose, NaCl), 100 mM (caffeine) and 10 mM (berberine and lobeline).&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/55868/55868fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55868/55868fig7.jpg" /> 
Figure 7: Intracellular Recordings from GRN Axons. (A, green box panel) Voltage traces recorded from a GRN with sharp glass intracellular electrodes (resistance of 80 - 120 M&#937;) placed into the maxillary nerve, elicited by 5 consecutive stimulations with 1 M sucrose (Suc). (B) Raster plots of the responses of two GRNs, including responses shown in panel A (green box panel), to two different tastants delivered in sequence (100 mM gray font color; 1 M black font color) recorded with sharp glass intracellular electrodes placed into the maxillary nerve (7 trials/tastant and 3 trials/water are shown). Tastant delivery time is indicated by red-shaded areas in both panels. The onset and offset of the stimulus was monitored by the color sensor, as indicated by the red voltage traces at the bottom of each panel. <a href="https://cloudflare.jove.com/files/ftp_upload/55868/55868fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about New Methods to Study Gustatory Coding at JoVE.com

New Methods to Study Gustatory Coding | Neuroscience | Video

New Methods to Study Gustatory Coding

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

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9.3K Views.  National Institutes of Health (NIH).  Wij presenteren drie nieuwe methoden om gustatorische codering te bestuderen . Met behulp van een simpel dier beschrijven de mot Manduca sexta ( Manduca ) een dissectieprotocol, het gebruik van extracellulaire tetrodes om de activiteit van meerdere gustatorreceptorneuronen op te nemen en een systeem voor het leveren en bewaken van nauwkeurig ingestelde pulsen van tastanten. 

Video: New Methods to Study Gustatory Coding

Psychophysical Tracking Method to Measure Taste Preferences in Children and Adults

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to adulthood. The method, which is identical for children, adolescents, and adults, is of short duration (&#60; 15 min), does not rely on sustained attention or place demands on memory (which would yield spurious age differences), and minimizes the impact of language development, making this method amenable to the cognitive limitations of pediatric populations. In this whole-mouth tasting method, subjects are asked to taste (without swallowing) pairs of solutions of different sucrose concentrations and to point to the solution they prefer. Each subsequent pair contains the participant&#39;s preceding preferred concentration and an adjacent stimulus concentration. The procedure continues until the subject chooses either a given concentration of sucrose when paired with both a higher and a lower concentration, or the highest or lowest concentration two consecutive times. Subjects are prevented from reaching response criteria on the basis of first or second position bias by the two-series design of the method, which counterbalances the order of solution presentation within each pair between the series (the weaker concentration is presented first in Series 1, second in Series 2). The geometric mean of the two sucrose concentrations chosen in Series 1 and 2 is an estimate of the participant&#39;s most preferred level of sucrose. Sucrose preference as determined with this laboratory-based measure has been shown to be associated with preference for sugars in foods and beverages and with taste receptor genotype, family history of alcoholism, and race/ethnicity, as well as depressive symptomatology among pediatric populations. The method has real-world relevance and has been applied to determine most preferred level of other tastes (e.g., salt), making it a valuable psychophysical tool.

Sweet taste has powerful hedonic appeal among people of all ages, particularly children. Described herein is a reliable and valid method that can be used to determine the level of sweetness most preferred, making it a valuable psychophysical tool for scientists.

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to ...

Behavior

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Medicine

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...

A Novel Method for Enhanced Analysis of Insect Taste Neuron Activity

Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential food source, compounds from the food source enter through the pore and activate neurons within. For over 50 years, these responses have been recorded using a technique called tip recording. However, this method has major limitations, including the inability to measure neural activity before or after stimulus contact and the requirement for tastants to be soluble in aqueous solutions. We describe here a technique that we call base recording, which overcomes these limitations. Base recording allows the measurement of taste neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It can be used to study hydrophobic compounds such as long-chain pheromones that have very low solubility in water. In summary, base recording offers the advantages of single-sensillum electrophysiology as a means of measuring neuronal activity - high spatial and temporal resolution, without the need for genetic tools - and overcomes key limitations of the traditional tip recording technique.

Author Spotlight: Advances in Chemoreception &#8211; From Insect Odor Receptors to Non-Coding RNAs

A rarely used method of electrophysiological recording, base recording, allows analysis of features of taste coding that cannot be examined by conventional recording methods. Base recording also allows the analysis of taste responses to hydrophobic stimuli that cannot be studied using traditional electrophysiological methods.

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential ...

In Vivo Calcium Imaging of Taste-Induced Neural Responses in Adult Drosophila

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model organism, Drosophila melanogaster. A key strength of this methodology is its ability to record taste-induced neural responses in awake animals without the need for anesthesia. This approach employs binary expression systems (e.g., Gal4-UAS) to express the calcium indicator GCaMP in specific neurons of interest. This protocol describes a procedure in which flies expressing GCaMP are mounted with the labellum securely positioned, enabling fluorescence in the brain to be recorded at millisecond resolution under a confocal microscope while a solution is applied to the labellum, stimulating all labellar taste sensilla. The examples provided focus on calcium responses in primary gustatory receptor neurons of D. melanogaster. However, this approach can be adapted to record from other neurons of interest within the brain of Drosophilids or other insect species. This imaging method enables researchers to simultaneously record collective calcium responses from groups of gustatory neurons across the labellum, complementing electrophysiological tip recordings that quantify action potentials from individual neurons. The in vivo calcium imaging technique outlined here has been instrumental in uncovering molecular and cellular mechanisms of chemosensation, identifying unique temporal response patterns in primary taste neurons, investigating mechanisms of gustatory modulation, and exploring taste processing in downstream circuits.

A calcium imaging approach enables the recording of taste-induced responses in the brains of awake flies while a solution is applied to the labellum. Primary gustatory responses in Drosophila melanogaster are used as an example, but this protocol can be adapted to study downstream neurons or other species.

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model ...

Gustation

Gustation is a chemical sense that, along with olfaction (smell), contributes to our perception of taste. It starts with the activation of receptors by chemical compounds (tastants) dissolved in the saliva. The saliva and filiform papillae on the tongue distribute the tastants and increase their exposure to the taste receptors.

Taste receptors are found on the surface of the tongue as well as on the soft palate, the pharynx, and the upper esophagus. On the tongue, taste receptors are contained within structures called taste buds. The taste buds are embedded within papillae, which are visible on the tongue surface. There are three types of papillae that contain taste buds and their receptors. Circumvallate papillae are the largest papillae and are located near the back of the tongue. Foliate papillae resemble folds on the side of the tongue. Fungiform papillae are found across the front three-quarters of the tongue but are less concentrated in the middle of the tongue.

There are five basic tastes: salty, sour, sweet, bitter, and savory (or umami). The perception of salty taste is caused by tastants that release sodium ions upon dissolution. Sour taste, by contrast, is produced by the release of hydrogen ions from dissolved acidic tastants. Salty and sour tastants produce a neural response by depolarizing the membrane directly (salty tastants) or via ion channel changes (sour tastants).

Sweet, bitter, and savory compounds activate G-protein-coupled receptors. Chemicals perceived as sweet, like glucose and fructose, typically have hydroxyl and carbonyl groups. Numerous diverse molecules stimulate bitter receptors, and over 30 receptors for bitter taste have been identified. The amino acid glutamate activates savory receptors. The addition of monosodium glutamate, or MSG, to foods enhances the savory flavor.

Cranial nerves carry taste information from receptors in taste buds to the medulla in the brainstem. From there, the information travels to the thalamus and is then relayed to the primary gustatory cortex. This gustatory cortex also receives other sensory information from the tongue, like thermal and mechanical stimulation. The incorporation of the different sensory inputs provides a hedonic quality to flavor, allowing it to be perceived as pleasant or unpleasant.

Tastes and Mechanism of Gustation

Gustation is a chemical sense that, along with olfaction (smell), contributes to our perception of taste. It starts with the activation of receptors by ...

KEY TERMS AND CONCEPTS

Taste Buds and Receptors

Gustation, or the sense of taste, is intrinsically linked to the anatomical structures located on the tongue. This organ&#39;s surface, along with the entirety of the oral cavity, is adorned with stratified squamous epithelium. Evident on the tongue are elevated structures known as papillae (singular = papilla), which house the mechanisms for the transduction of gustatory stimuli. Four distinct types of papillae exist, each identified by their unique morphological attributes: the circumvallate, foliate, filiform, and fungiform papillae. Nestled within the papillae architecture are taste buds, which harbor specific gustatory receptor cells dedicated to the transduction of taste stimuli. These cells exhibit sensitivity to the chemical composition of ingested food, releasing neurotransmitters commensurate with the chemical concentration of the food. The released neurotransmitters can stimulate sensory neurons in the cranial nerves: facial, glossopharyngeal, and vagus.
The human gustatory system, responsible for perceiving taste, is an intricate matrix of processes that combine to discern food flavors. On the microscopic level, gustatory cells are activated by chemical constituents in our food, triggering the release of neurotransmitters. These chemicals relay signals to the brain, where taste perception, such as &#39;sweet,&#39; is formed. Despite its rapid execution, this system&#39;s complexity is evident in its various stages: chemical detection by gustatory receptors, accurate and swift signal transmission via nerves, and efficient information processing by the brain. Furthermore, taste perception is not a solitary phenomenon but is influenced by other sensory inputs like olfactory cues and tactile sensations. For instance, sweet-smelling food might be perceived as bitter if the texture is undesirable. This exemplifies the gustatory system&#39;s remarkable intricacy, hinting at integrating multiple sensory modalities to generate a precise flavor profile. Besides contributing to the hedonic enjoyment of food, taste perception plays a crucial role in assessing food safety. A comprehensive understanding of the biology and sensory perception surrounding taste can provide insights into our body&#39;s food processing mechanisms and flavor experiences, aiding in making informed, health-conscious dietary selections and enhancing our appreciation of taste subtleties.
Commonly Known Gustatory Dysfunction
1) Ageusia refers to the complete loss of taste, while hypogeusia is a reduced ability to taste sweet, sour, bitter, salty, and umami flavors. Conversely, dysgeusia is a condition characterized by a distorted sense of taste.
Common symptoms of these disorders include difficulty identifying specific tastes or experiencing an unpleasant or strange taste in the mouth. They may also cause changes in appetite and dietary habits, potentially leading to weight gain or loss.
2) Phantogeusia is a less common but equally impactful gustatory disorder. It involves tasting something that is not present, often described as a persistent, phantom flavor. This can be particularly distressing and disruptive to daily life, making eating and drinking unpleasant experiences.
Causes of Gustatory Dysfunction
The causes of gustatory disorders are diverse. They range from aging, which naturally diminishes taste sensitivity, to more specific factors, such as medication side effects, infections, and head injuries. Other potential causes include radiation therapy, certain neurological conditions like Parkinson&#39;s or Alzheimer&#39;s disease, and nutritional deficiencies.
Treatment Options
Treatment for gustatory disorders largely depends on the underlying cause. A doctor may adjust the dosage or switch to a different drug if medication is the culprit. Infections may be treated with antibiotics, while nutritional deficiencies can be addressed through diet modifications or supplements.
Surgical interventions may be considered in some cases, particularly if a nerve injury or tumor is causing the taste disorder. However, these are usually last-resort options due to their invasive nature and potential risks.
Pharmacological interventions are another treatment avenue. These may involve drugs that aim to enhance taste sensation or manage symptoms. For instance, zinc supplements have shown promise in improving taste function in some individuals.
The Future of Gustatory Disorder Treatment
Research into gustatory disorders is ongoing, with studies exploring new treatment options and seeking to understand these conditions&#39; underlying mechanisms better. While progress has been made, much remains to be discovered. By continuing to advance our knowledge in this area, we hope to improve the lives of those living with these challenging disorders.
In conclusion, gustatory disorders are complex conditions that can significantly impact an individual&#39;s ability to enjoy food and maintain a healthy diet. Understanding their causes, symptoms, and treatments is essential for providing adequate care and improving patients&#39; quality of life.

Taste buds&#8212;the sensory organs of the taste&#8212;are oval-shaped structures consisting of two major types of epithelial cells.
Gustatory epithelial ...

New Methods to Study Gustatory Coding

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