Name: new methods to study gustatory coding
Uploaded: 2017-06-29T15:00:00
Duration: 10 min 59 s
Description: Watch this Scientific Journal Video about New Methods to Study Gustatory Coding at JoVE.com

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.

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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.

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	<tbody>
		<tr>
			<td colspan="4">Dissection and specimen preparation</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, 19G x 1-1/2&#34;&#160;</td>
			<td>&#160;MONOJET</td>
			<td>888200144</td>
			<td>For aplying air to remove the hair from the moth.</td>
		</tr>
		<tr>
			<td>Modeling Clay-Van Aken Plastalina&#160;</td>
			<td>DickBlick</td>
			<td>33268</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish-100 x 15 mm&#160;</td>
			<td>VWR International</td>
			<td>89000-304</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip (1-200 &#181;L)</td>
			<td>USA Scientific</td>
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			<td>For removing fat and non nervous tissue.</td>
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		<tr>
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			<td>14096</td>
			<td>For removing fat and non nervous tissue.</td>
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			<td>Collagenase/Dispase</td>
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			<td>Name</td>
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			<td>Name</td>
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			<td colspan="4">Tastant delivery system</td>
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			<td>White Translucent Nylon Tubing OD 1/4&#34;, ID 1/8&#34;</td>
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			<td>Rigid tube that connects the four main elements of the system.</td>
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			<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)&#160;</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&#160;</td>
			<td>Bioscience Tools</td>
			<td>PS-16H</td>
			<td>For tastant delivery. This system includes pinch valves, tubing, manifold, solution cylinders, valve controler and fitting accesories.</td>
		</tr>
		<tr>
			<td>Polypropylene tubing, ID 0.034&#34;, ID 0.050&#34;</td>
			<td>Becton, Dickinson &#38; Co</td>
			<td>427421</td>
			<td>&#160;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.&#160;</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&#34; OD 1/8&#34;</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>&#160;FS-V31M</td>
			<td>For monitoring tastant delivery.&#160;</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&#160;</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 moths proboscis into the proboscis hole from the rigid tube.</td>
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			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Electrophysiology Equipment</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 precicely insert the tetrodes into the animals 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&#160;</td>
			<td>TDS2014</td>
			<td></td>
		</tr>
		<tr>
			<td>16-channle pre-amplifier and amplifier</td>
			<td>16 Channel MA-800 Amplifier System&#160;</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: 3.32GHz, 3GB. Processor: Intel Core&#160;2 Duo. Graphic card: integrated Intel GMA X4500.&#160;</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Tastants</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

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 ...

The overall goal of these methods is to study gustatory coding in a simple animal mandi-casecsta by recording the activity of multiple receptor neurons in vivo, while delivering and monitoring precisely timed pulses of tastants. These methods can help answer key questions about gustation, such as how tasters are encoded by populations of gustatory receptor neurons. The main advantage of this technique is that it allows in vivo recordings for multiple gustatory neurons before, during, and after precisely delivered tastant stimuli.Individuals new to this method might find it challenging because the dissection procedure is intricate. Because of this, we hope that the visual demonstration of the method will be helpful. These techniques were developed by Sam Writer when he was a graduate student in my lab, along with Alejandra, Kui Sun will demonstrate these procedures.Three days after eclosion, chose a moth of either sex that has a generally healthy appearance. The wings should be fully extended, and the proboscis and antennae should be well formed and intact. In a fume hood, insert the selected moth into a restraining tube until the head is exposed.Then, immobilize the moth by inserting tissue paper into the other end of the tube. Now, using pressurized air delivered from a syringe, remove the hair from the exposed head. Then, place the tube on a clay platform in a Petri dish.Orient the head dorsal side up. Next, protect the antennae and the proboscis. First, make covers for each structure from 200 microliter pipette tips cut into half centimeter lengths.To manipulate the proboscis, prepare a hook from a piece of wire. Extend the proboscis into one of the covers until the most proximal part of the proboscis is protected. Secure the tube to the dorsal part of the head capsule using firm batik wax.Next, carefully sheet the antennae and secure the tubes with batik wax. Now, stabilize the brain by cutting the buckle compressor muscle. Under the dissection microscope, use microdissection tools to open the head capsule by making a small cut just below the proboscis.To confirm the muscle is cut, offer sucrose solution to the distal two thirds of the proboscis, and for five minutes make sure that the proboscis does not extend. Then, close the head capsule using a layer of melted batik wax. To perfuse the brain with saline, build a wax cup around the ventral side of the head.Using an electric waxer, start building a wax cup along the front of the head moving towards the back. Keep the proboscis and antennae tubes inside the cup. Continue building the cup outward and upward until it reaches the level of the eyes.Using forceps, take the labial palps and attach them to the wax cup by using melted wax. Next, seal any openings in the wax cup and tubes using a thing layer on binary epoxy. Mix the epoxy using a toothpick.First, cover the outer and inner surfaces of the cup. Second, fill the part of the restraining tube surrounding the neck so the head is held more firmly. After 20 minutes, check the cup for leaks.To begin the brain surgery, first cut off the labial palps. To open the ventral side of the head capsule, cut the cuticle along the back and front of the head and around the eyes. Then, cut along the back of the head and make more cuts along the front of the head near the proboscis.Then, gently lift the flap of cuticle with forceps to expose the brain. Now, perform the most critical step, exposing the SEZ and maxillary nerves. Slowly and carefully remove fat tissue and the trachea.Rinse the brain frequently with fresh saline. Be very careful, because it is very easy to crush the maxillary nerves. When the SEZ and the maxillary nerves are finally visible, they will be covered by a thin sheath.To remove this sheath, drain any excess saline and then weaken the sheath by applying a 10%collagenase dispase solution. After soaking for five minutes, rinse out the solution several times with saline. Then, after several rinses with saline gently remove the sheath using superfine microdissection forceps.Lastly, secure a saline drip line into the perfusion cup. This section describes how to build and use a tastants delivery system. The system consists of a testing tube with several openings.The openings provide entrance for the animal's proboscis, the tastants manifold, a color sensor, and they allow for the flor through of water. To build this system, first prepare a testing tube for the proboscis. Make a proboscis sized hole using a soldering iron.Then, build a screened in section within the tube to hold the proboscis in place. Cut five millimeters above the hole, place the mesh there with wax, and reconnect the tubes using wax and epoxy. Use a pressurized perfusion system to deliver the tastants.Join as many tastant tubes as needed to a manifold. To connect the tastant output tube, make a hole about one centimeter above the mesh, then secure the output tube to it with epoxy. In each tastant, include an artificial dye to be detected by a color sensor.Attach a color sensor to the apparatus at a hole one half centimeter about the mesh, and secure it there using epoxy. Use a peristaltic pump to deliver distilled water into the system via silicon tubing. Collect the waste water with a large container.Use a pneumatic pump to deliver the tastants with puffs of air. Now, begin pumping clean water through the tubing, and deliver pulses of tastant to test the color sensor. Now, load the distill two thirds of the moth proboscis into the testing tube.Using forceps, extend the proboscis and gently push it into the hole. Then, seal the proboscis in place with soft dental wax and a layer of epoxy. Now, connect the saline perfusion line and set the perfusion flow.Then, immerse the ground electrode of the twisted wire tetrode into the saline bath. Then, using a micromanipulator, position the tetrode next to the maxillary nerve, and slowly advance it into the nerve. After detecting spiking signals, let the preparation stabilize for at least 10 minutes before taking recordings.Recordings were made from gustatory receptor neurons before, during, and after tastant delivery using the described protocol. The onset and offset of a one second pulse of each tastant was monitored by the color sensor. The tastants elicited spikes that were detected by each of the four channels.Within the spike trains, individual action potentials could be observed. Spike sorting software was used to assign these spikes to unique neurons. The response of three different gustatory receptor neurons to six tastants delivered in sequence reveals different levels of baseline activity and selectivity to the tastants.Also, responses had different timing. Some are locked to the duration of the stimulus, while others outlast the stimulus. Intracellular recordings could also be made from gustatory receptor axons using conventional sharp glass electrodes.The intracellular recordings were very similar to responses observed using the described tetrode technique, but the tetrode recordings were easier to make, longer lasting, and more robust. After watching this video, you should be able to expose the maxillary nerves and the sub-esophagal zone in mandi-casecsta, and be able to build a tastant delivery system with precise temporal control. While performing this protocol, wear laboratory safety gloves, and a face mask because the fine powdery scaled shed by mandi-casecsta can be allergenic.Once mastered, this procedure can be completed in three and a half hours. Following this procedure, simultaneous recordings from multiple brain areas can also be performed to answer additional questions such as, how gustatory receptor neuron responses to tastants are transformed when they are transmitted to the postsynaptic target neurons.

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