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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>
			<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)&#160;</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)&#160;</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 nervious 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Saline perfusion system</td>
		</tr>
		<tr>
			<td>Extension set with rate flow regulator</td>
			<td>&#160;B Braun Medical Inc.</td>
			<td>V5200</td>
			<td></td>
		</tr>
		<tr>
			<td>IV administration set with Y injection site</td>
			<td>&#160;B Braun Medical Inc.</td>
			<td>V1402</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Tastant delivery system</td>
		</tr>
		<tr>
			<td>White Translucent Nylon Tubing OD 1/4&#34;, ID 1/8&#34;</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)&#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>
		</tr>
		<tr>
			<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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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). 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.

Video: New Methods to Study Gustatory Coding

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

How to Create and Use Binocular Rivalry

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent representation. However, when the eyes are presented with images that are sufficiently different from each other, an interesting thing happens: Rather than fusing the two images into a combined conscious percept, what transpires is a pattern of perceptual alternations where one image dominates awareness while the other is suppressed; dominance alternates between the two images, typically every few seconds. This perceptual phenomenon is known as binocular rivalry. Binocular rivalry is considered useful for studying perceptual selection and awareness in both human and animal models, because unchanging visual input to each eye leads to alternations in visual awareness and perception. To create a binocular rivalry stimulus, all that is necessary is to present each eye with a different image at the same perceived location. There are several ways of doing this, but newcomers to the field are often unsure which method would best suit their specific needs. The purpose of this article is to describe a number of inexpensive and straightforward ways to create and use binocular rivalry. We detail methods that do not require expensive specialized equipment and describe each method's advantages and disadvantages. The methods described include the use of red-blue goggles, mirror stereoscopes and prism goggles.

Binocular rivalry occurs when the eyes are presented with different images at the same location: one image dominates while the other is suppressed, and dominance alternates periodically. Rivalry is useful for investigating perceptual selection and visual awareness. Here we describe several easy methods for creating and using binocular rivalry stimuli.

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent ...

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Measuring the Subjective Value of Risky and Ambiguous Options using Experimental Economics and Functional MRI Methods

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition termed "risky". In other cases when probabilities cannot be estimated, this is a condition described as "ambiguous". While most people are averse to both risk and ambiguity1,2, the degree of those aversions vary substantially across individuals, such that the subjective value of the same risky or ambiguous option can be very different for different individuals. We combine functional MRI (fMRI) with an experimental economics-based method3 to assess the neural representation of the subjective values of risky and ambiguous options4. This technique can be now used to study these neural representations in different populations, such as different age groups and different patient populations.
In our experiment, subjects make consequential choices between two alternatives while their neural activation is tracked using fMRI. On each trial subjects choose between lotteries that vary in their monetary amount and in either the probability of winning that amount or the ambiguity level associated with winning. Our parametric design allows us to use each individual's choice behavior to estimate their attitudes towards risk and ambiguity, and thus to estimate the subjective values that each option held for them. Another important feature of the design is that the outcome of the chosen lottery is not revealed during the experiment, so that no learning can take place, and thus the ambiguous options remain ambiguous and risk attitudes are stable. Instead, at the end of the scanning session one or few trials are randomly selected and played for real money. Since subjects do not know beforehand which trials will be selected, they must treat each and every trial as if it and it alone was the one trial on which they will be paid. This design ensures that we can estimate the true subjective value of each option to each subject. We then look for areas in the brain whose activation is correlated with the subjective value of risky options and for areas whose activation is correlated with the subjective value of ambiguous options.

Using functional MRI and behavioral methods to determine the neural representation of the subjective value of risky and ambiguous options in the human brain.

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...