This work was supported by grants from NIH (DC014423 and DC016224) and the Defense Advanced Research Project Agency RealNose Project.&#160;YF stayed at Duke University with financial support from JSPS Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (R2801). We thank Sahar Kaleem for editing of the manuscript.

1. Hana3A Cells Culture
<ol>
	<li>Prepare M10 (Minimum Essential Medium (MEM) plus 10 % v/v fetal bovine serum (FBS)) and M10PSF (M10 plus 100 &#181;g/mL penicillin-streptomycin and 1.25 &#181;g/mL amphotericin B).</li>
	<li>Culture the cells in 10 mL of M10PSF in a 100 mm cell culture dish in an incubator set at 37 &#176;C and 5% carbon dioxide (CO2).</li>
	<li>Divide the cells every 2 days at a 20% ratio: when 100% confluence of cells (approximately 1.1 x 107 cells) is observed under a phase-contrast microscope, aspire the media and wash the cells gently with 10 mL of phosphate-buffered saline (PBS).</li>
	<li>Aspirate PBS and add 3 mL of 0.05% trypsin-ethylene diamine tetraacetic acid (EDTA, 0.48 mM). Let act for approximately 1 min, until the cells dissociate from the plate.</li>
	<li>Add 5 mL of M10 to inactivate the trypsin and eventually detach the cells still attached to the plate by pipetting up and down.</li>
	<li>Transfer the volume (8 mL) to a 15 mL tube and centrifuge at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells into 5 mL of M10PSF by pipetting up and down to break any cell mass. Avoid creating bubbles in the tube.</li>
	<li>Transfer 1 mL of the resuspended cells solution in a new 100 mm cell culture dish and add 9 mL of fresh M10PSF. Incubate at 37 &#176;C and 5% CO2.</li>
</ol>
2. Preparation of the Cells for Transfection
<ol>
	<li>Evaluate the confluence, or the number of cells, by observing them under a phase-contrast microscope. At least 10% confluence (approximately 1.1 x 106 cells) is needed for one plate.</li>
	<li>Aspirate the media and wash the cells gently with 10 mL of PBS. Aspirate PBS and add 3 mL of EDTA. Let act for approximately 1 min at room temperature, until the cells dissociate from the plate.</li>
	<li>Add 5 mL of M10 to inactivate the trypsin and eventually detach the cells still attached to the plate by pipetting up and down. Transfer the volume (8 mL) to a 15 mL tube and centrifuge at 200 x g for 5 min.</li>
	<li>Aspirate the supernatant and resuspend the cells into 5 mL of M10PSF by pipetting up and down to break any cell mass. Avoid creating bubbles in the tube.</li>
	<li>Depending on the number of plates to be transfected, transfer an appropriate amount of cells in a reservoir with the proper corresponding volume of M10PSF. One 96-well plate should be plated with 1/10 of a 100% confluent 100 mm dish (approximately 1.1 x 106 cells) diluted in M10PSF to reach a total volume of 6 mL. For one 96-well plate starting with a 100% confluence 100 mm dish, add 500 &#181;L from the 5 mL of resuspended cells to 5.5 mL of fresh M10PSF. Mix the cells and M10PSF without generating air bubbles.</li>
	<li>Pipette 50 &#181;L of the suspended cells into each well of the 96-well plate using a multichannel pipette. Incubate overnight at 37 &#176;C and 5 % CO2.</li>
</ol>
3. Plasmid Transfection
<ol>
	<li>Observe the 96-well plate under a phase-contrast microscope to assure a cell confluence between 30% and 50%.</li>
	<li>Prepare a first transfection mix that contains the plasmids common to the entire plate (RTP1S, OR and Glosensor protein, see Table of Materials) following the volumes in Table 1. Notice that the quantity of Rho-tagged OR should be divided by the number of ORs if several ORs. 
	NOTE: We strongly advice to add an empty vector negative control (here Rho-pCI) and any positive control (OR known to respond to the tested odorant) to the experiment plan.</li>
	<li>Prepare a second transfection mix containing 500 &#181;L of MEM and 20 &#181;L of Lipofectamine 2000 reagent (valid for one 96-well plate, see Table of Materials). Add the second mix to the first one, and gently mix by pipetting up and down and incubate for 15 min at room temperature. Add 5 mL of M10 and mix gently.</li>
	<li>Replace the M10PSF in the previously platted 96-well plate by 50 &#181;L of the final transfection media. Incubate in an incubator set to 37 &#176;C and 5% CO2 and vacuum the chamber of the luminometer overnight following the procedure described in step 6.</li>
</ol>
4. Substrate Incubation
<ol>
	<li>Observe the 96-well plate under a phase-contrast microscope to assure a cell confluence between 60% and 100%. Prepare a stimulation solution of Hank&#8217;s Balanced Salt Solution (HBSS) containing 10 mM of hydroxyethyl piperazineethanesulfonic acid (HEPES) and 5 mM of D-glucose.</li>
	<li>Dilute 75 &#181;L of the cAMP reagent (see Table of Materials) solution to 2.75 mL of the stimulation solution. Remove the transfection medium from the 96-well plate and wash the cells by adding 50 &#181;L of fresh stimulation solution to each well.</li>
	<li>Remove the stimulation solution and add 25 &#181;L of cAMP reagent solution prepared in step 4.2 to each well. Incubate the 96-well plate at room temperature in a dark and odor-free environment (for example, a clean empty drawer far away from chemicals or any odorant source) for 2 h.</li>
</ol>
5. Odorant Stimulation
<ol>
	<li>First, equilibrate the luminometer chamber with volatile odorant molecules. Dilute the odorant to the desired concentration in 10 mL of mineral oil (Figure 2A). Before the end of the cAMP reagent incubation time, add 25 &#181;L of the odorant solution in a new 96-well plate (not the one containing the cells). Incubate this odorant plate at room temperature in the luminometer chamber for 5 min (Figure 2B) (no luminometer recording is required here).</li>
	<li>Set the luminometer to record the luminescence with 0 s of delay during 20 cycles of plate measurement of 90 s with 0.7 s of interval between cycles.</li>
	<li>Right before reading the plate, remove the odorant plate from the chamber. Add 25 &#181;L of odorant between the wells of the 96-well plate containing the cells (do not add the odorant in the wells containing the cells) and quickly start the luminescence measurement of all wells for 20 cycles within 30 min (Figure 2C).</li>
</ol>
6. Removal of remaining odorant inside the&#160;Luminometer
<ol>
	<li>Open the door of the luminometer. Insert the tube connected to the vacuum pump.</li>
	<li>Vacuum odorants in the reading chamber extensively (at least 2 h, preferably overnight) between two odorants to avoid cross contamination of odor volatiles from one experiment to another. Replace with fresh air by sending compressed air during 5 min before incubating the next odorant.</li>
</ol>
7. Data Analysis
<ol>
	<li>Export the data from the luminometer software.</li>
	<li>Average the replicates of the same OR for each recording time. Calculate the normalized OR response to any eventual control (e.g., Empty vector, Figure 3A and representative results section) by dividing the control averaged value to the OR averaged value at each recording time (Figure 3B and representative results section).</li>
	<li>Normalize the each OR response to their basal activity by dividing the averaged OR response at 0 s to each recording time response (See Figure 3C and representative results section).</li>
	<li>Calculate the area under the curve of each OR to obtain a single OR response value. To do so, sum all the luminescence values of each recording time for each OR.</li>
</ol>

Y.F., H. K and H.M. filed a patent application relevant to this work on 27 October 2016.

The perception of odor is fundamentally dependent on the activation of ORs. Consequently, understanding of their functionality is required to crack the complex mechanisms that the brain use to perceive its volatile chemical environment. However, the understanding of this process has been hampered by the difficulties in establishing a robust method to screen the OR repertoire for functionality against odorants in vitro. Cell surface and heterologous expression of ORs has been partially solved by the creation of t...

The sense of smell allows terrestrial animals to interact with their volatile chemical environment to drive behaviors and emotions. Fundamentally, the odor detection process begins with the very first interaction of odorant molecules with the olfactory system, at the level of odorant receptors (ORs)1. In mammals, ORs are individually expressed in olfactory sensory neurons (OSNs) located in the olfactory epithelium2. They belong to the G-protein coupled receptor (GPCR) family and more precisely to the rhodopsin-like sub-family (also called class A). ORs couple with the stimulatory G protein Golf whose activation leads to cAMP production followed by the opening of cyclic nucleotide gated channels and the generation of action potentials. It is accepted that an odor percept relies on a specific pattern of activated ORs3,4 and therefore odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. And through such combinatorial coding, it is postulated that organisms can detect and discriminate between a myriad of volatile odor molecules. One of the keys to understanding how odors are perceived is to understand how and which ORs are activated by a given odor.
In an attempt to elucidate odorant-OR interactions, in vitro functional assays have played an essential role. The identification of agonist odorous ligands for orphan ORs (OR de-orphanization) has been a very active field for the past twenty years, through the use of various in vitro, ex vivo and in vivo functional assays5,6,7,8,9,10,11,12,13,14,15,16,17.
In vitro assay systems are best suited for the detailed functional characterization of ORs, including identifying the functional domains and critical residues of ORs, as well as potential engineering applications. However, further development of valuable in vitro systems for ORs has been a challenge, in part due to difficulty with culturing OSNs and functional expression of ORs in heterologous cells. The first challenge had been to establish protocols that allowed for the cell surface expression of functional ORs in the mapping of odorant-OR interactions. A number of independent groups have utilized various approaches5,6,7,8,9,10,11,12,14,18,19,20. One of the earliest achievements was made by Krautwurst et al. in tagged the N-terminus of ORs with a shortened sequence of rhodopsin (Rho-tag) and observed an improved surface expression in human embryonic kidney (HEK) cells13. Variations made to the tag attached to the OR sequence is still a path explored for improving OR expression and functionality19,21. Saito et al. then identified receptor-transporting protein 1 (RTP1) and RTP2 which facilitate OR trafficking.22 A shorter version of RTP1, called RTP1S, has also been shown to be even more effective than the original protein23. The development of a cell line (Hana3A) which stably expresses Golf, REEP1, RTP1, and RTP2 24, coupled with the use of cyclic adenosine monophosphate (cAMP) reporters has enabled identification of odorant-OR interactions. The mechanism by which the RTP family of proteins promotes cell surface expression of ORs remains to be determined.
One caveat of these established methods is that they rely on odorant stimulation in liquid phase, meaning that odorants are pre-dissolved into a stimulation medium and stimulate cells by replacing the medium. This is very distinct from the physiological conditions where odorant molecules reach the olfactory epithelium in vapor phase and activate ORs by dissolution into the nasal mucosa. To more closely resemble physiologically relevant stimulus exposure, Sanz et al.20 proposed an assay based on vapor stimulation by applying a drop of odorant solution to hang beneath the inner face of a plastic film placed on the top of cell wells. They recorded the calcium responses by monitoring fluorescence intensity. This method was the first to use air-phase odorant stimulation, but it did not allow a large screening of OR activation.
Here, we developed a new method that enables real-time monitoring of in vitro OR activation via vapor phase odorant stimulation by the Glosensor assay (Figure 1). This assay has been used previously in the context of liquid odorant stimulation18,19,25,26,27,28,29,30,31. The monitoring chamber of the luminometer is first equilibrated with vaporized odorant prior to plate reading (Figure 1A). Odorant molecules are then solvated into the buffer, bathing Hana3A cells expressing the OR of interest, RTP1S and the Glosensor proteins (Figure 1B). If the odorant is an agonist of the OR, the OR will switch to an activated conformation and bind the Golf, activating the adenylyl cyclase (AC), and ultimately cause cAMP levels to rise. This rising cAMP will bind to and activate the Glosensor protein to generate luminescence catalyzing luciferin. This luminescence is then recorded by the luminometer and enables OR activation monitoring. This method is of high interest in the context of OR deorphanization as it brings in vitro systems closer to the natural perception of odors.

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			<td height="22" style="height:22px;width:205px;">0.05 % trypsin-EDTA</td>
			<td style="width:93px;">Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td style="width:449px;">0.05% Trypsin - EDTA (1x), phenol red - store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm cell culture dish&#160;</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td>100 mm x 20 mm cell culture dish&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL tube</td>
			<td>BD Falcon</td>
			<td>352099</td>
			<td style="width:449px;">17 mm x 120 mm conical tubes</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:205px;">96-well plate</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">3843</td>
			<td style="width:449px;">96 well, with LE lid white with clear bottom Poly-D-lysine coated Polystyrene</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Amphotericin</td>
			<td style="width:93px;">Gibco</td>
			<td style="width:119px;">15290-018</td>
			<td style="width:449px;">Amphotericin B 250 &#181;g/mL - store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge machine</td>
			<td>Jouan</td>
			<td>C312</td>
			<td style="width:449px;">Centrifuge machine with swinging bucket rotor for 15 mL</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Class II Type A/B3 fumehood</td>
			<td>NUAIRE</td>
			<td>NU-407-500</td>
			<td style="width:449px;">fumehood for cell culturing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:205px;">FBS</td>
			<td style="width:93px;">Gibco</td>
			<td style="width:119px;">16000-044</td>
			<td style="width:449px;">Fetal Bovine Serum - store at -20&#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:205px;">GloSensor cAMP Reagent</td>
			<td style="width:93px;">Promega</td>
			<td style="width:119px;">E1290</td>
			<td style="width:449px;">GloSensor cAMP Reagent luminescent protein substrate - store at -20&#176;C</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Incubator 37 &#176;C; 5 % CO2</td>
			<td>Fisher Scientific</td>
			<td>11-676-604</td>
			<td style="width:449px;">Incubator for cell culturing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamine 2000 reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td style="width:449px;">Lipofectamine 2000 Reagent 1mg/ml transfection reagent - store at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luminometer POLARstar OPTIMA</td>
			<td>BMG LABTECH</td>
			<td>discontinued</td>
			<td style="width:449px;">96 well plate reader for luminescence</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:205px;">Mineral oil</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">M8410</td>
			<td style="width:449px;">Solvent for odorants - store at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:205px;">Minimum Essential Medium (MEM)</td>
			<td style="width:93px;">Corning cellgro</td>
			<td style="width:119px;">10-010-CV</td>
			<td style="width:449px;">Minimum Essential Medium Eagle with Earle&#8217;s salts &#38; L-glutamine - store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:205px;">Penicillin/Streptomycin</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td style="width:119px;">P4333</td>
			<td style="width:449px;">Penicillin-Streptomycin solution stabilized with 10,000 U of penicillin and 10 mg streptomycin - store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:205px;">pGlosensor</td>
			<td style="width:93px;">Promega</td>
			<td style="width:119px;">E2301</td>
			<td style="width:449px;">pGloSensor-22F cAMP luminescent protein plasmid - store at 4&#176;C</td>
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		<tr height="20">
			<td height="20" style="height:20px;">phase contrast microscope</td>
			<td>Leica</td>
			<td>090-131.001</td>
			<td style="width:449px;">phase contrast microscope with x4, x10, x20 objectives</td>
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			<td height="40" style="height:40px;width:205px;">RTP1S</td>
			<td style="width:93px;">H. Matsunami lab</td>
			<td style="width:119px;">-</td>
			<td style="width:449px;">100 ng/&#181;L plasmid - store at 4&#176;C</td>
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real-time in vitro monitoring of odorant receptor activation by an odorant in the vapor phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

We screened the response of three mouse ORs, Olfr746, Olfr124 and Olfr1093 using cinnamaldehyde vapor stimulation (Figure 3). Simultaneously, we used an empty vector control (Rho-pCI) to assure that the odorant-induced activities of the tested ORs were specific (Figure 3A). The real-time activation of the ORs upon vapor odorant stimulus was monitored over 20 measurement cycles. The data for each well were first normalized to the empty vector con...

Watch this Scientific Journal Video about Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase at JoVE.com

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

real-time-vitro-monitoring-odorant-receptor-activation-an-odorant

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JoVE Journal

Neuroscience

6.9K Views.  Duke University Medical Center. Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Video: Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

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

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

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

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

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

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

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

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

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

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

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

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

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

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Enhancing Peripheral Nerve Regeneration with nsPEF-Stimulated Schwann Cells

Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse Electric Field (nsPEF) is an emerging method applicable in nerve electrical stimulation that has been demonstrated to be effective in stimulating cell proliferation and other biological processes. Aiming to assess whether SCs undergo significant changes under nsPEF and help explore the potential for new peripheral nerve regeneration methods, cultured RSC96 cells were subjected to nsPEF stimulation at 5 kV and 10 kV, followed by continued cultivation for 3-4 days. Subsequently, some relevant factors expressed by SCs were assessed to demonstrate the successful stimulation, including the specific marker protein, neurotrophic factor, transcription factor, and myelination regulator. The representative results showed that nsPEF significantly enhanced the proliferation and migration of SCs and the ability to synthesize relevant factors that contribute positively to the regeneration of peripheral nerves. Simultaneously, lower expression of GFAP indicated the benign prognosis of peripheral nerve injuries. All these outcomes show that nsPEF has great potential as an efficient treatment method for peripheral nerve injuries by stimulating SCs.

Author Spotlight: Innovative Use of nsPEF to Boost Peripheral Nerve Regeneration

Here, we present a protocol for applying nanosecond pulse electric field (nsPEF) to stimulate Schwann cells in vitro. The synthesis and secretion ability of relevant factors and cell behavior changes validated the successful stimulation using nsPEF. The study gives a positive view of the peripheral nerve regeneration method.

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse ...