Name: localization of odorant receptor genes in locust antennae by rna in situ hybridization
Uploaded: 2017-07-13T13:00:00.000+00:00
Duration: 9 min 30 s
Description: Watch this Scientific Journal Video about Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization at JoVE.com

This work is supported by a grant&#160;from National Natural Science Foundation of China (No.31472037). Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation.

NOTE: To limit RNA degradation, prepare solutions using wet-autoclaved distilled water (at 121 &#176;C for 60 min) and also wet-autoclave materials.
1. Preparation of RNA ISH Antisense and Sense Probes
<ol>
	<li>Target gene amplification and purification
	<ol>
		<li>First, produce a 387 bp double-stranded fragment of L. migratoria OR1 (LmigOR1, GenBank: JQ766965) from the plasmid containing the full-length cDNA of LmigOR1 with a Taq DNA Polymerase that adds adenines to both ends of the fragments.
		<ol>
			<li>Use a 100 &#181;L final reaction volume, containing 50 &#181;L of 2x reaction mix, 4 &#181;L each of sense and antisense primers (Table 1), 1 &#181;L of plasmid template, and 41 &#181;L of RNase-free H2O. Use the following PCR protocol: 94 &#176;C for 5 min, followed by 30 cycles of 94 &#176;C for 30 s, 55 &#176;C for 30 s and 72 &#176;C for 1 min, followed by 72 &#176;C for 10 min.</li>
			<li>Repeat these steps to produce the 1,303 bp double-stranded fragment of LmigOR2 (GenBank: JQ766966) and 1,251 bp double-stranded fragment of LmigORco (GenBank: JN989549). The primers used in these experiments are presented in Table 1.</li>
			<li>Run PCR products on 1.2% agarose gels in 1x Tris-acetate-EDTA buffer and visualize using ethidium bromide staining.</li>
		</ol>
		</li>
		<li>Extract these PCR products using a gel extraction kit.
		<ol>
			<li>Following electrophoresis, excise DNA bands from the gel and place the gel slices in a 1.5 mL tube. Then, add 100 &#181;L of binding buffer (Buffer PN) per 0.1 g of gel slice. Vortex and incubate at 50 &#176;C until the gel slice is completely dissolved.</li>
			<li>Insert a CA2 adsorption column into the collection tube and add 500 &#181;L of balance buffer (Buffer BL). This improves the absorption capability and stability of the silica membrane. Centrifuge at 12,400 x g for 1 min.</li>
			<li>Transfer the dissolved gel mixture to the adsorption column assembly and incubate&#160;at room temperature for 2 min. Then, centrifuge tubes at 12,400 x g for 1 min. Discard the flow-through and reinsert the adsorption column into collection tube.</li>
			<li>Add 600 &#181;L of wash solution (ethanol added, Buffer PW) twice. Centrifuge at 12,400 x g for 1 min. Discard flow-through and reinsert the adsorption column into collection tube.</li>
			<li>Empty the collection tube and recentrifuge the column assembly at 12,400 x g for 2 min to allow evaporation of any residual ethanol.</li>
			<li>Carefully transfer adsorption column to a clean 1.5 mL centrifuge tube. Air-dry the pellet for 5-10 min and redissolve the DNA in a suitable volume (e.g., 30 &#181;L) of nuclease-free water.</li>
			<li>Incubate at RT for 2 min. Centrifuge at 12,400 x g for 2 min. Discard adsorption column and store the DNA at 4 &#176;C or -20 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Construct the recombinant plasmids
	<ol>
		<li>Individually ligate the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2&#160;and 1,251 bp fragment of LmigORco into a T vector that contains promoters for T7 (upstream) and SP6 (downstream) RNA polymerases adjacent to the inserted DNA using T4 DNA ligase. Prepare the following 10 &#181;L reaction: 5 &#181;L of 2x ligation buffer, 1 &#181;L of T vector, 3 &#181;L (&#8764;100 ng) of the inserted gene&#39;s DNA and 1 &#181;L of T4 DNA ligase, and incubate O/N at 4 &#176;C.</li>
		<li>Add 5 &#181;L of each recombinant plasmid containing the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2&#160;and 1,251 bp fragment of LmigORco separately into 50 &#181;L of competent Escherichia coli DH5&#945; cells in sterile 1.5 mL tubes.
		<ol>
			<li>Mix gently and put the tubes into ice for 30 min and then incubate them for 90 s at 42 &#176;C. Transfer them back into ice for 3 min.</li>
			<li>Add 450 &#181;L of Luria-Bertani (LB) liquid medium without ampicillin to every tube and incubate them in a shaker at 150 rpm for 1 h at 37 &#176;C to restore the E. coli&#160;DH5&#945;.</li>
			<li>Take a 100 &#181;L aliquot of each transformant and use them to inoculate LB solid substrate plates containing 50 &#181;g/mL ampicillin, 24 &#181;g/mL isopropyl &#946;-D-1-thiogalactopyranoside (IPTG), and 40 &#181;g/mL 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-gal).</li>
			<li>Incubate these plates in a constant incubator at 37 &#176;C for 18 h. Screen the target recombinant plasmids using the blue-white screening method. Sequence the target recombinant plasmids using Sanger sequencing and identify the three OR genes&#39; insert directions.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Select restriction endonucleases to linearize recombinant plasmids
	<ol>
		<li>Use restriction endonucleases that not only digest in the multiple cloning site of the vector but also do not cut the insert DNA. In this experiment, all 3 genes are inserted into the T vector in the direction corresponding to that of the vector.</li>
		<li>Use Nco I restriction endonuclease to linearize the recombinant plasmids containing the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2 and 1,251 bp fragment of LmigORco to produce the antisense probes. Use Spe I restriction endonuclease to produce the sense probes. 
		NOTE: If the insert direction corresponds to that of the vector, then a unique restriction site from the end of T7 is selected to linearize the recombinant plasmid and SP6 RNA polymerase is used to prepare the antisense probe. A unique restriction site from the end of SP6 is selected to linearize the recombinant plasmid, and the T7 RNA polymerase is used to prepare the sense probe. Otherwise, if the insert direction does not correspond to the vector&#39;s direction, then a unique restriction site from the end of SP6 is selected to linearize the recombinant plasmid and T7 RNA polymerase is used to prepare the antisense probe. The unique restriction site from the end of T7 is selected to linearize the recombinant plasmid, and the SP6 RNA polymerase is used to prepare sense probe.</li>
	</ol>
	</li>
	<li>Purifying the linearized recombinant plasmids
	<ol>
		<li>Digest 20 &#181;g each of the three recombinant plasmids containing the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2 and 1,251 bp fragment of LmigORco using the Nco I restriction endonuclease in a 100 &#181;L reaction that contains 10 &#181;L of 10x buffer, 40 &#181;L of 0.5 &#181;g/&#181;L plasmid, 3 &#181;L&#160;of Nco I and 47 &#181;L of RNase-free H2O, followed by a 3 h incubation at 37 &#176;C.</li>
		<li>Similarly, digest 20 &#181;g of each of these three recombinant plasmids using the Spe I restriction endonuclease.</li>
		<li>Purify these digested plasmids using a gel extraction kit as described in 1.1.2. Determine the concentrations of these extracted linearized recombinant plasmids by spectrophotometry and adjust to 0.5 &#181;g/&#181;L.</li>
	</ol>
	</li>
	<li>Prepare antisense and sense probes
	<ol>
		<li>Use the T7/SP6 RNA transcription system to generate antisense and sense probes. SP6 RNA polymerase is used to transcribe double-stranded RNA for DIG- and biotin-labeled antisense probes for these three OR genes using linearized recombinant plasmids as templates in vitro. The T7 RNA polymerase is used to produce sense probes. Perform the reaction in a 20 &#181;L final volume, containing 2 &#181;L of 10x NTP mix, 2 &#181;L of 10X transcription buffer, 1 &#181;L of RNase inhibitor, 2 &#181;L of T7 or SP6 RNA polymerase, 4 &#181;L of 0.5 &#181;g/&#181;L linearized plasmid and 9 &#181;L RNase-free H2O, followed by a 3 h incubation at 37 &#176;C.</li>
		<li>Incubate the DIG- and biotin-labeled antisense and sense probes for 30 min with 1 &#181;L DNase at 37 &#176;C. Add 2.5 &#181;L of 5 M LiCl and 75 &#181;L of absolute ethanol, then incubate the reactions for 30 min at -70 &#176;C.</li>
		<li>Centrifuge at 15,000 x g for 30 min at 4 &#176;C. Carefully decant the supernatant. Add 150 &#181;L of 70% ethanol to wash the precipitant. Prepare the 70% ethanol (1 L) by adding 95% ethanol (736.8 mL) to sterile distilled water (263.2 mL).</li>
		<li>Centrifuge at 15,000 x g for 30 min at 4 &#176;C again and air-dry the pellet for 5-10 min at RT. Add 25 &#181;L of RNase-free H2O to dissolve the RNA antisense and sense probes.</li>
		<li>If the length of the inserted gene is longer than 1 kb, subsequently fragment RNA antisense and sense probes to an average length of &#65374;300 bp by incubation in a bicarbonate-carbonate buffer solutions. Perform the reaction in a 50 &#181;L final volume, containing 25 &#181;L of the RNA probe and 25 &#181;L of the bicarbonate-carbonate buffer solution (80 mM NaHCO3, 120 mM Na2CO3, pH 10.2) at 60 &#176;C. The hydrolysis time required is given by a previously published formula17.</li>
		<li>Add 5 &#181;L of 10% acetic acid to stop the reaction. In this experiment, fragment the LmigOR2 and LmigORco antisense and sense probes for 24 and 23 min, respectively. 
		t = (Lo-Lf)/kXLoXLf 
		Lo= the initial fragment lengths in kb 
		Lf=the final fragment lengths in kb 
		k=the rate constant for hydrolysis, approximately 0.11 kb-1min-1 
		t=the hydrolysis time in min</li>
		<li>Finally, add 250 &#181;L of hybridization buffer and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of Cryostat Sections
<ol>
	<li>Precool the freezing microtome to -24 &#176;C.</li>
	<li>Select new molting adult locusts that are active and have intact antennae. Cut the antennae into 2-3 mm pieces using sterile razors.</li>
	<li>Put O.C.T. compound on a freezing microtome holder and put one or two samples on the compound horizontally. Then, transfer the holder into the freezing microtome at -24 &#176;C to equilibrate until the compound freezes. Take out the holder and cover the samples with a little compound. Transfer the holder into the freezing microtome at -24 &#176;C again for at least 10 min (Figure 1). 
	NOTE: Avoid getting bubbles in the compound.</li>
	<li>Fix the holder with the samples into the freezing microtome, then section the frozen samples into 12 &#181;m-thick slices at -24 &#176;C.</li>
	<li>Thaw mount the slices one by one on slides (25 &#160;x 75 mm; nuclease-free) and air dry for 10 min.</li>
</ol>
3. Fixing Sections
<ol>
	<li>After preparing cryostat sections, put the slides in a plastic container (&#65374;100 &#160;x 40 &#160;x 80 mm), and fix the tissues by incubating the slides in 4% paraformaldehyde solution (PFA) for 30 min at 4 &#176;C.</li>
	<li>Wash the slides in 1X Phosphate-Buffered Saline (PBS) for 1 min.</li>
	<li>To eliminate the alkaline proteins, transfer the slides to 0.2 M HCl for 10 min.</li>
	<li>To eliminate the surface protein of nucleic acid, transfer the slides to 1XPBS with 1% Triton X-100 for 2 min.</li>
	<li>Wash the slides twice for 30 s in 1x PBS.</li>
	<li>Finally, rinse the slides in formamide solution for 10 min at 4 &#176;C.</li>
</ol>
4. Hybridization
<ol>
	<li>Prepare the antisense and sense probes
	<ol>
		<li>Use hybridization buffer to dilute RNA antisense or sense probes in the 1.5 mL nuclease-free tubes. In this experiment, for all of the antisense and sense probes (LmigOR1, LmigOR2, and LmigOrco), add 1 &#181;L of probe to 99 &#181;L hybridization buffer per slide. Generally, the use of 1:100 dilutions of antisense probes produces significant positive signals and low backgrounds using this protocol.</li>
		<li>For chromogenic detection, dilute DIG-labeled probes individually.</li>
		<li>For TSA detection, dilute DIG-labeled LmigOR1 with biotin-labeled LmigOrco probes or DIG-labeled LmigOR2 with biotin-labeled LmigOR1 probes&#160;together in the hybridization buffer.</li>
		<li>Heat the dilutions for 10 min at 65 &#176;C and put them on ice for at least 5 min.</li>
	</ol>
	</li>
	<li>Hybridization
	<ol>
		<li>Drain the slides and add 100 &#181;L of diluted antisense and sense probes (Step 4.1) to the tissue sections. Then, place coverslips (24 mm x 50 mm; nuclease-free) on the tissue sections.</li>
		<li>Place the covered slides horizontally into a humid box (&#8764;300 &#160;x 180 &#160;x 50 mm3; Figure 2) and incubate at 55 &#176;C for 22 h. Add formamide solution or 1X PBS to the bottom of the box to keep the environment moist, but do not submerge the slides in the liquid.</li>
	</ol>
	</li>
	<li>Washing and blocking.
	<ol>
		<li>After hybridization, remove the coverslips carefully. Wash the slides twice for 30 min in 0.1x Saline Sodium Citrate (SSC) at 60 &#176;C. 
		NOTE: Washing means putting the slides in a slide holder that is then placed into a plastic container and gently agitated on a rocker (&#65374;50 rpm; Figure 2).</li>
		<li>Rinse the slides in 1x Tris-Buffered Saline (TBS) for 30 s.</li>
		<li>Add 1 mL of 1% blocking reagent in TBS supplemented with 0.03% Triton X-100 on each slide, and incubate for 30 min. Then, discard the blocking solution.</li>
	</ol>
	</li>
	<li>Immunohistochemistry.
	<ol>
		<li>For chromogenic detection, use blocking reagent in TBS to dilute 750 units (U)/mL anti-digoxigenin AP-conjugated antibody to 1.5 U/mL AP solution. Add 100 &#181;L of AP solution per covered (24 mm x 50 mm) slide.</li>
		<li>For TSA detection, use blocking reagent in TBS to dilute 750 U/mL of anti-digoxigenin AP- conjugated antibody and anti-biotin streptavidin HRP- conjugated antibody to the AP/HRP solution. Add 100 &#181;L of AP/HRP solution per covered (24 mm x 50 mm) slide.</li>
		<li>Incubate the slides in a humid box (Figure 2) for 60 min at 37 &#176;C. Use the formamide solution or 1X PBS to keep box moist but not soggy.</li>
	</ol>
	</li>
</ol>
5. Staining
<ol>
	<li>Remove the coverslips carefully. Wash the slides three times for 5 min in 1x TBS supplemented with 0.05% Tween-20. Rinse the slides in DAP-buffer (chromogenic detection: pH 9.5; TSA detection: pH 8.0) for 5 min.</li>
	<li>Staining (chromogenic detection)
	<ol>
		<li>Add 100 &#181;L of NBT (375 &#181;g/mL)/BCIP (188 &#181;g/mL) substrate solution (diluted in DAP; pH 9.5) to every slide. Carefully place coverslips (24&#160;x 50 mm) onto the slides.</li>
		<li>Incubate the slides in a humid box with substrate solution for 10 min - O/N at 37 &#176;C. 
		NOTE: Check the development by looking at the slides from time to time under the microscope.</li>
		<li>When the development is ready, stop the reaction by transferring the slides into water.</li>
	</ol>
	</li>
	<li>Staining (TSA detection)
	<ol>
		<li>Use a syringe to move the HNPP (100 &#181;g/mL)/Fast Red (250 &#181;g/mL) substrate from the syringe filter (0.22 &#181;m; Figure 2).</li>
		<li>Add 100 &#181;L of HNPP/Fast Red substrate per slide covered with a coverslip (24&#160;x 50 mm). Incubate the slides for 30 min with HNPP/Fast Red substrate at RT.</li>
		<li>Remove the coverslips carefully, and wash the slides three times for 5 min in 1X TBS supplemented with 0.05% Tween-20.</li>
		<li>Use 100 &#181;L of TSA substrate/covered (24&#160;x 50 mm2) slide. Incubate the slides with TSA substrate for 10 min at RT.</li>
		<li>Remove the coverslips carefully, and wash the slides three times for 5 min in 1x TBS supplemented with 0.05% Tween-20.</li>
	</ol>
	</li>
	<li>Embed the slides in PBS/glycerol (1:3). 
	NOTE: After finishing these procedures, the slides should be observed as soon as possible because the fluorescent signal will quench quickly.</li>
</ol>
6. Observation
<ol>
	<li>Chromogenic detection
	<ol>
		<li>Observe tissue sections using an optical microscope. Choose the 10X, 20X, and 40X objective lenses to observe the results of chromogenic detection.</li>
		<li>Use DP-BSW software to analyze the results and capture the images.</li>
	</ol>
	</li>
	<li>TSA detection
	<ol>
		<li>Observe tissue sections using a confocal microscope. DIG-labeled genes should be observed under 543 nm light, presenting a red color, and biotin-labeled genes should be observed under 488 nm light, presenting a green color. When they emerge, they present yellow color.</li>
		<li>Choose the 20X and 40X objective lenses to observe the results of TSA detection, respectively.</li>
		<li>Use FV1000 software to analyze the results and capture the images.</li>
	</ol>
	</li>
</ol>

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Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate.</a> Histochemistry. 98 (4), 217-228 (1992).</li><li>Leary, J. J., Brigati, D. J., Ward, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+colorimetric+method+for+visualizing+biotin-labeled+DNA+probes+hybridized+to+DNA+or+RNA+immobilized+on+nitrocellulose:+Bio-blots.">Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.</a> Proc Natl Acad Sci U S A. 80 (13), 4045-4049 (1983).</li><li>Hallem, E. A., Ho, M. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+odor+coding+in+the+Drosophila+antenna.">The molecular basis of odor coding in the Drosophila antenna.</a> Cell. 117 (7), 965-979 (2004).</li><li>Jones, W. D., Nguyen, T. A., Kloss, B., Lee, K. J., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+conservation+of+an+insect+odorant+receptor+gene+across+250+million+years+of+evolution.">Functional conservation of an insect odorant receptor gene across 250 million years of evolution.</a> Curr Biol. 15 (4), R119-R121 (2005).</li></ol>

The authors have nothing to disclose or any other conflicts of interest.

It is hard to perform RNA ISH to localize OR genes in insect antennae because the expression levels of OR genes, except ORco, are very low and preserving histological slices of insect antennae is very difficult. In addition, TSA detection is also very tricky. To address these problems, the following measures should be taken. The antennae are selected from newly molting adult locusts that have thin and soft antennal cuticles, which maintain their morphology on the slide. The frozen samples are sectioned into 12 &#181;m-thick slices. Highly sensitive and specific biotin- and DIG-labeled probes are used. Detergents, such as Tween-20 and Triton X-100, are used to decrease the background in many steps. In TSA detection, a highly expressed gene, relative to another lowly expressed gene, should be labeled with biotin-16-UTP. The slides should be observed as soon as possible because the fluorescent signals will quench quickly.
DIG- and biotin-labeled probes have the advantages of longer shelf lives, higher signal to noise ratios and better cellular resolutions than radioactive probes18,19. The protocol presented in this paper has some advantages over whole mount in situ hybridization. This protocol easily identifies the localizations of genes at the cellular level but cannot be easily used to investigate gene distribution patterns, which is more readily performed with whole mount in situ hybridization.
To identify an OR gene, two approaches were taken. One was the chromogenic detection in consecutive sections, and the other was fluorescent detection in one section. ORco is co-expressed with a specific OR as an ORN marker10,20,21. Cells expressing LmigOR1 or LmigOR2 were both located to clusters of LmigORco-expressing cells that unambiguously verified them as ORs (Figure 4 and Figure 6). Using the same approaches, we found that LmigOR1 and LmigOR2 are not co-expressed in one sensillum. The results of these two approaches are corroborative.
This protocol was also successfully used to localize LmigORco, SgreORco, SgreIR8a and SgreIR25a in locust antennae10,14. Recently LmigOR3 was localized to trichoid sensilla neurons in L. migratoria using the same protocol15. This protocol was used to localize two sensory neural membrane proteins SgreSNMP1 and SgreSNMP2 in the antennae of S. gregaria16. Thus, this protocol reliably localized chemosensory-related genes in locust antennae, which not only verified these candidate genes, but also localized these genes to specific cells housed in different types of sensilla.
In conclusion, this highly effective protocol of RNA ISH is specifically described to localize OR genes, as well as other genes expressed at low levels, in insect antennae.

Insect antennae, which are the most important chemosensory organs, are covered with many hair-like structures &#8211; called sensilla &#8211; that are innervated by Olfactory Receptor Neurons (ORNs). On the membrane of insect ORNs, Odorant Receptors (ORs), a type of protein containing seven transmembrane domains, are expressed with a coreceptor (ORco) to form a heteromer that functions as an odorant-gated ion channel1,2,3. Different ORs respond to different combinations of chemical compounds4,5,6.
Locusts (Locusta migratoria) mainly rely on olfactory cues to trigger important behaviors7. Locust ORs are key factors for understanding molecular olfactory mechanisms. Localizing a specific locust OR gene to the neuron of a morphologically specific sensillum type by RNA In Situ&#160;Hybridization (RNA ISH) is the first step in exploring the ORs function.
RNA ISH uses a labeled complementary RNA probe to measure and localize a specific RNA sequence in section of tissue, cells or whole mounts in situ, providing insights into physiological processes and disease pathogenesis. Digoxigenin-labeled (DIG-labeled) and biotin-labeled RNA probes have been widely used in RNA hybridization. RNA labeling with digoxigenin-11-UTP or biotin-16-UTP can be prepared by in vitro transcription with SP6 and T7 RNA polymerases. DIG- and biotin-labeled RNA probes have the following advantages: non-radioactive; safe; stable; highly sensitive; highly specific; and easy to produce using PCR and in vitro transcription. DIG- and biotin-labeled RNA probes can be chromogenically and fluorescently detected. DIG-labeled RNA probes can be detected with anti-digoxigenin Alkaline Phosphatase (AP)-conjugated antibodies that can be visualized either with the highly sensitive chemiluminescent substrates nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate toluidine salt (NBT/BCIP) using an optical microscope or with 2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate (HNPP) coupled with 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (Fast Red) using a confocal microscope. Biotin-labeled RNA probes can be detected with anti-biotin streptavidin Horse Radish Peroxidase (HRP)-conjugated antibodies that can be visualized with fluorescein-tyramides using a confocal microscope. Thus, double-color fluorescent in situ hybridization can be performed to detect two target genes in one slice using DIG- and biotin-labeled RNA probes.
RNA ISH with DIG- and/or biotin-labeled probes has been successfully used to localize olfactory-related genes, such as OR, ionotropic receptor, odorant-binding protein and sensory neuron membrane protein, in insect antennae of, but not limited to, Drosophila melanogaster, Anopheles gambiae, L. migratoria and the desert locust Schistocera gregaria8,9,10,11,12,13,14,15,16. However, there are two substantial challenges when performing RNA ISH for insect ORs: (1) OR genes (except ORco) are expressed at low levels and only in a few cells, making signal detection very difficult, and (2) preserving insect tissue for histology, such that the morphology is preserved and the background noise is low, can be challenging. In this paper a detailed and effective protocol describing RNA ISH for localizing OR genes in insect antennae is presented, including both chromogenic and Tyramide Signal Amplification (TSA) detection.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2&amp;times;TSINGKE&lt;sup&gt;TM&lt;/sup&gt; Master Mix</td><td>TSINGKE, China</td><td>TSE004</td><td></td></tr><tr><td>RNase-free H&lt;sub&gt;2&lt;/sub&gt;O</td><td>TIANGEN, China</td><td>RT121-02</td><td></td></tr><tr><td>REGULAR AGAROSE G-10</td><td>BIOWEST, SPAIN</td><td>91622</td><td></td></tr><tr><td>Binding buffer</td><td>TIANgel Midi Purification Kit, TIANGEN, China</td><td>DP209-02</td><td></td></tr><tr><td>Balance buffer</td><td>TIANgel Midi Purification Kit, TIANGEN, China</td><td>DP209-02</td><td></td></tr><tr><td>Wash solution</td><td>TIANgel Midi Purification Kit, TIANGEN, China</td><td>DP209-02</td><td></td></tr><tr><td>T Vector</td><td>Promega, USA</td><td>A362A</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>Promega, USA</td><td>M180A</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; DH5&amp;alpha;</td><td>TIANGEN, China</td><td>CB101</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma, USA</td><td>A-6140</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside</td><td>Inalco, USA</td><td>1758-1400</td><td></td></tr><tr><td>5-bromo-4-chloro-3-indolyl-&amp;beta;-D-galactopyranoside</td><td>SBS Genetech, China</td><td>GX1-500</td><td></td></tr><tr><td>&lt;em&gt;Nco&lt;/em&gt; I</td><td>BioLabs, New England</td><td>R0193S</td><td></td></tr><tr><td>&lt;em&gt;Spe&lt;/em&gt; I</td><td>BioLabs, New England</td><td>R0133M</td><td></td></tr><tr><td>DIG RNA Labeling Kit</td><td>Roche, Switzerland</td><td>11175025910</td><td></td></tr><tr><td>Biotin RNA Labeling Kit</td><td>Roche, Switzerland</td><td>11685597910</td><td></td></tr><tr><td>DNase</td><td>DIG RNA Labeling Kit, Roche, Switzerland</td><td>11175025910</td><td></td></tr><tr><td>LiCl</td><td>Sinopharm, China</td><td>10012718</td><td></td></tr><tr><td>Ethanol</td><td>Sinopharm, China</td><td>10009257</td><td></td></tr><tr><td>Acetic acid</td><td>BEIJING CHEMICAL REGENTS COMPANY, China</td><td>10000292</td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura Finetek Europe, Zoeterwoude, Netherlands</td><td>4583</td><td></td></tr><tr><td>Slides</td><td>TINA JIN HAO YANG BIOLOGCAL MANUFACTURE CO., LTE, China</td><td>FISH0010</td><td></td></tr><tr><td>HCl</td><td>Sinopharm, China</td><td>80070591</td><td></td></tr><tr><td>Millex</td><td>Millipore, USA</td><td>SLGP033RS</td><td></td></tr><tr><td>Tween 20</td><td>AMRESCO, USA</td><td>0777-500ML</td><td></td></tr><tr><td>Nitroblue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl-phosphate toluidine salt</td><td>Roche, Switzerland</td><td>11175041910</td><td></td></tr><tr><td>Glycerol</td><td>Sinopharm, China</td><td>10010618</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1&amp;times;Tris-acetate-EDTA</td><td>Sigma, USA</td><td>V900483-1KG</td><td>0.04 mol/L Tris-Base</td></tr><tr><td>1&amp;times;Tris-acetate-EDTA</td><td>BEIJING CHEMICAL REGENTS COMPANY, China</td><td>10000292</td><td>0.12% acetic acid</td></tr><tr><td>1&amp;times;Tris-acetate-EDTA</td><td>Sigma, USA</td><td>03677</td><td>Ethylenediaminetetraacetic acid disodium salt (EDTA)</td></tr><tr><td>Luria-Bertani (LB) liquid medium</td><td>Sinopharm, China</td><td>10019392</td><td>10 g/L NaCl</td></tr><tr><td>Luria-Bertani (LB) liquid medium</td><td>MERCK, Germany</td><td>VM335231</td><td>10 g/L Peptone from casein (Tryptone)</td></tr><tr><td>Luria-Bertani (LB) liquid medium</td><td>MERCK, Germany</td><td>VM361526</td><td>5 g/L Yeast extract</td></tr><tr><td>LB solid substrate plate</td><td>Sinopharm, China</td><td>10019392</td><td>10 g/L NaCl</td></tr><tr><td>LB solid substrate plate</td><td>MERCK, Germany</td><td>VM335231</td><td>10 g/L Peptone from casein (Tryptone)</td></tr><tr><td>LB solid substrate plate</td><td>MERCK, Germany</td><td>VM361526</td><td>5 g/L Yeast extract</td></tr><tr><td>LB solid substrate plate</td><td>WISENT ING, Canada</td><td>800-010-CG</td><td>15 g/L Agar Bacteriological Grade</td></tr><tr><td>10&amp;times;Phosphate-Buffered Saline (PBS, pH 7.1)</td><td>Sinopharm, China</td><td>10019392</td><td>8.5% NaCl</td></tr><tr><td>10&amp;times;Phosphate-Buffered Saline (PBS, pH 7.1)</td><td>Sigma, USA</td><td>V900041-500G</td><td>14 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>10&amp;times;Phosphate-Buffered Saline (PBS, pH 7.1)</td><td>Sigma, USA</td><td>V900268-500G</td><td>80 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>10&amp;times;Tris-Buffered Saline (TBS, pH 7.5)</td><td>Sigma, USA</td><td>V900483-1KG</td><td>1 M Tris-Base</td></tr><tr><td>10&amp;times;Tris-Buffered Saline (TBS, pH 7.5)</td><td>Sinopharm, China</td><td>10019392</td><td>1.5 M NaCl</td></tr><tr><td>Detection Buffer (DAP) &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; chromogenic detection pH 9.5&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TSA detection pH 8.0</td><td>Sigma, USA</td><td>V900483-1KG</td><td>100 mM Tris-Base</td></tr><tr><td>Detection Buffer (DAP) &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; chromogenic detection pH 9.5&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TSA detection pH 8.0</td><td>Sinopharm, China</td><td>10019392</td><td>100 mM NaCl</td></tr><tr><td>Detection Buffer (DAP) &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; chromogenic detection pH 9.5&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TSA detection pH 8.0</td><td>Sigma, USA</td><td>V900020-500G</td><td>50 mM MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>20&amp;times;saline-sodium citrate (pH 7.0)</td><td>Sinopharm, China</td><td>10019392</td><td>3 M NaCl</td></tr><tr><td>20&amp;times;saline-sodium citrate (pH 7.0)</td><td>Sigma, USA</td><td>V900095-500G</td><td>0.3 M Na-Citrate</td></tr><tr><td>4% paraformaldehyde solution (PFA, pH 9.5)</td><td>Sigma, USA</td><td>V900894-100G</td><td>4% paraformaldehyde</td></tr><tr><td>Sodium Carbonate Buffer</td><td>Sigma, USA</td><td>V900182-500G</td><td>0.1 M NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium Carbonate Buffer (pH 10.2)</td><td>Sigma, USA</td><td>V900182-500G</td><td>80 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium Carbonate Buffer (pH 10.2)</td><td>Sigma, USA</td><td>S7795-500G</td><td>120 mM Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Formamide Solution (pH 10.2)</td><td>MPBIO, USA</td><td>FORMD002</td><td>50% Deionized Formamide</td></tr><tr><td>Formamide Solution (pH 10.2)</td><td></td><td></td><td>5&amp;times;saline-sodium citrate</td></tr><tr><td>Blocking Buffer in TBS</td><td>Roche, Switzerland</td><td>11175041910</td><td>1% Blot</td></tr><tr><td>Blocking Buffer in TBS</td><td>AMRESCO, USA</td><td>0694-500ML</td><td>0.03% Triton X-100</td></tr><tr><td>Blocking Buffer in TBS</td><td></td><td></td><td>1&amp;times;Tris buffered saline</td></tr><tr><td>Alkaline phosphatase solution</td><td>Roche, Switzerland</td><td>11175041910</td><td>1.5 U/mL anti-digoxigenin alkaline phosphatase conjugated antibody</td></tr><tr><td>Alkaline phosphatase solution</td><td></td><td></td><td>Blocking Buffer in TBS</td></tr><tr><td>Alkaline phosphatase/ horse radish peroxidase solution</td><td>Roche, Switzerland</td><td>11175041910</td><td>1.5 U/mL anti-digoxigenin alkaline phosphatase conjugated antibody</td></tr><tr><td>Alkaline phosphatase/ horse radish peroxidase solution</td><td>TSA kit, Perkin Elmer, USA</td><td>NEL701A001KT</td><td>1% anti-biotin streptavidin horse radish peroxidase- conjugated antibody</td></tr><tr><td>Alkaline phosphatase/ horse radish peroxidase solution</td><td></td><td></td><td>Blocking Buffer in TBS</td></tr><tr><td>Hybridization Buffer</td><td>MPBIO, USA</td><td>FORMD002</td><td>50% Deionized Formamide</td></tr><tr><td>Hybridization Buffer</td><td></td><td></td><td>2&amp;times;saline-sodium citrate</td></tr><tr><td>Hybridization Buffer</td><td>Sigma, USA</td><td>D8906-50G</td><td>10% dextran sulphate</td></tr><tr><td>Hybridization Buffer</td><td>invitrogen, USA</td><td>AM7119</td><td>20 &amp;micro;g/mL yeast t-RNA</td></tr><tr><td>Hybridization Buffer</td><td>Sigma, USA</td><td>D3159-10G</td><td>0.2 mg/mL herring sperm DNA</td></tr><tr><td>2-hydroxy-3-naphtoic acid-2&#039;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td><td>Roche, Switzerland</td><td>11758888001</td><td>1% 2-hydroxy-3-naphtoic acid-2&#039;-phenylanilide phosphate (10 mg/mL)</td></tr><tr><td>2-hydroxy-3-naphtoic acid-2&#039;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td><td>Roche, Switzerland</td><td>11758888001</td><td>1% 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (25 mg/mL)</td></tr><tr><td>2-hydroxy-3-naphtoic acid-2&#039;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td><td></td><td></td><td>Detection Buffer</td></tr><tr><td>Tyramide signal amplification substrate</td><td>TSA kit, Perkin Elmer, USA</td><td>NEL701A001KT</td><td>2% fluorescein-tyramides</td></tr><tr><td>Tyramide signal amplification substrate</td><td>TSA kit, Perkin Elmer, USA</td><td>NEL701A001KT</td><td>Amplification Diluent</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instrument&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Freezing microtome</td><td>Leica, Nussloch, Germany</td><td>Jung CM300 cryostat</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo SCIENTIFIC, USA</td><td>NANODROP 2000</td><td></td></tr><tr><td>Optical microscope</td><td>Olympus, Tokyo, Japan</td><td>Olympus IX71microscope</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus, Tokyo, Japan</td><td>Olympus BX45 confocal microscope</td><td></td></tr></tbody></table>

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.

With chromogenic detection, a small subset of the antennal cells in every adult antennal section was denoted by the DIG-labeled LmigOR1 and LmigOR2 antisense probes (Figure 3). RNA ISH on consecutive sections to localize LmigOR1 and LmigOR2 showed that antennal cells expressing the two genes were located in ORN clusters expressing LmigORco, indicating that the putative LmigOR1 and LmigOR2 were actually expressed in ORNs (Figure 4a-4d). Occasionally, labeled dendritic-like structures were visualized (Figure 4e-4f). LmigOR1 and LmigOR2 were both localized to neurons in the basiconic sensilla (Figure 5a-5b), but they were not co-expressed in individual sensilla, indicating that they were present in different basiconic sensillium subtypes (Figure 5c-5d).
In TSA detection, double-color fluorescent in situ hybridization showed that LmigOR1-expressing cells (red color) were located to ORN clusters expressing LmigORco (green color), indicating that the putative LmigOR1 was expressed in ORNs (Figure 6a). A close view of the boxed areas in Figure 6a are shown in Figure 6b. LmigOR1- expressing neurons (green color, Figure 7a) and LmigOR2-expressing neuron (red color, Figure 7b) were located to different basiconic sensillium subtypes (Figure 7c).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55924/55924fig1.jpg" /> 
Figure 1: Preparation of Insect Antennal Sections for RNA In Situ Hybridization. (a) The freezing microtome; (b-c) The samples are embedded in freezing O.C.T. Compound. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55924/55924fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55924/55924fig2.jpg" /> 
Figure 2: Experimental Items for RNA In Situ&#160;Hybridization. (a) The slide holder and the plastic container. (b) The humid box. (c) The rocker. (d) The membrane filter. (e) Confocal microscope. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55924/55924fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55924/55924fig3.jpg" /> 
Figure 3: Cellular Localization of LmigOR1 &#38; LmigOR2 in Olfactory Organs. (a) Overview of LmigOR1-expressing cells in a locust antennal segment. (b) Overview of LmigOR2-expressing cells in a locust antennal segment. Arrowheads indicate cells expressing LmigOR1 (a) and LmigOR2 (b). Scale bars = 100 &#181;m (a and b). Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55924/55924fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55924/55924fig4.jpg" /> 
Figure 4: Neuronal Identity of Antennal Cells Expressing LmigORs by Chromogenic Detections. (a-b) The labeling pattern of LmigOR1 (a) and LmigORco (b) antisense probe on consecutive sections of locust antenna. (c-d) The labeling pattern of LmigORco (c) and LmigOR2 (d) antisense probe on consecutive sections of locust antenna. (e-f) Illustration of occasionally labeled dendritic-like structures (indicated by red arrows). Scale bars = 50 &#181;m (a-d); 20 &#181;m (e and f). Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55924/55924fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55924/55924fig5.jpg" /> 
Figure 5: LmigOR1 &#38; LmigOR2&#160;are Expressed in ORNs Housed in Basiconic Sensilla. (a-b) Basiconic sensillum housed ORNs expressing LmigOR1 (a) and LmigOR2 (b). (c-d) The expression of LmigOR1 and LmigOR2 in distinct subset of antennal ORNs was verified on consecutive sections (c-d). Arrowheads denote antennal cells expressing LmigOR1 (a, c) and LmigOR2 (b, d). Ba: basiconic sensillum. Scale bars = 20 &#181;m (a and b); 50 &#181;m (c and d). Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55924/55924fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55924/55924fig6.jpg" /> 
Figure 6: Neuronal Identity of Antennal Cell Expressing LmigOR1 by TSA Detections. (a) Two-color in situ hybridization was performed on longitudinal antennal section to illustrate the expression of LmigOR1 (Red) and LmigORco (Green). Localization of LmigOR1-expressing cells in cell clusters expressing LmigORco confirmed its neural identity. (b) Close view of boxed areas in a. Occasionally labeled dendritic like structures were indicated by arrow. Circled areas indicate ORNs cluster expressing LmigORco and sharing the same sensillum. Scale bars = 50 &#181;m (a); 20 &#181;m (b). Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55924/55924fig6large.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/55924/55924fig7.jpg" /> 
Figure 7: LmigOR1 &#38; LmigOR2 were Located to Different Basiconic Sensilla ORNs by TSA Detection. Fluorescent signals were visualized using detection systems, indicating LmigOR1-labeled neurons by green fluorescence (a) and LmigOR2 positive cells by red fluorescence (b) were expressed in different basiconic sensilla subtypes (c). Arrowheads denote antennal cells expressing LmigOR1 and LmigOR2. Scale bars = 20 &#181;m. Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55924/55924fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Sequences</td>
		</tr>
		<tr>
			<td>Lmig OR1-probe-s</td>
			<td>5&#8217;-AAGGGGTGGGAGACGGCCTG-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig OR1-probe-as</td>
			<td>5&#8217;-CAGCTCCTCCCCAACGACAGC-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig OR2-probe-s</td>
			<td>5&#8217;-ATGGGTGAGCGTGGAGAGGC-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig OR2-probe-as</td>
			<td>5&#8217;-GGTCATCGCTGTGGACGTGG-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig Orco-probe-s</td>
			<td>5&#8217;-CTCGTCTGACAGCGTAACTCAC-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig Orco-probe-as</td>
			<td>5&#8217;-AAGACGCAGAAGAGGAAGACCT-3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 1: The Sequences of the PCR Primers.

Watch this Scientific Journal Video about Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization at JoVE.com

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.

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 ...

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8.0K Views.  China Agricultural University. 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.

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

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

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

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

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

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

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

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

Developmental Biology

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Electroantennography-based Bio-hybrid Odor-detecting Drone using Silkmoth Antennae for Odor Source Localization

Small drones with chemical or biosensor devices that can detect airborne odorant molecules have attracted considerable attention owing to their applicability in environmental and security monitoring and search-and-rescue operations. Small drones with commercial metal-oxide-semiconductor (MOX) gas sensors have been developed for odor source localization; however, their real-time-odor-detection performance has proven inadequate. However, biosensing technologies based on insect olfactory systems exhibit relatively high sensitivity, selectivity, and real-time response with respect to odorant molecules compared to commercial MOX gas sensors. In such devices, excised insect antennae function as portable odorant biosensor elements and have been found to deliver excellent sensing performance. This study presents experimental protocols for odorant-molecule detection in the air using a small autonomous bio-hybrid drone based on a mountable electroantennography (EAG) device incorporating silkmoth antennae.
We developed a mountable EAG device including sensing/processing parts with a Wi-Fi module. The device was equipped with a simple sensor enclosure to enhance the sensor directivity. Thus, odor source localization was conducted using the spiral-surge algorithm, which does not assume an upwind direction. The experimental bio-hybrid odor-detecting drone identified real-time odorant-concentration differences in a pseudo-open environment (outside a wind tunnel) and localized the source. The developed drone and associated system can serve as an efficient odorant molecule-detection tool and a suitable flight platform for developing odor source localization algorithms owing to its high programmability.

This study introduces experimental protocols for a bio-hybrid odor-detecting drone based on silkmoth antennae. The operation of an experimental electroantennogram device with silkmoth antennae is presented, in addition to the structure of a bio-hybrid drone designed for odor source localization using the spiral-surge algorithm.

Small drones with chemical or biosensor devices that can detect airborne odorant molecules have attracted considerable attention owing to their ...

Engineering

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

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. ...

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially for the detection of non-volatile chemical cues through sensilla chaetica (previously named terminal sensilla or crested sensilla). There is now increasing evidence that these palps also have an olfactory function. An odorant receptor (LmigOR2) and an odorant-binding protein (LmigOBP1) have been localized in the neurons and accessory cells, respectively, in the sensilla basiconica of the palps. Single sensillum recording (SSR) is used for recording the responses of odorant receptor neurons, which is an effective method for screening active ligands on specific odorant receptors. SSR is used in functional studies of odorant receptors in palp sensilla. The structure of the sensilla basiconica located on the dome of the palps differs somewhat from the structure of those on the antennae. Therefore, when performing an SSR elicited by odorants, some specific advice may be helpful for obtaining optimum results. In this paper, a detailed and highly effective protocol for an SSR from insect palp sensilla basiconica is introduced.

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

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

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Chapters in this video

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

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

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Electroantennography-based Bio-hybrid Odor-detecting Drone using Silkmoth Antennae for Odor Source Localization

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

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

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization