The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch.

Atopic dermatitis (AD) is a chronic itch and inflammatory disorder of the skin that affects one in ten people. Patients suffering from severe AD eventually progress to develop asthma and allergic rhinitis, in a process known as the "atopic march." Signaling between epithelial cells and innate immune cells via the cytokine thymic stromal lymphopoietin (TSLP) is thought to drive AD and the atopic march. Here, we report that epithelial cells directly communicate to cutaneous sensory neurons via TSLP to promote itch. We identify the ORAI1/NFAT calcium signaling pathway as an essential regulator of TSLP release from keratinocytes, the primary epithelial cells of the skin. TSLP then acts directly on a subset of TRPA1-positive sensory neurons to trigger robust itch behaviors. Our results support a model whereby calcium-dependent TSLP release by keratinocytes activates both primary afferent neurons and immune cells to promote inflammatory responses in the skin and airways.

Transcriptional profiling of identified neurons in leech.

BACKGROUND: While leeches in the genus Hirudo have long been models for neurobiology, the molecular underpinnings of nervous system structure and function in this group remain largely unknown. To begin to bridge this gap, we performed RNASeq on pools of identified neurons of the central nervous system (CNS): sensory T (touch), P (pressure) and N (nociception) neurons; neurosecretory Retzius cells; and ganglia from which these four cell types had been removed. RESULTS: Bioinformatic analyses identified 3565 putative genes whose expression differed significantly among the samples. These genes clustered into 9 groups which could be associated with one or more of the identified cell types. We verified predicted expression patterns through in situ hybridization on whole CNS ganglia, and found that orthologous genes were for the most part similarly expressed in a divergent leech genus, suggesting evolutionarily conserved roles for these genes. Transcriptional profiling allowed us to identify candidate phenotype-defining genes from expanded gene families. Thus, we identified one of eight hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as a candidate for mediating the prominent sag current in P neurons, and found that one of five inositol triphosphate receptors (IP3Rs), representing a sub-family of IP3Rs absent from vertebrate genomes, is expressed with high specificity in T cells. We also identified one of two piezo genes, two of ~ 65 deg/enac genes, and one of at least 16 transient receptor potential (trp) genes as prime candidates for involvement in sensory transduction in the three distinct classes of leech mechanosensory neurons. CONCLUSIONS: Our study defines distinct transcriptional profiles for four different neuronal types within the leech CNS, in addition to providing a second ganglionic transcriptome for the species. From these data we identified five gene families that may facilitate the sensory capabilities of these neurons, thus laying the basis for future work leveraging the strengths of the leech system to investigate the molecular processes underlying and linking mechanosensation, cell type specification, and behavior.

High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics.

To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next-generation sequencing data. By using this approach, we sequenced longitudinally collected acute myeloid leukemia (AML) tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.

A natural polymorphism alters odour and DEET sensitivity in an insect odorant receptor.

Blood-feeding insects such as mosquitoes are efficient vectors of human infectious diseases because they are strongly attracted by body heat, carbon dioxide and odours produced by their vertebrate hosts. Insect repellents containing DEET (N,N-diethyl-meta-toluamide) are highly effective, but the mechanism by which this chemical wards off biting insects remains controversial despite decades of investigation. DEET seems to act both at close range as a contact chemorepellent, by affecting insect gustatory receptors, and at long range, by affecting the olfactory system. Two opposing mechanisms for the observed behavioural effects of DEET in the gas phase have been proposed: that DEET interferes with the olfactory system to block host odour recognition and that DEET actively repels insects by activating olfactory neurons that elicit avoidance behaviour. Here we show that DEET functions as a modulator of the odour-gated ion channel formed by the insect odorant receptor complex. The functional insect odorant receptor complex consists of a common co-receptor, ORCO (ref. 15) (formerly called OR83B; ref. 16), and one or more variable odorant receptor subunits that confer odour selectivity. DEET acts on this complex to potentiate or inhibit odour-evoked activity or to inhibit odour-evoked suppression of spontaneous activity. This modulation depends on the specific odorant receptor and the concentration and identity of the odour ligand. We identify a single amino-acid polymorphism in the second transmembrane domain of receptor OR59B in a Drosophila melanogaster strain from Brazil that renders OR59B insensitive to inhibition by the odour ligand and modulation by DEET. Our data indicate that natural variation can modify the sensitivity of an odour-specific insect odorant receptor to odour ligands and DEET. Furthermore, they support the hypothesis that DEET acts as a molecular 'confusant' that scrambles the insect odour code, and provide a compelling explanation for the broad-spectrum efficacy of DEET against multiple insect species.

Microfluidic droplet enrichment for targeted sequencing.

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications.

TRPA1: A gatekeeper for inflammation.

Tissue damage evokes an inflammatory response that promotes the removal of harmful stimuli, tissue repair, and protective behaviors to prevent further damage and encourage healing. However, inflammation may outlive its usefulness and become chronic. Chronic inflammation can lead to a host of diseases, including asthma, itch, rheumatoid arthritis, and colitis. Primary afferent sensory neurons that innervate target organs release inflammatory neuropeptides in the local area of tissue damage to promote vascular leakage, the recruitment of immune cells, and hypersensitivity to mechanical and thermal stimuli. TRPA1 channels are required for neuronal excitation, the release of inflammatory neuropeptides, and subsequent pain hypersensitivity. TRPA1 is also activated by the release of inflammatory agents from nonneuronal cells in the area of tissue injury or disease. This dual function of TRPA1 as a detector and instigator of inflammatory agents makes TRPA1 a gatekeeper of chronic inflammatory disorders of the skin, airways, and gastrointestinal tract.

RNA-Seq following PCR-based sorting reveals rare cell transcriptional signatures.

BACKGROUND: Rare cell subtypes can profoundly impact the course of human health and disease, yet their presence within a sample is often missed with bulk molecular analysis. Single-cell analysis tools such as FACS, FISH-FC and single-cell barcode-based sequencing can investigate cellular heterogeneity; however, they have significant limitations that impede their ability to identify and transcriptionally characterize many rare cell subpopulations. RESULTS: PCR-activated cell sorting (PACS) is a novel cytometry method that uses single-cell TaqMan PCR reactions performed in microfluidic droplets to identify and isolate cell subtypes with high-throughput. Here, we extend this method and demonstrate that PACS enables high-dimensional molecular profiling on TaqMan-targeted cells. Using a random priming RNA-Seq strategy, we obtained high-fidelity transcriptome measurements following PACS sorting of prostate cancer cells from a heterogeneous population. The sequencing data revealed prostate cancer gene expression profiles that were obscured in the unsorted populations. Single-cell expression analysis with PACS was subsequently used to confirm a number of the differentially expressed genes identified with RNA sequencing. CONCLUSIONS: PACS requires minimal sample processing, uses readily available TaqMan assays and can isolate cell subtypes with high sensitivity. We have now validated a method for performing next-generation sequencing on mRNA obtained from PACS isolated cells. This capability makes PACS well suited for transcriptional profiling of rare cells from complex populations to obtain maximal biological insight into cell states and behaviors.

Insect olfactory receptors are heteromeric ligand-gated ion channels.

In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. Here we provide evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes showed extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment.

Scalable detection of technically challenging variants through modified next-generation sequencing.

BACKGROUND: Some clinically important genetic variants are not easily evaluated with next-generation sequencing (NGS) methods due to technical challenges arising from high- similarity copies (e.g., PMS2, SMN1/SMN2, GBA1, HBA1/HBA2, CYP21A2), repetitive short sequences (e.g., ARX polyalanine repeats, FMR1 AGG interruptions in CGG repeats, CFTR poly-T/TG repeats), and other complexities (e.g., MSH2 Boland inversions). METHODS: We customized our NGS processes to detect the technically challenging variants mentioned above with adaptations including target enrichment and bioinformatic masking of similar sequences. Adaptations were validated with samples of known genotypes. RESULTS: Our adaptations provided high-sensitivity and high-specificity detection for most of the variants and provided a high-sensitivity primary assay to be followed with orthogonal disambiguation for the others. The sensitivity of the NGS adaptations was 100% for all of the technically challenging variants. Specificity was 100% for those in PMS2, GBA1, SMN1/SMN2, and HBA1/HBA2, and for the MSH2 Boland inversion; 97.8%-100% for CYP21A2 variants; and 85.7% for ARX polyalanine repeats. CONCLUSIONS: NGS assays can detect technically challenging variants when chemistries and bioinformatics are jointly refined. The adaptations described support a scalable, cost-effective path to identifying all clinically relevant variants within a single sample.

Clonal Evolution and Changes in Two AML Patients Detected with A Novel Single-Cell DNA Sequencing Platform.

Next-generation sequencing (NGS) is used to detect gene variants in genetically complex cell populations of cancer patient samples. Traditional bulk analysis can only provide average variant allele frequencies of the targeted genes across all sampled cells. It fails to resolve mutational co-occurrences and may miss rare cancer cells. Genome analysis at the single cell level offers the opportunity to more fully resolve clonal architecture. Peripheral blood mononuclear cells were sampled from acute myeloid leukemia patients longitudinally and single-cell DNA sequencing libraries were generated with a novel droplet-based microfluidics approach. Molecular profiling of single nucleotide variants across thousands of cells revealed genetic chimerism in patients after bone marrow transplantation (BMT). Importantly, hierarchical clustering analysis of single nucleotide variants (SNVs) uncovered a distinct oncogenic clone of cells carrying mutated tumor-suppressor and/or oncogene(s). This novel single-cell DNA sequencing approach enabled precise monitoring of engraftment and revealed clonal evolution of oncogenic cells during the progression and treatment of the disease.

Clonal Selection with RAS Pathway Activation Mediates Secondary Clinical Resistance to Selective FLT3 Inhibition in Acute Myeloid Leukemia.

Gilteritinib is a potent and selective FLT3 kinase inhibitor with single-agent clinical efficacy in relapsed/refractory FLT3-mutated acute myeloid leukemia (AML). In this context, however, gilteritinib is not curative, and response duration is limited by the development of secondary resistance. To evaluate resistance mechanisms, we analyzed baseline and progression samples from patients treated on clinical trials of gilteritinib. Targeted next-generation sequencing at the time of AML progression on gilteritinib identified treatment-emergent mutations that activate RAS/MAPK pathway signaling, most commonly in NRAS or KRAS. Less frequently, secondary FLT3-F691L gatekeeper mutations or BCR-ABL1 fusions were identified at progression. Single-cell targeted DNA sequencing revealed diverse patterns of clonal selection and evolution in response to FLT3 inhibition, including the emergence of RAS mutations in FLT3-mutated subclones, the expansion of alternative wild-type FLT3 subclones, or both patterns simultaneously. These data illustrate dynamic and complex changes in clonal architecture underlying response and resistance to mutation-selective tyrosine kinase inhibitor therapy in AML. SIGNIFICANCE: Comprehensive serial genotyping of AML specimens from patients treated with the selective FLT3 inhibitor gilteritinib demonstrates that complex, heterogeneous patterns of clonal selection and evolution mediate clinical resistance to tyrosine kinase inhibition in FLT3-mutated AML. Our data support the development of combinatorial targeted therapeutic approaches for advanced AML.See related commentary by Wei and Roberts, p. 998.This article is highlighted in the In This Issue feature, p. 983.

Lgr6 is a stem cell marker in mouse skin squamous cell carcinoma.

The G-protein-coupled receptors LGR4, LGR5 and LGR6 are Wnt signaling mediators, but their functions in squamous cell carcinoma (SCC) are unclear. Using lineage tracing in Lgr5-EGFP-CreERT2/Rosa26-Tomato and Lgr6-EGFP-CreERT2/Rosa26-Tomato reporter mice, we demonstrate that Lgr6, but not Lgr5, acts as an epithelial stem cell marker in SCCs in vivo. We identify, by single-molecule in situ hybridization and cell sorting, rare cells positive for Lgr6 expression in immortalized keratinocytes and show that their frequency increases in advanced SCCs. Lgr6 expression is enriched in cells with stem cell characteristics, and Lgr6 downregulation in vivo causes increased epidermal proliferation with expanded lineage tracing from epidermal stem cells positive for Lgr6 expression. Surprisingly, mice with germline knockout of Lgr6 are predisposed to SCC development, through a mechanism that includes compensatory upregulation of Lgr5. These data provide a model for human patients with germline loss-of-function mutations in Wnt pathway genes, including RSPO1 or LGR4, who show increased susceptibility to squamous tumor development.

Microarray and large-scale in silico--based identification of genes functionally related to Haptoglobin and/or Hemopexin.

Haptoglobin and Hemopexin are plasma acute phase proteins that bind with high-affinity hemoglobin and heme, respectively. They play a key role in the protection against oxidative stress and inflammation. To dissect in more detail the mechanism of action of Haptoglobin and Hemopexin, it is important to identify their downstream effectors as well as genes functionally related to them. To this end, we performed a cDNA microarray analysis to compare gene expression profiles of the liver of Haptoglobin and Hemopexin single and double null mice to that of wild-type controls. Then, to extract the best candidates considered to be functionally related to Haptoglobin and/or Hemopexin from microarray-derived gene lists, we used a bioinformatic approach consisting in the screening of published microarray data for genes showing coexpression with Haptoglobin or Hemopexin. This strategy allowed us to identify a group of genes coexpressed with Haptoglobin or Hemopexin and transcriptionally modulated by their lack. These genes present a high probability to be functionally related to Haptoglobin and Hemopexin. Based on literature data, we picked up from this group of genes the ras suppressor Rsu1, the member of the G-protein signal transduction family Gnai2, and the cytokine Mdk as the best candidates mediating the anti-inflammatory action of Haptoglobin and Hemopexin.

Harshlight: a "corrective make-up" program for microarray chips.

BACKGROUND: Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans do show similar artifacts, which might affect subsequent analysis. Although all but the starkest blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs), few tools are available to help with the detection of those defects. RESULTS: We develop a novel tool, Harshlight, for the automatic detection and masking of blemishes in HDONA microarray chips. Harshlight uses a combination of statistic and image processing methods to identify three different types of defects: localized blemishes affecting a few probes, diffuse defects affecting larger areas, and extended defects which may invalidate an entire chip. CONCLUSION: We demonstrate the use of Harshlight can materially improve analysis of HDONA chips, especially for experiments with subtle changes between samples. For the widely used MAS5 algorithm, we show that compact blemishes cause an average of 8 gene expression values per chip to change by more than 50%, two of them by more than twofold; our masking algorithm restores about two thirds of this damage. Large-scale artifacts are successfully detected and eliminated.

HTR7 Mediates Serotonergic Acute and Chronic Itch.

Chronic itch is a prevalent and debilitating condition for which few effective therapies are available. We harnessed the natural variation across genetically distinct mouse strains to identify transcripts co-regulated with itch behavior. This survey led to the discovery of the serotonin receptor HTR7 as a key mediator of serotonergic itch. Activation of HTR7 promoted opening of the ion channel TRPA1, which in turn triggered itch behaviors. In addition, acute itch triggered by serotonin or a selective serotonin reuptake inhibitor required both HTR7 and TRPA1. Aberrant serotonin signaling has long been linked to a variety of human chronic itch conditions, including atopic dermatitis. In a mouse model of atopic dermatitis, mice lacking HTR7 or TRPA1 displayed reduced scratching and skin lesion severity. These data highlight a role for HTR7 in acute and chronic itch and suggest that HTR7 antagonists may be useful for treating a variety of pathological itch conditions.

CLOE: identification of putative functional relationships among genes by comparison of expression profiles between two species.

BACKGROUND: Public repositories of microarray data contain an incredible amount of information that is potentially relevant to explore functional relationships among genes by meta-analysis of expression profiles. However, the widespread use of this resource by the scientific community is at the moment limited by the limited availability of effective tools of analysis. We here describe CLOE, a simple cDNA microarray data mining strategy based on meta-analysis of datasets from pairs of species. The method consists in ranking EST probes in the datasets of the two species according to the similarity of their expression profiles with that of two EST probes from orthologous genes, and extracting orthologous EST pairs from a given top interval of the ranked lists. The Gene Ontology annotation of the obtained candidate partners is then analyzed for keywords overrepresentation. RESULTS: We demonstrate the capabilities of the approach by testing its predictive power on three proteomically-defined mammalian protein complexes, in comparison with single and multiple species meta-analysis approaches. Our results show that CLOE can find candidate partners for a greater number of genes, if compared to multiple species co-expression analysis, but retains a comparable specificity even when applied to species as close as mouse and human. On the other hand, it is much more specific than single organisms co-expression analysis, strongly reducing the number of potential candidate partners for a given gene of interest. CONCLUSIONS: CLOE represents a simple and effective data mining approach that can be easily used for meta-analysis of cDNA microarray experiments characterized by very heterogeneous coverage. Importantly, it produces for genes of interest an average number of high confidence putative partners that is in the range of standard experimental validation techniques.

The star-nosed mole reveals clues to the molecular basis of mammalian touch.

Little is known about the molecular mechanisms underlying mammalian touch transduction. To identify novel candidate transducers, we examined the molecular and cellular basis of touch in one of the most sensitive tactile organs in the animal kingdom, the star of the star-nosed mole. Our findings demonstrate that the trigeminal ganglia innervating the star are enriched in tactile-sensitive neurons, resulting in a higher proportion of light touch fibers and lower proportion of nociceptors compared to the dorsal root ganglia innervating the rest of the body. We exploit this difference using transcriptome analysis of the star-nosed mole sensory ganglia to identify novel candidate mammalian touch and pain transducers. The most enriched candidates are also expressed in mouse somatosesensory ganglia, suggesting they may mediate transduction in diverse species and are not unique to moles. These findings highlight the utility of examining diverse and specialized species to address fundamental questions in mammalian biology.

Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex.

Olfactory receptors (Ors) convert chemical signals--the binding of odors and pheromones--to electrical signals through the depolarization of olfactory sensory neurons. Vertebrates Ors are G-protein-coupled receptors, stimulated by odors to produce intracellular second messengers that gate ion channels. Insect Ors are a heteromultimeric complex of unknown stoichiometry of two seven transmembrane domain proteins with no sequence similarity to and the opposite membrane topology of G-protein-coupled receptors. The functional insect Or comprises an odor- or pheromone-specific Or subunit and the Orco co-receptor, which is highly conserved in all insect species. The insect Or-Orco complex has been proposed to function as a novel type of ligand-gated nonselective cation channel possibly modulated by G-proteins. However, the Or-Orco proteins lack homology to any known family of ion channel and lack known functional domains. Therefore, the mechanisms by which odors activate the Or-Orco complex and how ions permeate this complex remain unknown. To begin to address the relationship between Or-Orco structure and function, we performed site-directed mutagenesis of all 83 conserved Glu, Asp, or Tyr residues in the silkmoth BmOr-1-Orco pheromone receptor complex and measured functional properties of mutant channels expressed in Xenopus oocytes. 13 of 83 mutations in BmOr-1 and BmOrco altered the reversal potential and rectification index of the BmOr-1-Orco complex. Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco) altered both current-voltage relationships and K(+) selectivity. We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined. Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the heteromeric insect Or-Orco complex.

Post-fasting olfactory, transcriptional, and feeding responses in Drosophila.

The sensation of hunger after a period of fasting and of satiety after eating is crucial to behavioral regulation of food intake, but the biological mechanisms regulating these sensations are incompletely understood. We studied the behavioral and physiological adaptations to fasting in the vinegar fly (Drosophila melanogaster). Here we show that both male and female flies increased their rate of food intake transiently in the post-fasted state. Although the basal feeding rate was higher in females than males, the magnitude of the post-fasting feeding response was the same in both sexes. Flies returned to a stable baseline feeding rate within 12 h after return to food for males and 24 h for females. This modulation in feeding was accompanied by a significant increase in the size of the crop organ of the digestive system, suggesting that fasted flies responded both by increasing their food intake and storing reserve food in their crop. Flies demonstrated increased behavioral attraction to an attractive odor when food-deprived. Expression profiling of head, body, and chemosensory tissues by microarray analysis revealed 415 genes regulated by fasting after 24 h and 723 genes after 48 h, with downregulated genes outnumbering upregulated genes in each tissue and fasting time point. These transcriptional changes showed rich temporal dynamics and affected genes across multiple functional gene ontology categories. These observations suggest that a coordinated transcriptional response to internal physiological state may regulate both ingestive behaviors and chemosensory perception of food.

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 sites. 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 channels. 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 odorant. 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 OSNs. 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.