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 learning^4,5. 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 ORNs^6,7. 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.     

Local interneuron diversity in the primary olfactory center of the moth <Emphasis Type="Italic">Manduca sexta</Emphasis>

Local interneurons (LNs) play important roles in shaping and modulating the activity of output neurons in primary olfactory centers. Here, we studied the morphological characteristics, odor responses, and neurotransmitter content of LNs in the antennal lobe (AL, the insect primary olfactory center) of the moth Manduca sexta. We found that most LNs are broadly tuned, with all LNs responding to at least one odorant. 70% of the odorants evoked a response, and 22% of the neurons responded to all the odorants tested. Some LNs showed excitatory (35%) or inhibitory (33%) responses only, while 33% of the neurons showed both excitatory and inhibitory responses, depending on the odorant. LNs that only showed inhibitory responses were the most responsive, with 78% of the odorants evoking a response. Neurons were morphologically diverse, with most LNs innervating almost all glomeruli and others innervating restricted portions of the AL. 61 and 39% of LNs were identified as GABA-immunoreactive (GABA-ir) and non-GABA-ir, respectively. We found no correlations between odor responses and GABA-ir, neither between morphology and GABA-ir. These results show that, as observed in other insects, LNs are diverse, which likely determines the complexity of the inhibitory network that regulates AL output.     

Physiology and glomerular projections of olfactory receptor neurons on the antenna of female Heliothis virescens (Lepidoptera: Noctuidae) responsive to behaviorally relevant odors

The neurophysiology and antennal lobe projections of olfactory receptor neurons housed within short trichoid sensilla of female Heliothis virescens F. (Noctuidae: Lepidoptera) were investigated using a combination of cut-sensillum recording and cobalt-lysine staining techniques. Behaviorally relevant odorants, including intra- and inter-sexual pheromonal compounds, plant and floral volatiles were selected for testing sensillar responses. A total of 184 sensilla were categorized into 25 possible sensillar types based on odor responses and sensitivity. Sensilla exhibited both narrow (responding to few odors) and broad (responding to many odors) response spectra. Sixty-six percent of the sensilla identified were stimulated by conspecific odors; in particular, major components of the male H. virescens hairpencil pheromone (hexadecanyl acetate and octadecanyl acetate) and a minor component of the female sex pheromone, (Z)-9-tetradecenal. Following characterization of the responses, olfactory receptor neurons within individual sensilla were stained with cobalt lysine (N=39) and traced to individual glomeruli in the antennal lobe. Olfactory receptor neurons with specific responses to (Z)-9-tetradecenal, a female H. virescens sex pheromone component, projected to the female-specific central large female glomerulus (cLFG) and other glomeruli. Terminal arborizations from sensillar types containing olfactory receptor neurons sensitive to male hairpencil components and plant volatiles were also localized to distinct glomerular locations. This information provides insight into the representation of behaviorally relevant odorants in the female moth olfactory system. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Maturation of odor representation in the honeybee antennal lobe

The antennal lobe (AL) is the first center for processing odors in the insect brain, as is the olfactory bulb (OB) in vertebrates. Both the AL and the OB have a characteristic glomerular structure; odors sensed by olfactory receptor neurons are represented by patterns of glomerular activity. Little is known about when and how an odor begins to be perceived in a developing brain. We address this question by using calcium imaging to monitor odor-evoked neural activity in the ALs of bees of different ages. We find that odor-evoked neural activity already occurs in the ALs of bees as young as 1 or 2 days. In young bees, the responses to odors are relatively weak and restricted to a small number of glomeruli. However, different odors already evoke responses in different combinations of glomeruli. In mature bees, the responses are stronger and are evident in more glomeruli, but continue to have distinct odor-dependent signatures. Our findings indicate that the specific glomerular patterns for odors are conserved during the development, and that odor representations are fully developed in the AL during the first 2 weeks following emergence.     

Distribution of neuropeptides in the antennal lobes of male Spodoptera littoralis

Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.     

Ensemble Response in Mushroom Body Output Neurons of the Honey Bee Outpaces Spatiotemporal Odor Processing Two Synapses Earlier in the Antennal Lobe

Neural representations of odors are subject to computations that involve sequentially convergent and divergent anatomical connections across different areas of the brains in both mammals and insects. Furthermore, in both mammals and insects higher order brain areas are connected via feedback connections. In order to understand the transformations and interactions that this connectivity make possible, an ideal experiment would compare neural responses across different, sequential processing levels. Here we present results of recordings from a first order olfactory neuropile – the antennal lobe (AL) – and a higher order multimodal integration and learning center – the mushroom body (MB) – in the honey bee brain. We recorded projection neurons (PN) of the AL and extrinsic neurons (EN) of the MB, which provide the outputs from the two neuropils. Recordings at each level were made in different animals in some experiments and simultaneously in the same animal in others. We presented two odors and their mixture to compare odor response dynamics as well as classification speed and accuracy at each neural processing level. Surprisingly, the EN ensemble significantly starts separating odor stimuli rapidly and before the PN ensemble has reached significant separation. Furthermore the EN ensemble at the MB output reaches a maximum separation of odors between 84–120 ms after odor onset, which is 26 to 133 ms faster than the maximum separation at the AL output ensemble two synapses earlier in processing. It is likely that a subset of very fast PNs, which respond before the ENs, may initiate the rapid EN ensemble response. We suggest therefore that the timing of the EN ensemble activity would allow retroactive integration of its signal into the ongoing computation of the AL via centrifugal feedback.     

Local interneurons and information processing in the olfactory glomeruli of the moth Manduca sexta

Intracellular recordings were made from the major neurites of local interneurons in the moth antennal lobe. Antennal nerve stimulation evoked 3 patterns of postsynaptic activity: (i) a short-latency compound excitatory postsynaptic potential that, based on electrical stimulation of the antennal nerve and stimulation of the antenna with odors, represents a monosynaptic input from olfactory afferent axons (71 out of 86 neurons), (ii) a delayed activation of firing in response to both electrical- and odor-driven input (11 neurons), and (iii) a delayed membrane hyperpolarization in response to antennal nerve input (4 neurons).Simultaneous intracellular recordings from a local interneuron with short-latency responses and a projection (output) neuron revealed unidirectional synaptic interactions between these two cell types. In 20% of the 30 pairs studied, spontaneous and current-induced spiking activity in a local interneuron correlated with hyperpolarization and suppression of firing in a projection neuron. No evidence for recurrent or feedback inhibition of projection neurons was found. Furthermore, suppression of firing in an inhibitory local interneuron led to an increase in firing in the normally quiescent projection neuron, suggesting that a disinhibitory pathway may mediate excitation in projection neurons. This is the first direct evidence of an inhibitory role for local interneurons in olfactory information processing in insects. Through different types of multisynaptic interactions with projection neurons, local interneurons help to generate and shape the output from olfactory glomeruli in the antennal lobe.Abbreviations AL antennal lobe - EPSP excitatory postsynaptic potential - GABA -aminobutyric acid - IPSP inhibitory postsynaptic potential - LN local interneuron - MGC macroglomerular complex - OB olfactory bulb - PN projection neuron - TES N-tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid     

Friends and foes from an ant brain's point of view--neuronal correlates of colony odors in a social insect

Background

Successful cooperation depends on reliable identification of friends and foes. Social insects discriminate colony members (nestmates/friends) from foreign workers (non-nestmates/foes) by colony-specific, multi-component colony odors. Traditionally, complex processing in the brain has been regarded as crucial for colony recognition. Odor information is represented as spatial patterns of activity and processed in the primary olfactory neuropile, the antennal lobe (AL) of insects, which is analogous to the vertebrate olfactory bulb. Correlative evidence indicates that the spatial activity patterns reflect odor-quality, i.e., how an odor is perceived. For colony odors, alternatively, a sensory filter in the peripheral nervous system was suggested, causing specific anosmia to nestmate colony odors. Here, we investigate neuronal correlates of colony odors in the brain of a social insect to directly test whether they are anosmic to nestmate colony odors and whether spatial activity patterns in the AL can predict how odor qualities like “friend” and “foe” are attributed to colony odors.

Methodology/Principal Findings

Using ant dummies that mimic natural conditions, we presented colony odors and investigated their neuronal representation in the ant Camponotus floridanus. Nestmate and non-nestmate colony odors elicited neuronal activity: In the periphery, we recorded sensory responses of olfactory receptor neurons (electroantennography), and in the brain, we measured colony odor specific spatial activity patterns in the AL (calcium imaging). Surprisingly, upon repeated stimulation with the same colony odor, spatial activity patterns were variable, and as variable as activity patterns elicited by different colony odors.

Conclusions

Ants are not anosmic to nestmate colony odors. However, spatial activity patterns in the AL alone do not provide sufficient information for colony odor discrimination and this finding challenges the current notion of how odor quality is coded. Our result illustrates the enormous challenge for the nervous system to classify multi-component odors and indicates that other neuronal parameters, e.g., precise timing of neuronal activity, are likely necessary for attribution of odor quality to multi-component odors.     

昆虫气味认知的嗅觉神经结构及分子机制

大多数昆虫主要通过气味认知感知外界环境的变化,维持生命活动。探究昆虫气味认知的嗅觉系统神经结构及分子机制,对于完善气味认知神经生物学理论及利用其原理进行仿生学研究等有重要的科学意义。近年,关于昆虫气味认知科学研究有了很大的进展。本文从昆虫神经生物学的视角详细综述了近年关于昆虫气味认知的嗅觉神经结构、分子机制及气味信号的神经传导途径等方面的基本理论及最新研究成果。综述结果显示:昆虫对气味的认知是通过嗅觉神经系统的触角感器、触角叶(AL)、蕈形体(MB)等脑内多层信号处理神经结构来实现的。当外界气味分子进入触角感器内后,由感器内特定的气味识别蛋白(OBP)将气味分子运载到达嗅觉感受神经元(ORN)树突膜上的受体位点,气味分子与表达特定气味的受体(OR)结合产生电信号,并以动作电位的形式通过ORN的轴突传到脑内的触角叶。在触角叶经过嗅觉纤维球对气味信息选择性加工处理,再由投射神经元(PNs)将初步的识别和分类的气味信息传到蕈形体和外侧角(LH)等神经中枢,实现对气味的识别和认知。虽然,近年昆虫气味认知神经生物学的研究有了很大的进步,但是,我们认为目前的研究成果还不能完全阐明昆虫气味认知的神经机制,还有很多问题,例如,触角叶上众多的嗅觉纤维球是如何对嗅觉感受神经元传入的气味信息进行编码处理的?等有待进一步深入研究。为了搞清这些疑难问题,我们认为需要提高现有的实验技术水平,加强电生理学和分子神经生物学相结合的实验研究,从分子水平探究气味认知的神经机制可能是未来研究的热点。     

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 sensitivity^5-6, 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 substances^9-11. 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.     

Physiology and antennal lobe projections of olfactory receptor neurons from sexually isomorphic sensilla on male <Emphasis Type="Italic">Heliothis virescens</Emphasis>

The neurophysiology and antennal lobe projections of olfactory receptor neurons (ORNs) within sexually isomorphic short trichoid sensilla of male Heliothis virescens (Noctuidae: Lepidoptera) were investigated using cut-sensillum recording and cobalt-lysine staining. A total of 202 sensilla were sorted into 14 possible sensillar categories based on odor responses and physiology of ORNs within. Seventy-two percent of the sensilla identified contained ORNs stimulated by conspecific odors. In addition, a large number of ORNs were specifically sensitive to ß-caryophyllene, a plant-derived volatile (N = 41). Axons originating from ORNs associated with individual sensilla were stained with cobalt lysine (N = 67) and traced to individual glomeruli in the antennal lobe. ORNs with responses to female sex pheromone components exhibited similar axonal projections as those previously described from ORNs in long sensilla trichodea in male H. virescens. Antennal lobe axonal arborizations of ORNs sensitive to hairpencil components were also located in glomeruli near the base of the antennal nerve, whilst those sensitive to plant odorants projected to more medial glomeruli. Comparisons with ORNs described from female H. virescens supports the notion that glomeruli at the base of the antennal nerve are associated with conspecific and interspecific odorants, whereas those located medially are associated with plant volatiles.     

Modulation by octopamine of olfactory responses to nonpheromone odorants in the cockroach, Periplaneta americana L

Olfactory receptor cells in insects are modulated by neurohormones. Recordings from cockroach olfactory sensilla showed that a subset of sensory neurons increase their responses to selected nonpheromone odorants after octopamine application. With octopamine application, recordings demonstrated increased firing rates by the short but not the long alcohol-sensitive sensilla to the nonpheromone volatile, hexan-1-ol. Within the same sensillum, individual receptor cells are shown to be modulated independently from each other, indicating that the octopamine receptors reside in the receptor not in the accessory cells. A uniform decrease in the amplitude of electroantennogram, which is odorant independent, is suggested to reflect the rise in octopamine concentration in the antennal hemolymph. Perception of general odorants measured as behavioral responses changed qualitatively under octopamine treatment: namely, repulsive hexan-1-ol became neutral, whereas neutral eucalyptol became attractive. Octopamine induced a change in male behavioral responses to general odors that were essentially the same as in the state of sexual arousal. Our findings suggest that sensitivity to odors having different biological significances is modulated selectively at the peripheral as well as other levels of olfactory processing.     

昆虫触角叶的结构

触角叶是昆虫脑内初级嗅觉中心,通过触角神经与触角联系。触角叶主要由嗅觉受体神经元、局域中间神经元、投射神经元和远心神经元构成。这些神经元的形态多样,其形态变化与其功能和昆虫嗅觉行为相关。这些神经元在触角叶内交织形成神经纤维网,在突触联系紧密的地方形成纤维球,纤维球通常排列在触角叶外周。通常,昆虫触角叶内纤维球的数量、大小和位置相对固定,并且几乎每个小球都可以被识别和命名。不同种类、性别和品级的昆虫中,纤维球的数量、大小和排列方式各不相同。触角叶结构神经元组成和纤维球的多样性,与各种昆虫嗅觉行为的特异性相对应。     

Transplant Antennae and Host Brain Interact to Shape Odor Perceptual Space in Male Moths

Behavioral responses to odors rely first upon their accurate detection by peripheral sensory organs followed by subsequent processing within the brain’s olfactory system and higher centers. These processes allow the animal to form a unified impression of the odor environment and recognize combinations of odorants as single entities. To investigate how interactions between peripheral and central olfactory pathways shape odor perception, we transplanted antennal imaginal discs between larval males of two species of moth Heliothis virescens and Heliothis subflexa that utilize distinct pheromone blends. During metamorphic development olfactory receptor neurons originating from transplanted discs formed connections with host brain neurons within olfactory glomeruli of the adult antennal lobe. The normal antennal receptor repertoire exhibited by males of each species reflects the differences in the pheromone blends that these species employ. Behavioral assays of adult transplant males revealed high response levels to two odor blends that were dissimilar from those that attract normal males of either species. Neurophysiological analyses of peripheral receptor neurons and central olfactory neurons revealed that these behavioral responses were a result of: 1. the specificity of H. virescens donor olfactory receptor neurons for odorants unique to the donor pheromone blend and, 2. central odor recognition by the H. subflexa host brain, which typically requires peripheral receptor input across 3 distinct odor channels in order to elicit behavioral responses.     

Representation of a mixture of pheromone and host plant odor by antennal lobe projection neurons of the silkmoth Bombyx mori

Pheromone-source orientation behavior can be modified by coexisting plant volatiles. Some host plant volatiles enhance the pheromonal responses of olfactory receptor neurons and increase the sensitivity of orientation behavior in the Lepidoptera species. Although many electrophysiological studies have focused on the pheromonal response of olfactory interneurons, the response to the mixture of pheromone and plant odor is not yet known. Using the silkmoth, Bombyx mori, we investigated the physiology of interneurons in the antennal lobe (AL), the primary olfactory center in the insect brain, in response to a mixture of the primary pheromone component bombykol and cis-3-hexen-1-ol, a mulberry leaf volatile. Application of the mixture enhanced the pheromonal responses of projection neurons innervating the macroglomerular complex in the AL. In contrast, the mixture of pheromone and cis-3-hexen-1-ol had little influence on the responses of projection neurons innervating the ordinary glomeruli whereas other plant odors dynamically modified the response. Together this suggests moths can process plant odor information under conditions of simultaneous exposure to sex pheromone.     

A large-scale model of the locust antennal lobe

The antennal lobe (AL) is the primary structure within the locust’s brain that receives information from olfactory receptor neurons (ORNs) within the antennae. Different odors activate distinct subsets of ORNs, implying that neuronal signals at the level of the antennae encode odors combinatorially. Within the AL, however, different odors produce signals with long-lasting dynamic transients carried by overlapping neural ensembles, suggesting a more complex coding scheme. In this work we use a large-scale point neuron model of the locust AL to investigate this shift in stimulus encoding and potential consequences for odor discrimination. Consistent with experiment, our model produces stimulus-sensitive, dynamically evolving populations of active AL neurons. Our model relies critically on the persistence time-scale associated with ORN input to the AL, sparse connectivity among projection neurons, and a synaptic slow inhibitory mechanism. Collectively, these architectural features can generate network odor representations of considerably higher dimension than would be generated by a direct feed-forward representation of stimulus space.     

Peripheral and central olfactory tuning in a moth

Animals can be innately attracted to certain odorants. Because these attractants are particularly salient, they might be expected to induce relatively strong responses throughout the olfactory pathway, helping animals detect the most relevant odors but limiting flexibility to respond to other odors. Alternatively, specific neural wiring might link innately preferred odors to appropriate behaviors without a need for intensity biases. How nonpheromonal attractants are processed by the general olfactory system remains largely unknown. In the moth Manduca sexta, we studied this with a set of innately preferred host plant odors and other, neutral odors. Electroantennogram recordings showed that, as a population, olfactory receptor neurons (ORNs) did not respond with greater intensity to host plant odors, and further local field potential recordings showed that no specific amplification of signals induced by host plant odors occurred between the first olfactory center and the second. Moreover, when odorants were mutually diluted to elicit equally intense output from the ORNs, moths were able to learn to associate all tested odorants equally well with food reward. Together, these results suggest that, although nonpheromonal host plant odors activate broadly distributed responses, they may be linked to attractive behaviors mainly through specific wiring in the brain.     

Physiological and morphological characterization of honeybee olfactory neurons combining electrophysiology,calcium imaging and confocal microscopy

The insect antennal lobe is the first brain structure to process olfactory information. Like the vertebrate olfactory bulb the antennal lobe is substructured in olfactory glomeruli. In insects, glomeruli can be morphologically identified, and have characteristic olfactory response profiles. Local neurons interconnect glomeruli, and output (projection) neurons project to higher-order brain centres. The relationship between their elaborate morphology and their physiology is not understood. We recorded electrophysiologically from antennal lobe neurons, and iontophoretically injected a calcium-sensitive dye. We then measured their spatio-temporal calcium responses to a variety of odours. Finally, we confocally reconstructed the neurons, and identified the innervated glomeruli. An increase or decrease in spiking frequency corresponded to an intracellular calcium increase or decrease in the cell. While intracellular recordings generally lasted between 10 and 30 min, calcium imaging was stable for up to 2 h, allowing a more detailed physiological analysis. The responses indicate that heterogeneous local neurons get input in the glomerulus in which they branch most strongly. In many cases, the physiological response properties of the cells corresponded to the known response profile of the innervated glomerulus. In other words, the large variety of response profiles generally found when comparing antennal lobe neurons is reduced to a more predictable response profile when the innervated glomerulus is known.Abbreviations ACT antenno-cerebralis-tract - AL antennal lobe - AP action potential - l-ACT lateral ACT - LN local neuron - LPL lateral protocerebral lobe - m-ACT medial ACT - MB mushroom body - OSN olfactory sensory neuron - PN projection neuron - T1 tract 1 of the antennal nerve     

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 channels^{4, 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 odorant^{6, 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 OSNs^{1, 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.Open in a separate windowClick here to view.^{(78M, flv)}