Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines

The pectines of scorpions are ventral bilateral appendages supporting 10⁴–10⁵ chemosensory sensilla called pegs. Each peg contains 10–18 sensory neurons, some of which show ultrastructural evidence of axo-axonic synapses with other sensory neurons in the same sensillum. In extracellular recordings from single-peg sensilla, individual sensory units can be distinguished by impulse waveform and firing frequency. Cross-correlation analysis of impulse activity showed that at least two of these units, types `A1' and `A2', are inhibited during the 100-ms period immediately following activity of a third unit, type `B'. This interaction between sensory units in a single sensillum also occurs in surgically isolated pectines, indicating that it does not involve efferent feedback from the central nervous system. Other sensillar neurons appear to have excitatory interactions. Thus, in scorpion pectine, chemosensory information undergoes some form of processing within individual sensilla prior to its relay to the CNS, making this an unusually accessible preparation for study of first-order chemosensory processing events. Accepted: 12 April 1997     

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species ^1,2. These intraspecies signals, the pheromones, as well as signals from some predators ³, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity ⁴. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR ^5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively ^6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli ^4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs ^3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations ^12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.Sensory stimulation elicits strong Ca²⁺ influx in VSNs that is indicative of receptor activation ^4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs ^15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca²⁺ imaging experiments. This method has eliminated the dye loading process used in previous studies ^4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.     

Labial tip sensilla of Blissus leucopterus leucopterus (Hemiptera: Blissidae): Ultrastructure and behavior

The cuticular sensory receptors that are found on the apex of the labium of hemipterans play an important role in their feeding behavior. In this study we describe the ultrastructure, number, and distribution of sensilla on the labium apex of the chinch bug, Blissus leucopterus leucopterus. Each apical field of sensilla on the labium contains 11 uniporous peg sensilla and one sensillum chaeticum. The uniporous peg sensilla are innervated by 4–5 bipolar neurons that send dendrites in the lumen of each peg. Three neurons are associated with each sensillum chaeticum, two neurons have dendrites in the lumen of the sensillum, and the third dendrite ends in a tubular body at the base of the sensillum. Behavioral tests that involve chemical blockage of the sensory receptors show the importance of the labial sensilla in feeding behavior. Both morphological and behavioral evidence indicate that the labial sensilla have a chemosensitive function.     

Diel Cycles in Chemosensory Behaviors of Free‐Ranging Rattlesnakes Lying in Wait for Prey

The sensory ecology of foragers is fundamentally influenced by changes in environmental conditions such as ambient light. Changes in ambient light may hinder the effectiveness of particular senses (e.g., impaired vision at night), but many predators rely on multiple sensory systems and may continue to forage despite changes in light availability. Exactly how predator behaviors and sensory systems compensate under changes in light availability in the field is not well understood. We used radio telemetry and portable video surveillance cameras to quantify the sit‐and‐wait chemosensory foraging behavior of free‐ranging red diamond (Crotalus ruber) and northern Pacific (Crotalus oreganus oreganus) rattlesnakes during day and night periods. The two most common behaviors we observed were chemosensory probes, a behavior we describe in detail for the first time, and mouth gapes. During chemosensory probes, rattlesnakes extend their head beyond their coil, explore the surrounding area while tongue‐flicking, and subsequently return to a stationary position inside their coil. Foraging rattlesnakes probed at significantly higher rates during nocturnal vs. diurnal hours. Similarly, mouth gaping occurred during a higher percentage of nocturnal vs. diurnal hours for foraging snakes. Nearly half of all mouth gapes were followed immediately with a chemosensory probe, suggesting that mouth gaping also serves a chemosensory function in this context. Our results suggest that chemical cues play an increasingly important role in mediating rattlesnake foraging behavior at night. Examining how abiotic factors, such as light availability, influence the sensory ecology of free‐ranging predators is essential for accurately characterizing their interactions with prey.     

Expression patterns of anoctamin 1 and anoctamin 2 chloride channels in the mammalian nose

Calcium-activated chloride channels are expressed in chemosensory neurons of the nose and contribute to secretory processes and sensory signal transduction. These channels are thought to be members of the family of anoctamins (alternative name: TMEM16 proteins), which are opened by micromolar concentrations of intracellular Ca²⁺. Two family members, ANO 1 (TMEM16A) and ANO 2 (TMEM16B), are expressed in the various sensory and respiratory tissues of the nose. We have examined the tissue specificity and sub-cellular localization of these channels in the nasal respiratory epithelium and in the five chemosensory organs of the nose: the main olfactory epithelium, the septal organ of Masera, the vomeronasal organ, the Grueneberg ganglion and the trigeminal system. We have found that the two channels show mutually exclusive expression patterns. ANO 1 is present in the apical membranes of various secretory epithelia in which it is co-localized with the water channel aquaporin 5. It has also been detected in acinar cells and duct cells of subepithelial glands and in the supporting cells of sensory epithelia. In contrast, ANO 2 expression is restricted to chemosensory neurons in which it has been detected in microvillar and ciliary surface structures. The different expression patterns of ANO 1 and ANO 2 have been observed in the olfactory, vomeronasal and respiratory epithelia. No expression has been detected in the Grueneberg ganglion or trigeminal sensory fibers. On the basis of this differential expression, we derive the main functional features of ANO 1 and ANO 2 chloride channels in the nose and suggest their significance for nasal physiology.     

The proteome of rat olfactory sensory cilia

Olfactory sensory neurons expose to the inhaled air chemosensory cilia which bind odorants and operate as transduction organelles. Odorant receptors in the ciliary membrane activate a transduction cascade which uses cAMP and Ca²⁺ for sensory signaling in the ciliary lumen. Although the canonical transduction pathway is well established, molecular components for more complex aspects of sensory transduction, like adaptation, regulation, and termination of the receptor response have not been systematically identified. Moreover, open questions in olfactory physiology include how the cilia exchange solutes with the surrounding mucus, assemble their highly polarized set of proteins, and cope with noxious substances in the ambient air. A specific ciliary proteome would promote research efforts in all of these fields. We have improved a method to detach cilia from rat olfactory sensory neurons and have isolated a preparation specifically enriched in ciliary membrane proteins. Using LC‐ESI‐MS/MS analysis, we identified 377 proteins which constitute the olfactory cilia proteome. These proteins represent a comprehensive data set for olfactory research since more than 80% can be attributed to the characteristic functions of olfactory sensory neurons and their cilia: signal processing, protein targeting, neurogenesis, solute transport, and cytoprotection. Organellar proteomics thus yielded decisive information about the diverse physiological functions of a sensory organelle.     

Antennal contact chemosensilla in Psylliodes chrysocephala responding to cruciferous allelochemicals

Sensilla chaetica, which protrude above all other sensilla on the antenna of Psylliodes chrysocephala L., the cabbage stem flea beetle, were investigated ultrastructurally and found to be innervated by five to six sensory neurones. A dendrite from one of these neurones terminates in a tubular body at the shaft base, whereas dendrites from the others run unbranched to a pore at the shaft tip. Such a structure typifies a sensillum with a combined gustatory/mechanosensory function. Electrophysiological recordings using the tip-recording technique confirmed that this sensillum contains one mechanosensory cell and several chemosensory cells. The chemosensory cells were responsive to host plant chemicals. Sensilla chaetica were also found to be responsive to glucosinolates. One of the sensilla chaetica emerges from a domed area of cuticle on antennomere six. This was found to be relatively less responsive to the chemical stimuli tested and more responsive to mechanical stimulation. It is suggested that the sensilla chaetica are contact chemosensilla, that respond to chemicals present in plant surface waxes when P. chrysocephala antennates a leaf.     

The antennal sensilla of the praying mantis Tenodera aridifolia: A new flagellar partition based on the antennal macro-, micro- and ultrastructures

In insects, the antenna consists of a scapus, a pedicellus, and a flagellum comprising many segments (flagellomeres). These segments possess many morphological types of sensory organs (sensilla) to process multimodal sensory information. We observed the sensilla on flagellomeres in praying mantis (Tenodera aridifolia) with both scanning and transmission electron microscopes. We classified the sensilla into six types: chaetic, campaniform, coelocapitular, basiconic, trichoid and grooved peg sensilla, and inferred their presumptive functions on the basis of their external and internal structures. In addition, based on their distribution, we newly divided the flagellum into 6 distinct parts. This new division leads to a better understanding about the sexual dimorphism and the antennal development in the mantises. The sexual difference in distribution of the grooved peg sensilla suggests that this type of sensilla may play a role in sex-pheromone detection in mantis, which is a rare case of double-walled sensilla mediating this function.     

Chemosensory signal transduction in Caenorhabditis elegans

Chemosensory neurons translate perception of external chemical cues, including odorants, tastants, and pheromones, into information that drives attraction or avoidance motor programs. In the laboratory, robust behavioral assays, coupled with powerful genetic, molecular and optical tools, have made Caenorhabditis elegans an ideal experimental system in which to dissect the contributions of individual genes and neurons to ethologically relevant chemosensory behaviors. Here, we review current knowledge of the neurons, signal transduction molecules and regulatory mechanisms that underlie the response of C. elegans to chemicals, including pheromones. The majority of identified molecules and pathways share remarkable homology with sensory mechanisms in other organisms. With the development of new tools and technologies, we anticipate that continued study of chemosensory signal transduction and processing in C. elegans will yield additional new insights into the mechanisms by which this animal is able to detect and discriminate among thousands of chemical cues with a limited sensory neuron repertoire.     

Antennal sensilla of whiteflies: Trialeurodes vaporariorum (Westwood), the glasshouse whitefly,and Aleyrodes proletella (Linnaeus), the cabbage whitefly, (Homoptera : Aleyrodidae). Part 2: Ultrastructure

A transmission electron microscope study of the antennal sensilla of the whitefly Trialeurodes vaporariorum and Aleyrodes proletella (Homoptera : Aleyrodidae) revealed that of the sensilla unique to the antennal flagellum (basiconic, coeloconic and small digitate-tipped sensory pegs), basiconic and coeloconic sensilla occur as subtypes. Four subtypes of basiconic cone sensilla occur on the flagella of T. vaporariorum and 3 on A. proletella. All the subtypes of basiconic sensilla have an ultrastructure typical of olfactory sensilla and probably have a primary olfactory function. Two subtypes of coeloconic sensilla occur on the flagella of both species. Their ultrastructure suggests primarily a chemosensory function. The digitate-tipped sensory peg of both species possesses a triad of neurones which have ultrastructural characteristics similar to the known thermo-/hygroreceptors of other insect species. The other sensilla, which occur on the antennae of the whiteflies, include cheatae, campaniform and subcuticular sensilla, all of which have an ultrastructure typical of mechanoreceptors.     

Chemosensory behavior of semi‐restrained Caenorhabditis elegans

A new behavioral assay is described for studying chemosensation in the nematode Caenorhabditis elegans. This assay presents three main characteristics: (1) the worm is restrained by gluing, preserving correlates of identifiable behaviors; (2) the amplitude and time course of the stimulus are controlled by the experimenter; and (3) the behavior is recorded quantitatively. We show that restrained C. elegans display behaviors comparable to those of freely moving worms. Moreover, the chemosensory response of wild‐type glued animals to changes in salt concentration is similar to that of freely moving animals. This glued‐worm assay was used to reveal new chemosensory deficits of the potassium channel mutant egl‐2. We conclude that the glued worm assay can be used to study the chemosensory regulation of C. elegans behavior and how it is affected by neuronal or genetic manipulations. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

The Role of Antennae in Mate Recognition in <Emphasis Type="Italic">Phoracantha semipunctata</Emphasis> (<Emphasis Type="Italic">Coleoptera: Cerambycidae</Emphasis>)

The mating behavior of the eucalyptus longhorned borer Phoracantha semipunctata was studied to understand its mate recognition system. Bioassays were conducted to determine the existence of a cuticular chemical on its body surface and how individuals perceived it. Males walked oriented to and attempted copulation with live conspecifics only upon antennal contact with their bodies. They showed similar responses to intact dead females and males, but failed to respond to washed bodies. Dummies carrying male extracts were more likely to elicit copulation attempts than control dummies. This constitutes behavioral evidence that unidentified chemical(s) on the body surface play a major role in mate recognition, and can only be perceived after antennal contact. Sensilla trichodea on the antennal flagellum are candidates for this contact chemoreception. They are distributed throughout the entire flagellum, especially along its margins and at the tip of the distal flagellomere, and share structural features with contact chemosensory sensilla of other insects.     

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues¹ and survival factors² located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons³. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons⁴. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro^5,6.Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol⁷) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival⁸.     

Comparative dissection of the peripheral olfactory system of the Chagas disease vectors Rhodnius prolixus and Rhodnius brethesi

American trypanosomiasis, or Chagas disease, is transmitted by both domestic and sylvatic species of Triatominae which use sensory cues to locate their vertebrate hosts. Among them, odorants have been shown to play a key role. Previous work revealed morphological differences in the sensory apparatus of different species of Triatomines, but to date a comparative functional study of the olfactory system is lacking. After examining the antennal sensilla with scanning electronic microscopy (SEM), we compared olfactory responses of Rhodnius prolixus and the sylvatic Rhodnius brethesi using an electrophysiological approach. In electroantennogram (EAG) recordings, we first showed that the antenna of R. prolixus is highly responsive to carboxylic acids, compounds found in their habitat and the headspace of their vertebrate hosts. We then compared responses from olfactory sensory neurons (OSNs) housed in the grooved peg sensilla of both species, as these are tuned to these compounds using single-sensillum recordings (SSRs). In R. prolixus, the SSR responses revealed a narrower tuning breath than its sylvatic sibling, with the latter showing responses to a broader range of chemical classes. Additionally, we observed significant differences between these two species in their response to particular volatiles, such as amyl acetate and butyryl chloride. In summary, the closely related, but ecologically differentiated R. prolixus and R. brethesi display distinct differences in their olfactory functions. Considering the ongoing rapid destruction of the natural habitat of sylvatic species and the likely shift towards environments shaped by humans, we expect that our results will contribute to the design of efficient vector control strategies in the future.     

Olfactory receptor gene evolution is unusually rapid across Tetrapoda and outpaces chemosensory phenotypic change

Chemosensation is the most ubiquitous sense in animals, enacted by the products of complex gene families that detect environmental chemical cues and larger-scale sensory structures that process these cues. While there is a general conception that olfactory receptor (OR) genes evolve rapidly, the universality of this phenomenon across vertebrates, and its magnitude, are unclear. The supposed correlation between molecular rates of chemosensory evolution and phenotypic diversity of chemosensory systems is largely untested. We combine comparative genomics and sensory morphology to test whether OR genes and olfactory phenotypic traits evolve at faster rates than other genes or traits. Using published genomes, we identified ORs in 21 tetrapods, including amphibians, reptiles, birds, and mammals and compared their rates of evolution to those of orthologous non-OR protein-coding genes. We found that, for all clades investigated, most OR genes evolve nearly an order of magnitude faster than other protein-coding genes, with many OR genes showing signatures of diversifying selection across nearly all taxa in this study. This rapid rate of evolution suggests that chemoreceptor genes are in “evolutionary overdrive,” perhaps evolving in response to the ever-changing chemical space of the environment. To obtain complementary morphological data, we stained whole fixed specimens with iodine, µCT-scanned the specimens, and digitally segmented chemosensory and nonchemosensory brain regions. We then estimated phenotypic variation within traits and among tetrapods. While we found considerable variation in chemosensory structures, they were no more diverse than nonchemosensory regions. We suggest chemoreceptor genes evolve quickly in reflection of an ever-changing chemical space, whereas chemosensory phenotypes and processing regions are more conserved because they use a standardized or constrained architecture to receive and process a range of chemical cues.