Dendrobaena veneta avoids ethyl pentanoate and ethyl hexanoate, two compounds produced by the soil fungus Geotrichum candidum.

Earthworms shape the biological and physicochemical qualities of the soil they choose to reside in, but our understanding of the specific chemicals that attract or repel a particular species of earthworm remains incomplete. Current research indicates that some species feed on and are attracted to fungi, such as Geotrichum candidum. In the present study, as part of our continuing effort to characterize mechanisms of earthworm chemosensation, we tested whether ethyl hexanoate and ethyl pentanoate, two compounds produced by G. candidum, are appetitive to the European nightcrawler (Dendrobaena veneta). In a soil T-maze, both of these compounds significantly repelled individual earthworms in a dosage-dependent manner, this result ran counter to our initial hypothesis. D. veneta also avoided ethyl hexanoate and ethyl pentanoate in an assay we specifically developed to test an earthworms aversion to chemical stimuli in soil. In both of these assays, ethyl hexanoate was aversive at lower concentrations than ethyl pentanoate. These findings further clarify our understanding of the chemical cues that trigger the decision of D. veneta to select a particular soil-environment, and emphasize that different earthworm species may react very differently to commonly encountered chemical stimuli.

Behavioral Aversion to AITC Requires Both Painless and dTRPA1 in Drosophila.

There has been disagreement over the functional roles of the painless gene product in the detection and subsequent behavioral aversion to the active ingredient in wasabi, allyl isothiocyanate (AITC). Originally, painless was reported to eliminate the behavioral aversion to AITC, although subsequent reports suggested that another trpA homolog, dTRPA1, was responsible for AITC aversion. We re-evaluated the role of the painless gene in the detection of AITC, employing several different behavioral assays. Using the proboscis extension reflex (PER) assay, we observed that AITC did not reduce PER frequencies in painless or dTRPA1 mutants but did in wild-type genotypes. Quantification of food intake showed a significant decline in food consumption in the presence of AITC in wild-type, but not painless mutants. We adapted an oviposition choice assay and found wild-type oviposit on substrates lacking AITC, in contrast to painless and dTRPA1 mutants. Lastly, tracking individual flies relative to a point source of AITC, showed a consistent clustering of wild-type animals away from the point source, which was absent in painless mutants. We evaluated expression patterns of both dTRPA1 and painless, which showed expression in distinct central and peripheral populations. We identified the transmitter phenotypes of subsets of painless and dTRPA1 neurons and found similar neuropeptides as those expressed by mammalian trpA expressing neurons. Using a calcium reporter, we observed AITC-evoked responses in both painless and dTRPA1 expressing neurons. Collectively, these results reaffirm the necessity of painless in nociceptive behaviors and suggest experiments to further resolve the molecular basis of aversion.

Naturally Produced Defensive Alkenal Compounds Activate TRPA1.

(E)-2-alkenals are aldehydes containing an unsaturated bond between the alpha and beta carbons. 2-alkenals are produced by many organisms for defense against predators and secretions containing (E)-2-alkenals cause predators to stop attacking and allow the prey to escape. Chemical ecologists have described many alkenal compounds with 3-20 carbons common, having varied positions of double bonds and substitutions. How do these defensive alkenals act to deter predators? We have tested the effects of (E)-2-alkenals with 6-12 carbons on transient receptor potential channels (TRP) commonly found in sensory neurons. We find that (E)-2-alkenals activate transient receptor potential ankyrin subtype 1 (TRPA1) at low concentrations-EC50s 10-100 µM (in 0 added Ca(2+) external solutions). Other TRP channels were either weakly activated (TRPV1, TRPV3) or insensitive (TRPV2, TRPV4, TRPM8). (E)-2-alkenals may activate TRPA1 by modifying cysteine side chains. However, target cysteines include others beyond the 3 in the amino-terminus implicated in activation, as a channel with cysteines at 621, 641, 665 mutated to serine responded robustly. Related chemicals, including the aldehydes hexanal and decanal, and (E)-2-hexen-1-ol also activated TRPA1, but with weaker potency. Rat trigeminal nerve recordings and behavioral experiments showed (E)-2-hexenal was aversive. Our results suggest that TRPA1 is likely a major target of these commonly used defensive chemicals.

Dissecting the role of TRPV1 in detecting multiple trigeminal irritants in three behavioral assays for sensory irritation.

Polymodal neurons of the trigeminal nerve innervate the nasal cavity, nasopharynx, oral cavity and cornea. Trigeminal nociceptive fibers express a diverse collection of receptors and are stimulated by a wide variety of chemicals. However, the mechanism of stimulation is known only for relatively few of these compounds. Capsaicin, for example, activates transient receptor potential vanilloid 1 (TRPV1) channels. In the present study, wildtype (C57Bl/6J) and TRPV1 knockout mice were tested in three behavioral assays for irritation to determine if TRPV1 is necessary to detect trigeminal irritants in addition to capsaicin. In one assay mice were presented with a chemical via a cotton swab and their response scored on a 5 level scale. In another assay, a modified two bottle preference test, which avoids the confound of mixing irritants with the animal's drinking water, was used to assess aversion. In the final assay, an air dilution olfactometer was used to administer volatile compounds to mice restrained in a double-chambered plethysmograph where respiratory reflexes were monitored. TRPV1 knockouts showed deficiencies in the detection of benzaldehyde, cyclohexanone and eugenol in at least one assay. However, cyclohexanone was the only substance tested that appears to act solely through TRPV1.

A TRPA1-dependent mechanism for the pungent sensation of weak acids.

Acetic acid produces an irritating sensation that can be attributed to activation of nociceptors within the trigeminal ganglion that innervate the nasal or oral cavities. These sensory neurons sense a diverse array of noxious agents in the environment, allowing animals to actively avoid tissue damage. Although receptor mechanisms have been identified for many noxious chemicals, the mechanisms by which animals detect weak acids, such as acetic acid, are less well understood. Weak acids are only partially dissociated at neutral pH and, as such, some can cross the cell membrane, acidifying the cell cytosol. The nociceptor ion channel TRPA1 is activated by CO(2), through gating of the channel by intracellular protons, making it a candidate to more generally mediate sensory responses to weak acids. To test this possibility, we measured responses to weak acids from heterologously expressed TRPA1 channels and trigeminal neurons with patch clamp recording and Ca(2+) microfluorometry. Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids. Notably, the degree of channel activation was predicted by the degree of intracellular acidification produced by each acid, suggesting that intracellular protons are the proximate stimulus that gates the channel. Responses to weak acids produced a Ca(2+)-independent inactivation that precluded further activation by weak acids or reactive chemicals, whereas preactivation by reactive electrophiles sensitized TRPA1 channels to weak acids. Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice. We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.

SYNAPSE, Symposium for Young Neuroscientists and Professors of the Southeast: A One-day, Regional Neuroscience Meeting Focusing on Undergraduate Research.

The Symposium for Young Neuroscientists and Professors of the Southeast (SYNAPSE; synapse.cofc.edu) was designed to encourage contacts among faculty and students interested in neuroscience. Since its inception in 2003, the SYNAPSE conference has consistently drawn faculty and undergraduate interest from the region. This unique meeting provides undergraduates with a valuable opportunity for neuroscience education; students interact with noted neuroscience faculty, present research results, obtain feedback from neuroscientists at other institutions, and form connections with other neuroscientists in the region. Additionally, SYNAPSE allows undergraduate students and faculty to attend workshops and panel discussions about issues related to professional skills and career options. The SYNAPSE conference currently travels among host institutions in the southeastern United States in two-year cycles. This article briefly describes the genesis of SYNAPSE and reviews SYNAPSE conferences from 2006 through 2010. The goal of this paper is to highlight key issues organizers have experienced launching, sustaining, and hosting this regional undergraduate neuroscience conference as well as assist faculty to develop similar conferences.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals.

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl-homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca(2+). Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either G alpha-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl-homoserine lactones serve as quorum-sensing molecules for gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

The anatomical and electrophysiological basis of peripheral nasal trigeminal chemoreception.

The trigeminal nerve (TN) provides sensory information from the eyes, nose, and mouth. A subset of trigeminal nerve fibers, particularly those containing the neuropeptides substance P and calcitonin gene-related peptide (CGRP), responds to chemical irritants in the environment. Axons in the ethmoid and nasopalatine branches of the trigeminal nerve innervate the nasal mucosa where they ramify repeatedly. TN endings extend close to the nasal epithelial surface stopping at the line of tight junctions only a few micrometers from the surface. A single ethmoid nerve axon may send branches to the nasal mucosa, olfactory bulb, and the spinal trigeminal complex. Traditionally, irritants are thought to stimulate free TN endings in the nasal epithelium. Recently, however, solitary chemoreceptor cells (SCCs) have been found scattered throughout the nasal cavity. The SCCs are contacted by TN fibers and may express T2R ''bitter-taste'' receptors alpha-gustducin, and TRPM5. Peripheral trigeminal electrophysiological recordings in response to irritants have been obtained from the mucosa (negative mucosal potential, NMP) and the nerve to analyze characteristics of trigeminal stimuli. Responses to a wide variety of irritants have been recorded from the ethmoid nerve. In general, the more lipid soluble the compound, the lower the threshold. Nerve recordings have also suggested several mechanisms by which irritants elicit responses. Bitter substances elicit responses from the ethmoid nerve and cause a change in respiration indicating stimulation via SCCs. SCCs themselves respond to chemical stimuli and may be contributing to the detection of nasal irritants.

TRPV1 receptors and nasal trigeminal chemesthesis.

The trigeminal nerve responds to a wide variety of irritants. Trigeminal nerve fibers express several receptors that respond to chemicals, including TRPV1 (vanilloid) receptors, acid-sensing ion channels, P2X (purinergic) receptors, and nicotinic acetylcholine receptors. In order to assess whether TRPV1 plays a role in responses to a broad array of substances, TRPV1 (along with green fluorescent protein) was expressed in human embyonic kidney cells (HEK) 293t cells which were then stimulated with diverse trigeminal irritants. Calcium imaging was used to measure responses to capsaicin, amyl acetate, cyclohexanone, acetic acid, toluene, benzaldehyde, (-)-nicotine, (R)-(+)-limonene, (R)-(-)-carvone, and (S)-(+)-carvone. Three irritants (acetic acid and the 2 carvones) stimulated nontransfected controls. Two irritants (capsaicin and cyclohexanone) stimulated only transfected cells. The response could be eliminated with capsazepine, a TRPV1 blocker. The 5 remaining irritants were nonstimulatory in both nontransfected and transfected cells. Because all the compounds tested on HEK cells elicited neural responses from the ethmoid branch of the trigeminal nerve in rats, the 5 nonstimulatory compounds must do so by a non-TRPV1 receptor. These results suggest that TRPV1 serves as a receptor for both cyclohexanone and capsaicin in trigeminal nerve endings.

Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration.

Inhalation of irritating substances leads to activation of the trigeminal nerve, triggering protective reflexes that include apnea or sneezing. Receptors for trigeminal irritants are generally assumed to be located exclusively on free nerve endings within the nasal epithelium, requiring that trigeminal irritants diffuse through the junctional barrier at the epithelial surface to activate receptors. We find, in both rats and mice, an extensive population of chemosensory cells that reach the surface of the nasal epithelium and form synaptic contacts with trigeminal afferent nerve fibers. These chemosensory cells express T2R "bitter-taste" receptors and alpha-gustducin, a G protein involved in chemosensory transduction. Functional studies indicate that bitter substances applied to the nasal epithelium activate the trigeminal nerve and evoke changes in respiratory rate. By extending to the surface of the nasal epithelium, these chemosensory cells serve to expand the repertoire of compounds that can activate trigeminal protective reflexes. The trigeminal chemoreceptor cells are likely to be remnants of the phylogenetically ancient population of solitary chemoreceptor cells found in the epithelium of all anamniote aquatic vertebrates.

Trigeminal collaterals in the nasal epithelium and olfactory bulb: a potential route for direct modulation of olfactory information by trigeminal stimuli.

The nasal epithelium is richly invested with peptidergic (substance P and calcitonin gene-related peptide [CGRP]) trigeminal polymodal nociceptors, which respond to numerous odorants as well as irritants. Peptidergic trigeminal sensory fibers also enter the glomerular layer of the olfactory bulb. To test whether the trigeminal fibers in the olfactory bulb are collaterals of the epithelial trigeminal fibers, we utilized dual retrograde labeling techniques in rats to identify the trigeminal ganglion cells innervating each of these territories. Nuclear Yellow was injected into the dorsal nasal epithelium, and True Blue was injected into the olfactory bulb of the same side. Following a survival period of 3-7 days, the trigeminal ganglion contained double-labeled, small (11.8 x 8.0 microm), ellipsoid ganglion cells within the ethmoid nerve region of the ganglion. Tracer injections into the spinal trigeminal complex established that these branched trigeminal ganglion cells also extended an axon into the brainstem. These results indicate that some trigeminal ganglion cells with sensory endings in the nasal epithelium also have branches reaching directly into both the olfactory bulb and the spinal trigeminal complex. These trigeminal ganglion cells are unique among primary sensory neurons in having two branches entering the central nervous system at widely distant points. Furthermore, the collateral innervation of the epithelium and bulb may provide an avenue whereby nasal irritants could affect processing of coincident olfactory stimuli.