High salt recruits aversive taste pathways

In the tongue, distinct classes of taste receptor cells detect the five basic tastes; sweet, sour, bitter, sodium salt and umami. Among these qualities, bitter and sour stimuli are innately aversive, whereas sweet and umami are appetitive and generally attractive to animals. By contrast, salty taste is unique in that increasing salt concentration fundamentally transforms an innately appetitive stimulus into a powerfully aversive one. This appetitive–aversive balance helps to maintain appropriate salt consumption, and represents an important part of fluid and electrolyte homeostasis. We have shown previously that the appetitive responses to NaCl are mediated by taste receptor cells expressing the epithelial sodium channel, ENaC, but the cellular substrate for salt aversion was unknown. Here we examine the cellular and molecular basis for the rejection of high concentrations of salts. We show that high salt recruits the two primary aversive taste pathways by activating the sour- and bitter-taste-sensing cells. We also demonstrate that genetic silencing of these pathways abolishes behavioural aversion to concentrated salt, without impairing salt attraction. Notably, mice devoid of salt-aversion pathways show unimpeded, continuous attraction even to very high concentrations of NaCl. We propose that the ‘co-opting’ of sour and bitter neural pathways evolved as a means to ensure that high levels of salt reliably trigger robust behavioural rejection, thus preventing its potentially detrimental effects on health

The Taste of Carbonation

Carbonated beverages are commonly available and immensely popular, but little is known about the cellular and molecular mechanisms underlying the perception of carbonation in the mouth. In mammals, carbonation elicits both somatosensory and chemosensory responses, including activation of taste neurons. We have identified the cellular and molecular substrates for the taste of carbonation. By targeted genetic ablation and the silencing of synapses in defined populations of taste receptor cells, we demonstrated that the sour-sensing cells act as the taste sensors for carbonation, and showed that carbonic anhydrase 4, a glycosylphosphatidylinositol-anchored enzyme, functions as the principal CO_2 taste sensor. Together, these studies reveal the basis of the taste of carbonation as well as the contribution of taste cells in the orosensory response to CO_2

A Novel GTP-binding Protein γ-Subunit, Gγ8, Is Expressed during Neurogenesis in the Olfactory and Vomeronasal Neuroepithelia (∗)

A novel heterotrimeric G-protein gamma-subunit has been cloned, and its function has been confirmed by expression and purification. This gamma-subunit is only detected in the olfactory epithelium, the vomeronasal epithelium and, to a lesser extent, the olfactory bulb. It is absent from all other tissues studied including the nasal respiratory epithelium. During development, expression of G gamma 8 in the olfactory epithelium parallels neurogenesis, peaking shortly after birth and declining in the adult. In situ hybridization studies localize expression of this novel gamma-subunit to the sensory neurons; hybridization is strongest in the region of the epithelium that contains immature neurons. Unlike proteins that are expressed only in mature olfactory neurons (e.g. olfactory marker protein or Golf alpha), expression of G gamma 8 in the olfactory epithelium is relatively unaffected by olfactory bulbectomy. In the vomeronasal epithelium expression of G gamma 8 is also highest in the developing neurons. Taken together, these findings are consistent with a very specific role for G gamma 8 in the development and turnover of olfactory and vomeronasal neurons

A Smell That Causes Seizure

<div><p>In mammals, odorants are detected by a large family of receptors that are each expressed in just a small subset of olfactory sensory neurons (OSNs). Here we describe a strain of transgenic mice engineered to express an octanal receptor in almost all OSNs. Remarkably, octanal triggered a striking and involuntary phenotype in these animals, with passive exposure regularly inducing seizures. Octanal exposure invariably resulted in widespread activation of OSNs but interestingly seizures only occurred in 30–40% of trials. We hypothesized that this reflects the need for the olfactory system to filter strong but slowly-changing backgrounds from salient signals. Therefore we used an olfactometer to control octanal delivery and demonstrated suppression of responses whenever this odorant is delivered slowly. By contrast, rapid exposure of the mice to octanal induced seizure in every trial. Our results expose new details of olfactory processing and provide a robust and non-invasive platform for studying epilepsy.</p></div

Octanal induced activation of OSNs in the MOE.

<p><i>In situ</i> hybridization was used to monitor expression of <i>c-fos</i> as a measure of neuronal activity in the MOE. Control (A) and OiS- (B) mice showed essentially no <i>c-fos</i> expression after exposure to mineral oil. Exposure of mice to filter paper carrying a 20 µl drop of 10% octanal in mineral oil (MO) induced moderate <i>c-fos</i> expression in a subset (25–40%) of OSNs of control animals (C) and strong expression in the vast majority (≥90%) of OiS-line OSNs (D–E). No systematic differences in the intensity or density of <i>c-fos</i> staining were observed between individual exhibiting no response (D) or strong (E) seizures. Scale bar, 50 µm.</p

Expression of <i>rI7</i> and <i>M72</i> in OiS-mice.

<p>(A) Schematic representation of the bidirectional <i>TetO</i> construct used to express two different ORs (<i>rI7</i> and <i>M72</i>) from a single locus. <i>In situ</i> hybridization demonstrated that approx. 90% of OSNs expressed <i>rI7</i> (B) and only approx. 5% of OSNs expressed <i>M72</i> (C); more detail is presented in supplemental data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041899#pone.0041899.s001" target="_blank">Fig. S1</a>). The projection pattern of OSNs expressing <i>tgOR</i>s in OiS-animals was monitored using the co-expressed marker gene (D–F). The MOB exhibited a normal distribution and size range of glomeruli. Whole-mount fluorescence of the dorsal OB revealed rI7 and GFP-expressing neurons innervate nearly all glomeruli (D). Although the M72 transgene is expressed in ∼5% of mature OSNs in OiS-mice, staining for LacZ revealed very few glomerular targets for M72-expressing neurons (E, arrowed). The image shown (lateral view) is typical of LacZ staining in this line with most stained fibers restricted to the ventro-caudal region of the bulb. (This is quite different from LacZ staining in other <i>tgOR-LacZ</i>-lines where targeted glomeruli are generally very clearly labeled even when an equal number of MOE neurons express LacZ thus this does not reflect restriction of LacZ to the cell bodies of OSNs.) (F) GFP-fluorescence (green) in a coronal section through the MOB of an OiS-mouse counterstained with DAPI (gray) demonstrates that all glomeruli contained rI7 and GFP-positive fibers. Scale bars: B & C, 100 µm; D & E, 1 mm; F, 200 µm. Medial (M), Anterior (A), and Ventral (V) directions in whole-mount images are indicated.</p

The spread of neuronal activity corresponds to severity of seizures in OiS-mice.

<p><i>In situ</i> hybridization for <i>c-fos</i> was used to monitor the spread of neuronal activity in the piriform cortex and other regions of the brain (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041899#pone-0041899-g005" target="_blank">Fig. 5</a>). Representative coronal sections at approx. Bregma +1.0 of control (A) and OiS-mouse brains (B, C) are shown; boxed area (piriform cortex) in each panel is shown magnified to the right. After exposure to 10% octanal, control mice showed <i>c-fos</i> expression in a sparse and randomly distributed population of cells in the piriform cortex and other regions of the brain (A). A similar pattern of neural activity was observed in OiS-mice if they exhibited no seizure-like symptoms (B). Mice exhibiting strong symptoms of seizures in response to octanal displayed a robust increase in <i>c-fos</i> expression in the piriform cortex with labeling of many neurons in all layers (C). Seizures also triggered massive neural activity in most other regions of the forebrain. (D) Quantitation of <i>c-fos</i> positive cells in the piriform cortex. Scale bars: A–C, 1 mm; 500 µm for magnified boxed area, data are mean ± s.e.m, n = 3 mice; **denotes <i>p</i><0.01.</p

<i>c-fos</i> expression in OiS mice under controlled octanal exposure.

<p>Controlled delivery of octanal using an olfactometer results in neural activity patterns that closely resemble those observed when mice are passively exposed to odor. <i>In situ</i> hybridization for <i>c-fos</i> was used to monitor the spread of neuronal activity after exposure of mice to defined concentrations and gradients of octanal. Control mice exposed to 5% octanal for 1 minutes in the olfactometer (A, D, G) exhibit <i>c-fos</i> expression patterns in the MOE (A), MOB (D) and brain (G), including piriform cortex (boxed, G) that closely resemble those observed when control animals were passively exposed to odorant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041899#pone-0041899-g002" target="_blank">Figs. 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041899#pone-0041899-g004" target="_blank">4</a>). OiS mice exposed to a gradient of 0–5% octanal over a period of 4 minutes in the olfactometer show activation of the MOE (B) but little change in <i>c-fos</i> expression in the MOB (E) or brain (H). In contrast, OiS mice that were exposed to 5% octanal for 1 minute and exhibited strong seizures not only showed pronounced activation of the MOE (C) but also of M/T and GC cells in the MOB (F) as well as much of the forebrain including piriform cortex (I); see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041899#pone.0041899.s003" target="_blank">Fig. S3</a> for quantitation. Scale bars: A–C, 50 µm; D–F, 200 µm; G–I, 1 mm.</p

Neuronal activation in the MOB after octanal stimulation.

<p>(A) Schematic diagram of the main olfactory bulb (MOB) showing the location of periglomerular (PG), mitral and tufted (M/T), and granular (GC) cells. (B–F) Representative images from <i>in situ</i> hybridization of sections through the MOB using a <i>c-fos</i> probe: mineral oil exposure of control (B) and OiS- (D) mice revealed background <i>c-fos</i> expression that was concentrated in the granule cell layer. Only slight increases in <i>c-fos</i> staining were observed when control mice were (C) challenged with octanal. In contrast, octanal exposure resulted in dramatic differences in MOB neuronal activity of OiS-mice that reflected whether the mouse exhibited symptoms of seizure (E, F). Animals that did not exhibit any symptoms displayed a <i>c-fos</i> expression pattern (E) that resembled <i>c-fos</i> expression in control mice (B, C) or in OiS animals exposed to mineral oil (D). In contrast, OiS-mice that showed strong seizures exhibited a prominent increase in <i>c-fos</i> expression in the M/T but no significant change in activity of PG cells (see G, H for quantitation). In addition, octanal induced seizures were characterized by prominent labeling of the entire granule cell layer. Scale bars: B–F, 500 µm; data are mean ± s.e.m, n = 3 mice; **denotes <i>p</i><0.01.</p