Sour Ageusia in Two Individuals Implicates Ion Channels of the ASIC and PKD Families in Human Sour Taste Perception at the Anterior Tongue

BACKGROUND:The perception of sour taste in humans is incompletely understood at the receptor cell level. We report here on two patients with an acquired sour ageusia. Each patient was unresponsive to sour stimuli, but both showed normal responses to bitter, sweet, and salty stimuli. METHODS AND FINDINGS:Lingual fungiform papillae, containing taste cells, were obtained by biopsy from the two patients, and from three sour-normal individuals, and analyzed by RT-PCR. The following transcripts were undetectable in the patients, even after 50 cycles of amplification, but readily detectable in the sour-normal subjects: acid sensing ion channels (ASICs) 1a, 1beta, 2a, 2b, and 3; and polycystic kidney disease (PKD) channels PKD1L3 and PKD2L1. Patients and sour-normals expressed the taste-related phospholipase C-beta2, the delta-subunit of epithelial sodium channel (ENaC) and the bitter receptor T2R14, as well as beta-actin. Genomic analysis of one patient, using buccal tissue, did not show absence of the genes for ASIC1a and PKD2L1. Immunohistochemistry of fungiform papillae from sour-normal subjects revealed labeling of taste bud cells by antibodies to ASICs 1a and 1beta, PKD2L1, phospholipase C-beta2, and delta-ENaC. An antibody to PKD1L3 labeled tissue outside taste bud cells. CONCLUSIONS:These data suggest a role for ASICs and PKDs in human sour perception. This is the first report of sour ageusia in humans, and the very existence of such individuals ("natural knockouts") suggests a cell lineage for sour that is independent of the other taste modalities

Cholecystokinin: an excitatory modulator of mitral/tufted cells in the mouse olfactory bulb.

Cholecystokinin (CCK) is widely distributed in the brain as a sulfated octapeptide (CCK-8S). In the olfactory bulb, CCK-8S is concentrated in two laminae: an infraglomerular band in the external plexiform layer, and an inframitral band in the internal plexiform layer (IPL), corresponding to somata and terminals of superficial tufted cells with intrabulbar projections linking duplicate glomerular maps of olfactory receptors. The physiological role of CCK in this circuit is unknown. We made patch clamp recordings of CCK effects on mitral cell spike activity in mouse olfactory bulb slices, and applied immunohistochemistry to localize CCKB receptors. In cell-attached recordings, mitral cells responded to 300 nM-1 µM CCK-8S by spike excitation, suppression, or mixed excitation-suppression. Antagonists of GABAA and ionotropic glutamate receptors blocked suppression, but excitation persisted. Whole-cell recordings revealed that excitation was mediated by a slow inward current, and suppression by spike inactivation or inhibitory synaptic input. Similar responses were elicited by the CCKB receptor-selective agonist CCK-4 (1 µM). Excitation was less frequent but still occurred when CCKB receptors were blocked by LY225910, or disrupted in CCKB knockout mice, and was also observed in CCKA knockouts. CCKB receptor immunoreactivity was detected on mitral and superficial tufted cells, colocalized with Tbx21, and was absent from granule cells and the IPL. Our data indicate that CCK excites mitral cells postsynaptically, via both CCKA and CCKB receptors. We hypothesize that extrasynaptic CCK released from tufted cell terminals in the IPL may diffuse to and directly excite mitral cell bodies, creating a positive feedback loop that can amplify output from pairs of glomeruli receiving sensory inputs encoded by the same olfactory receptor. Dynamic plasticity of intrabulbar projections suggests that this could be an experience-dependent amplification mechanism for tuning and optimizing olfactory bulb signal processing in different odor environments

Contribution of CCK_A receptors to mitral cell excitation.

<p><b>A–B.</b> CCK evokes an excitatory inward current when CCK_B receptors are blocked (compare to Figs. 6F–H). <b>A.... </b><i>Upper panel</i>: whole-cell voltage clamp recording from a mitral cell (CD-1 mouse) stimulated with 1 µM CCK-8S (lower horizontal bar), in the presence of 5 µM LY225910 (upper horizontal bar), a CCK_B-selective antagonist. An inward current response was accompanied by increased EPSC activity (LLDs). <i>Middle panel</i>: histogram of EPSC counts over time (20 s bins) showing excitatory effect of CCK. <i>Lower panel</i>: time course of slow inward current response estimated by plotting baseline values for detected EPSCs (gray circles). Black line: smoothed curve obtained by averaging window of 8 data points. <b>B.... </b><i>Upper panel</i>: voltage clamp recording of inward current and EPSC from same cell as in <b>A</b>, stimulated with 1 µM CCK-8S (horizontal bar) after washout of LY225910, showing shorter inward current followed by outward current. <i>Middle panel</i>: histogram of EPSC counts over time (20 s bins) showing excitation followed by reduction in activity. <i>Lower panel</i>: Time course of slow inward/outward current response, estimated as in <b>A.... </b><b>C.</b> Histogram of spike counts over time (20 s bins) for cell-attached recording from a mitral cell (CD-1 mouse) showing excitation (<i>e</i>) in response to 300 nM CCK-8S (horizontal bar), in the presence of 1 µM LY225910 (pretreated slice). <b>D.</b> Histogram of spike counts over time (20 s bins) for cell-attached recording from a mitral cell from a CCK_B knockout mouse showing excitation (<i>e</i>) and suppression (<i>s</i>) in response to 1 µM CCK-8S (horizontal bar). In this cell, spike rate rebounded noticeably after CCK washout. <b>E.... </b><i>Upper panel</i>: mean spike rate (baseline subtracted) for mitral cells from CCK_B knockout mice unresponsive to CCK-8S (<i>n</i> = 13). <i>Lower panel</i>: mean normalized spike rate for excitatory and excitatory-suppressive spike responses in CCK_B knockout mice evoked by CCK-8S (<i>n</i> = 3).</p

Whole-cell recording of mitral cell currents response to CCK-8S stimulation.

<p><b>A.</b> Whole-cell voltage-clamp recording of membrane current of a mitral cell during perfusion with 1 µM CCK-8S (horizontal bar), showing slow inward current response. <b>B.</b> Time course of slow inward current revealed by applying a low pass filter (0.10 Hz) to trace in <b>A.. </b><b>C.</b> Whole-cell current-clamp recording from same cell as in <b>A</b>, showing voltage response to reapplication of 1 µM CCK-8S (horizontal bar). Depolarization caused the cell to switch between a lower voltage down-state (<i>d</i>) with action potential firing, and a high voltage up-state (<i>u</i>) with fast membrane potential oscillations, which are known properties of mitral cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064170#pone.0064170-Desmaisons1" target="_blank">[80]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064170#pone.0064170-Rubin1" target="_blank">[82]</a>. <b>D.</b> Whole-cell voltage clamp recording from a second mitral cell during perfusion with 1 µM CCK-8S (horizontal bar), showing lack of slow inward current and increase in IPSC activity. <b>E.</b> Histogram of IPSC counts over time (20 s bins) for the trace in <b>F.</b> Inset: expanded segment from <b>D</b> showing IPSCs occurring during CCK stimulation. <b>F.</b> Whole-cell voltage clamp recording from a third mitral cell during perfusion with 1 µM CCK-8S (horizontal bar), showing both a slow inward current and an increase in IPSC activity. Inset: in vitro fluorescence image of cell loaded with Alexa 594 to reveal soma (<i>s</i>), apical dendrite (<i>d</i>) and glomerular tuft (<i>t</i>). Scale bar: 50 µm. <b>G.</b> Time course of slow inward current revealed by applying a low pass filter (0.10 Hz) to trace in <b>F.. </b><b>H.</b> Histogram of IPSC counts over time (20 s bins) for trace in <b>F.</b> Inset: expanded segment from <b>F</b> showing IPSCs occurring during CCK stimulation. <b>I.</b> Mean normalized change in IPSC rate of mitral cell voltage clamp responses to CCK-8S (<i>n</i> = 8 cells, calculated as described in Figs. 2E–F).</p

Cell-attached recording of mitral cell spike excitation by CCK-8S.

<p><b>A.</b> Calibrated timing of agonist application, derived from measurement of K⁺ junction current of micropipette in olfactory bulb slice. Valve actuation at 100 s (left arrow) switched aCSF flow to high K⁺ perfusion, and washout was subsequently initiated at 400 s (right arrow). Black bar indicates time period of switched perfusion. Ordinate is the relative change in K⁺ concentration, estimated from shift in junction current at room temperature, ΔI [pA], as: [K]₀ exp(0.004 ΔI), for 100 MΩ pipette. With a flow rate of 5 ml/min, the time lag in junction current shift was ∼80 s. Perfusion for 300 s was minimum time to attain final applied agonist concentration in the bath. <b>B.</b> Excitatory spike response of a mitral cell to bath perfusion of 1 µM CCK-8S, applied with time course shown in <b>A</b> (cell-attached patch recording of action currents, slice from CD-1 mouse). <b>C.</b> Histogram of spike rate over time (counts in 10 s bins) for the response in <b>B. D.</b> Plot of windowed spike rate vs. time, computed from spike times for response in <b>B</b>, with 50 s sliding window. Vertical line marks timing of CCK-8S perfusion switch. Horizontal line marks the mean pre-stimulus spike rate; gray band is 99% confidence limit (3× sd<i>_r</i>) around the mean. Calculated duration of excitatory spike response (<i>D</i>_e) is indicated.</p

Whole-cell recording of mitral cell voltage response to CCK-8S stimulation.

<p><b>A.</b> Whole-cell current clamp recording of membrane potential change of a mitral cell during perfusion with 1 µM CCK-8S (bar indicates 400 s period of switched perfusion) showing elevated firing correlated with a slow depolarizing potential of ∼16 mV amplitude. <b>B.</b> Slow depolarizing response revealed by applying low pass filter (0.10 Hz) to trace in <b>A.</b> Inset image: biocytin stain of recorded cell showing amputated apical dendrite (scale bar: 50 µm). <b>C.</b> Plot of windowed spike rate vs. time for response in <b>A</b>, showing several phases of spike excitation and suppression (<i>e</i>₁, <i>s</i>₁, <i>e</i>₂, <i>s</i>₂). All spike events were counted, including those with reduced amplitude (but doublets only once). The first suppressive phase (<i>s</i>₁) correlated with a period of spike inactivation at depolarization peak, the second (<i>s</i>₂) with a post-stimulus hyperpolarization. Inset traces: 1 s expanded segments of voltage record at different times (arrows): basal spike rate (200 s), rising phase (430 s), first peak (500 s), first minimum (570 s), second peak (650) and recovery (1000 s). <b>D–E.</b> Running average plots of spike rise times (<b>D</b>) decay times (<b>E</b>) for the response in <b>A</b> (mean ± standard deviation, in 10 s bins). <b>F.</b> Whole-cell current clamp recording of membrane potential from a second mitral cell with a spiking response to 1 µM CCK-8S perfusion (horizontal bar). <b>G.</b> Slow depolarizing response revealed by applying a low pass filter (0.10 Hz) to trace in <b>F.. </b><b>H.</b> Histogram of spike counts over time (20 s bins) for the trace in <b>F.. </b><b>I.</b> Histogram of IPSP counts over time (20 s bins) for the trace in <b>F.</b> Inset: expanded trace from <b>F</b> showing action potential and IPSPs (arrows) occurring during the CCK response.</p

Contribution of CCK_B receptors to mitral cell excitation.

<p><b>A–B.</b> CCK_B-selective agonist CCK-4 evokes excitatory and suppressive spike responses. <b>A.</b> Histogram of spike counts over time (10 s bins) for a cell-attached spike recording from a mitral cell (CD-1 mouse) stimulated by perfusion with CCK-4 (horizontal bar). CCK-4 evoked a transient excitatory phase (<i>e</i>), followed by spike suppression (<i>s</i>), and after removal of stimulus there was a rebound excitation. <b>B.</b> Mean normalized spike rate for all mitral cell responses evoked by CCK-4 in CD-1 mice (<i>n</i> = 7 cells, 4 excitatory, 2 mixed excitatory-suppressive, 1 suppressive). <b>C–D.</b> Stimulation by 1 µM CCK-8S causes spike excitation in CCK_A knockout mice. <b>C.</b> Histogram of spike counts over time (20 s bins) for cell-attached spike recording from a mitral cell from a CCK_A knockout mouse, showing excitation (<i>e</i>) in response to CCK stimulus (horizontal bar). <b>D.</b> Histogram of spike counts from the same cell after washout and reapplication of 1 µM CCK-8S (horizontal bar) in the presence of a CCK_B-selective antagonist (5 µM LY 225910) which blocked spike excitation. <b>E. </b><i>Upper panel</i>: mean spike rate (baseline subtracted) for mitral cells unresponsive to CCK-8S in the presence of LY225910 (<i>n</i> = 9). <i>Lower panel</i>: mean normalized spike rate for excitatory spike responses evoked by CCK-8S in the presence of LY225910 (<i>n</i> = 3 cells). All data from CD-1 mice. <b>F–J.</b> Inward current underlies spike excitation in the CCK_A knockout mouse. <b>F.</b> Whole-cell voltage clamp recording from a mitral cell from a CCK_A knockout mouse stimulated with 1 µM CCK-8S (horizontal bar), showing both slow inward current and an increase in EPSC activity, including currents underlying long lasting depolarizations (LLDs) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064170#pone.0064170-Carlson1" target="_blank">[55]</a>. <b>G.</b> Histogram of EPSC counts over time (20 s bins) for the trace in <b>F</b>, showing excitatory effect of CCK. <b>H.</b> Time course of slow inward current response to CCK for the trace in <b>F</b>, estimated by plotting baseline values for detected EPSCs (gray circles). Black line: smoothed curve obtained by averaging window of 8 data points. <b>I.</b> Whole-cell current clamp recording from same cell as in <b>F</b>, showing strong spike excitation caused by reapplication of 1 µM CCK-8S (horizontal bar). <b>J.</b> Histogram of spike counts over time (20 s bins) for the voltage trace in <b>I</b>, showing periods of spike frequency excitation (<i>e</i>) and suppression (<i>s</i>).</p

Cellular localization of CCK_B receptor immunoreactivity in mouse olfactory bulb.

<p><b>A.</b> Confocal fluorescent image of anti-CCK_B receptor antibody binding to a horizontal section of an olfactory bulb from a CD-1 mouse, visualized with Alexa Fluor 633-conjugated secondary antibody (red). Strong immunoreactivity appears in a superficial zone including the inner margins of the glomerular layer (GL) and the distal part of the external plexiform layer (EPL), and in a deeper zone corresponding to the mitral cell body layer (MCL). Scale bar: 80 µm. <b>B.</b> Composite image showing overlay of CCK_B immunoreactivity in <b>A</b> (red) with fluorescent DAPI-stained nuclei (blue) to highlight the positions of cell bodies. Similar cellular distributions of immunoreactivity were obtained in 29 sections from 5 mice. <b>C.</b> Confocal fluorescent image (Alexa Fluor 633) of horizontal section of olfactory bulb from a 129-Cckbr^tm1Kpn/J (CCK_B knockout) mouse, processed with the same antibody and protocol as in <b>A.</b> Scale bar: 40 µm. <b>D.</b> Composite image from the CCK_B knockout obtained by overlaying Alexa Fluor 633 fluorescence in <b>C</b> (red) with DAPI stained nuclei (blue). A similar absence of cell labeling was seen in 4 other sections of the bulb from the same mouse. <b>E.</b> Confocal fluorescent image of anti-CCK_B receptor binding to horizontal section from a CD-1 mouse, using B2 antibody of Mercer & Beart (2000) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064170#pone.0064170-Mercer3" target="_blank">[42]</a>(1∶100 dilution). Scale bar: 80 µm. <b>F.</b> Composite image combining <b>E</b> (red) with fluorescent DAPI stained nuclei (blue). <b>G.</b> Distribution of cell body diameters in the GL and EPL that were immuno-positive for CCK_B receptor. <b>H.</b> Distribution of cell body diameters in the glomerular layer and EPL that were immuno-positive for CCK_B receptor. Histograms in <b>G</b>–<b>H</b> are normalized to total cell count. Note: Small non-nucleated fluorescent filaments visible in some sections are due to non-specific background staining of erythrocytes and appear in controls not treated with primary antibody. Abbreviations: GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; GCL, granule cell layer.</p

Colocalization of CCK_B receptor immunoreactivity in mouse olfactory bulb.

<p><b>A–F.</b> Colocalization of immunoreactivity to CCK_B receptor and mitral/tufted cell nuclear marker Tbx21. <b>A–C.</b> Confocal fluorescent images of double labeled horizontal olfactory bulb section from a CD-1 mouse showing binding of antibody to CCK_B receptor (<b>A</b>) (red, Alexa Fluor 633), to Tbx21 (<b>B</b>) (green, Alexa Fluor 488), and the overlay of both images (<b>C</b>). Scale bar: 150 µm. <b>D–F.</b> Higher magnification view of mitral cell somata in the double labeled section, showing nuclear localization of Tbx21, and contrasting extranuclear distribution of CCK_B. Scale bar: 15 µm. <b>G–I.</b> Confocal fluorescent images of double labeled horizontal olfactory bulb section from a CD-1 mouse showing binding of antibody to CCK_B receptor (<b>G</b>) (green, Alexa Fluor 488), to CCK-8S (<b>H</b>) (red, Alexa Fluor 633), and the overlay of both images (<b>I</b>). In the overlay, yellow labeling in the GL/EPL layer indicates cellular colocalization of receptor and peptide, whereas separated green and red bands in MCL and IPL indicates spatial separation of receptor and peptide. Scale bar: 150 µm. Abbreviations: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064170#pone-0064170-g008" target="_blank">Figure 8</a>. Insets in <b>I</b>: upper inset shows magnified view of cell somata in the superficial EPL (putative superficial tufted cells) with double labeling for peptide and CCK_B receptor; lower inset shows somata in mitral cell body layer (putative mitral cells) with single labeling for CCK_B receptor; scale bar for both in upper inset: 15 µm.</p

Parameters characterizing mitral spike responses to CCK receptor agonists.

<p>First three rows list descriptive parameters under control conditions for three classes of CCK-8S spike response: excitatory, mixed excitatory-suppressive, and suppressive (data pooled for 300 nM and 1 µM CCK-8S; there was no significant difference between the two concentrations). For multiphasic responses caused by spike inactivation (e.g. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064170#pone-0064170-g003" target="_blank">Fig. 3C</a>), duration of excitation was calculated using the end of the second excitatory peak. Next two rows list parameters of spike responses to 1 µM CCK-8S recorded in the presence of blockers of fast synaptic transmission. Last three rows list parameters of responses to 1 µM CCK-4, for 3 spike response classes: excitatory, mixed excitatory-suppressive, and suppressive. Values are expressed as mean ± SEM. Latencies were measured from time of CCK perfusion switch (including ∼50–80 s perfusion lag) to time of deviation from mean basal spike rate (p<0.01), and durations were computed from time points when responses deviated above or below (p<0.01) mean basal spike rate. Rate delta values for CCK responses are peak increments in spike rate relative to basal spike rate (positive for excitatory, negative for suppressive responses).</p