Bile salts as olfactory and gustatory stimuli in the channel catfish

A chemotopic map of biologically relevant odorants (that include amino acids, bile salts and nucleotides) exists in the olfactory bulb (OB) and forebrain (FB) of channel catfish, Ictalurus punctatus (Chapter one). Neurons processing bile salt odorant information lie medially within these bilaterally symmetric structures; however, information as to how single neurons discriminate and process this odorant information is lacking. Chapters two and three of the dissertation identify the range of odorant bile salt molecules that excite these neurons [i.e. the excitatory molecular receptive range (EMRR)] within the bile salt chemotopic zones of the OB and FB. The results of the investigations of single bile salt responsive neurons within the OB indicate that these neurons are selectively excited by combinations of molecular features found on the side-chain and the steroid nucleus of bile salt molecules. Further, the results of the investigations of single bile salt responsive neurons within the FB indicate that their EMRRs are virtually identical to that of OB neurons suggesting that little modification of the neural olfactory quality code for these molecules occurred between the OB and the FB. Bile salts are known olfactory stimuli to teleosts, but only a single report (Yamashita et al. 2006) indicated that the taste system of a fish was sensitive to this class of stimuli. Chapter four investigates the gustatory sensitivity of the facial taste system to bile salts in the channel catfish. Bile salts were shown to be highly effective facial taste stimuli with estimated electrophysiological thresholds of approximately 10-11M-10-10M. Multiunit cross-adaptation experiments indicate that bile salts and amino acids bind to relatively independent receptor sites; however, nerve twig data and a few single fiber recordings suggest that both independent and shared neural pathways exist for the transmission of bile salt and amino acid information to the primary gustatory nucleus of the medulla. The findings of the present report aid in understanding how bile salt molecules are detected and initially processed by the olfactory and gustatory systems in catfish and further suggest that bile salt odorant information is not greatly transformed by central olfactory neurons (Chapter five)

Polyamines as olfactory stimuli in goldfish

The effects of polyamines as odorants to goldfish olfactory receptors were investigated by in vivo electrophysiological recordings. Electro-olfactogram (EOG) recordings indicated that polyamines (putrescine, cadaverine and spermine) are potent olfactory stimuli for goldfish with estimated electrophysiological thresholds of 10-100nM, similar to that for L-arginine, the most stimulatory amino acid. Although thresholds were similar, the magnitude of the EOG responses to intermediate and high concentrations of polyamines dwarfed those to amino acids and single amine containing compounds (amylamine and butylamine). The EOG responses to 0.1mM putrescine, cadaverine and spermine were, respectively, 4.2x, 4.3x and 10.3x that of the standard, 0.1mM L-arginine. Electrophysiological cross-adaptation experiments indicated the independence of polyamine receptor sites from those to L-amino acids (arginine, methionine, alanine, glutamate, lysine and ornithine), bile salts (Na+ taurocholate and taurolithocholate), single amine containing compounds, and ATP. Further, cross-adaptation experiments indicated that independent receptors exist for the different polyamines tested. During continuous application of forskolin (5-20micromolar), an adenylate cyclase activator, EOG responses to bile salts were eliminated, while responses to L-amino acids, polyamines and ATP were only partially attenuated. These results suggest that polyamine odorants, in contrast to bile salt, L-amino acid and nucleotide odorants are transduced by a non-cAMP second messenger pathway. Although polyamines result in large EOG responses, olfactory receptor neurons responding excitedly to polyamines are likely few in number, because polyamine application to the olfactory mucosa failed to increase integrated multi-unit activity recorded from the sensory surfaces of olfactory lamellae. Preliminary data suggest that olfactory bulb neurons that respond excitedly to L-amino acids are inhibited by polyamines. The present results indicate polyamines are potent odorants to goldfish, distinct from L-amino acids, bile salts and nucleotides

Naris occlusion upregulates GFP presence in the OB.

<p>A shows the representative coronal 18 µm section of the naris-occluded OB of a TRPM5 GFP animal (scale bar = 500 µm). The right OB in the image is ipsilateral to the occluded (Closed) naris, and the left OB is ipsilateral to the open naris. The section was immunostained with an antibody against GFP (green). As expected, the OB is smaller in the occluded side <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061990#pone.0061990-Brunjes3" target="_blank">[52]</a>. (B) Histogram of the number of pixels as a function of the fluorescence intensity (0–4095) after subtracting intensity taken from the external plexiform layer (EPL) just underneath the glomerular layer. GFP immunofluorescence is higher in the occluded side (ii) compared to open side (i). (C) Cumulative histogram for fluorescence intensity after subtraction of EPL fluorescence levels for all four animals examined. Occluded OBs (red) express GFP at significantly higher level than open OB (blue) (t-test for mean intensity, <i>p</i> = 0.0286, n = 4). D shows a 2D color map of the glomeruli displaying GFP immunofluorescence as a function of percentage distance from the dorsal most point (*) around the glomerular layer in the olfactory bulb in A. Three representative OB sections were taken from the rostral, medial and caudal one-third and analyzed for GFP immunofluorescence intensity around the glomerular layer. (E) Mean fluorescence intensity around the glomerular layer of the occluded (red) and the open OB (blue). Thin lines represent the standard error of the mean (SEM). The intensity was averaged for caudal, middle and rostral images. Occluded side of OB (red) significantly differs from the open side of the OB (blue) (<i>p</i><0.0001, N-Way ANOVA, n = 4). % peripheral distance was measured starting from the dorsal most point. d = dorsal, l = lateral, v = ventral, m = medial.</p

Naris occlusion upregulates GFP immunofluorescence in the occluded side of the OE of TRPM5-GFP mice.

<p>A. This panel shows a representative image of a 14 µm coronal section of the OE taken from a naris-occluded animal. The right side in the image is ipsilateral to the occluded naris (Closed) (scale bar  = 500 µm). GFP immunofluorescence is green. B. Averaged GFP immunofluorescence (intensity ranges from 0 to 1, with a gain set so that OSN GFP immunofluorescence ranged from 0 to 0.2). The left side in the image is ipsilateral to the open side of naris (Open). Averaged GFP immunofluorescence intensity in the OE in the septum (i–iii) and the lateral regions (iv) were compared between open and closed sides. Averaged fluorescence intensity was significantly higher in the closed side of epithelium in all locations. (i; <i>p</i> = 0.02, ii; <i>p</i> = 0.006, iii; <i>p</i> = 0.002, iv; <i>p</i> = 0.007, p value FDR corrected 0.05, paired t-test, n = 4).</p

CNGA2 knockout OB shows wider distribution of glomeruli displaying GFP immunofluorescence that differs from the WT OB.

<p>A shows a representative coronal section of the OB of CNGA2 knockout/TRPM5-GFP mouse (CNGA2 KO in the figure). Eighteen µm OB section was immunoreacted with antibody against GFP (green, scale bar = 500 µm). B shows the mean fluorescence intensity around the glomerular layer as a function of percent peripheral distance from the dorsal most point. Thin lines represent the SEM. The CNGA2 knockout OB (black) significantly differs from the open side of naris occluded OB (blue) of TRPM5-GFP animals (Open in the figure). (two way ANOVA for CNGA2 knockout/TRPM5-GFP vs. TRPM5-GFP open naris <i>F</i>_(1,49) = 189, <i>p</i><0.0001, n = 4–6). % peripheral distance was measured starting from the dorsal most point. d = dorsal, l = lateral, v  =  ventral, m = medial.</p

Naris occlusion upregulates the intensity of ciliary layer immunolabeling with a TRPM5 antibody.

<p>Immunolabeling for TRPM5 (red) and GFP (green) is shown in the naris open and occluded sides under two different magnifications. A. Immunolabeling in the open and closed nostrils in endoturbinate II (bar is 50 µm, D is dorsal and M is medial). Notice that there is more intense labeling for both GFP and TRPM5 in the olfactory epithelium soma layer (OS) and cilia (cil) respectively in the closed nostril and that within each turbinate the labeling was inhomogeneous (some areas in the section show higher intensity than others). In addition, as expected from our earlier study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061990#pone.0061990-Lin3" target="_blank">[27]</a> there is intense labeling of microvillar cells that are not being studied here (mic). B. GFP immunolabeling intensity in the olfactory soma layer vs. TRPM5 immunolabeling intensity in the ciliary layer (red) measured in 20 degree sections around the endoturbinates in A (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061990#s2" target="_blank">methods</a>). Intensity in the image ranged from 0 to 1. Blue: closed, red: open. The straight line is a best fit for all the points. The correlation coefficient is 0.72 (different from zero, p<0.0001). The intensity for both GFP and TRPM5 immunolabeling in the open nostril are statistically significantly different compared to intensity in the closed nostril (t-test, p<0.000001). C. Higher magnification figures for the TRPM5 (red) and GFP (green) immunohistochemistry (bar is 20 µm, no microvillar cells are found in these images). The two images for the epithelium in the closed naris were from areas that displayed different intensity for labeling for the two antibodies.</p

Underwater EOG response to DMP and 2-heptanone was affected by unilateral naris occlusion in TRPM5(-) epithelia.

<p>A; Example traces of EOG. The responses to DMP and isoamylacetate (ISO) are depicted in red and the response to IBMX is depicted in blue. The black bars above the traces indicate when the stimuli reach the epithelia and the duration of the stimulus application ( = 1 second). B; Putative pheromones (2,5-dimethylpyrazine (DMP) and 2-heptanone (2-HEP)), diluted urine (1∶200 or 1∶100) and general odorants (lilial and isoamylacetate) were tested for underwater EOG. What is reported is the ratio of the response to the pheromone/odorant divided by the response to IBMX [odor/IBMX]. In TRPM5(+) olfactory epithelia, naris occlusion had no effect on the ratio [odor/IBMX] (p value>0.05, paired t-test, FDR corrected). However, in TRPM5(-) olfactory epithelia, naris occlusion significantly reduced the ratio [odor/IBMX] to 2-heptanone or DMP (p<0.01, paired t-test, FDR corrected). The numbers in parentheses show the number of epithelia tested. The error bars are SEM.</p

Decreased odor-evoked OSNs activity leads to an elevated level of TRPM5 mRNA.

<p>The mRNA levels of TRPM5, OMP, and Gα_olf were compared between: A. left (open) and right (closed, occluded) OE of the naris-occluded TRPM5-GFP animals. B. CNGA2 knockout/TRPM5-GFP and TRPM5-GFP control OE. mRNA levels are normalized to levels of 18 S rRNA. In the occluded OE, there was significantly higher expression of mRNA of TRPM5 (<i>p</i> = 0.024, n = 10), OMP (<i>p</i> = 0.008, n = 10), and Gα_olf (<i>p</i> = 0.033, n = 10, paired t-test, p value FDR corrected <i>p</i> = 0.05).Untreated control animals showed no significant difference between left and right OE (<i>p</i>>0.05, paired t-test, n = 5, data not shown). The mRNA expression level of TRPM5 was significantly higher in CNGA2 knockout/TRPM5-GFP compared to TRPM5-GFP (<i>p</i> = 0.016, unpaired t-test, p value FDR corrected <i>p</i> = 0.0167, n = 5). There was no significant difference between CNGA2 knockout/TRPM5-GFP and TRPM5-GFP in mRNA expression levels for OMP and Gα_olf (<i>p</i>>0.05, unpaired t-test FDR corrected, n = 5). Error bars are SEM.</p