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Polyamines as olfactory stimuli in the goldfish Carassius auratus

S. H. Rolen^1,*, P. W. Sorensen², D. Mattson² and J. Caprio¹

¹ Department of Biological Sciences, Louisiana State University, Life Sciences Building Room 202, Baton Rouge, LA 70830, USA
² Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota, 200 Hodson Hall, 980 Folwell Avenue, St Paul, MN 55108, USA

View larger version (21K): [in a new window]   Fig. 3. Compiled electro-olfactogram (EOG) responses of goldfish to ascending concentrations of L-arginine, polyamines (putrescine, cadaverine and spermine) and single amine containing compounds (amylamine and butylamine). Responses were standardized to 0.1 mmol l–1 L-arginine, which evoked a response of 0.4±0.2 mV (mean ± S.D.). Values are means ± S.E.M. 3–6 fish were tested for each concentration series. Putrescine, cadaverine and spermine evoked responses significantly greater than L-arginine at all tested odorant concentrations 10–5 mol l–1 (Student's t-test; P<0.05).

View larger version (21K): [in a new window]   Fig. 1. Schematic diagram of the open field maze used in the attraction tests. See Materials and methods for details.

View larger version (19K): [in a new window]   Fig. 2. Typical electro-olfactogram (EOG) recordings to ascending concentrations of (A) L-arginine, (B) putrescine, (C) cadaverine and (D) spermine, performed in a single goldfish. Odorant concentrations (in mol l–1) eliciting each response are listed below each respective trace. C, charcoal filtered tapwater. Scale bars, 0.5 mV (vertical); 30 s (horizontal).

View larger version (22K): [in a new window]   Fig. 4. Integrated multiunit recordings obtained from a single electrode location in the sensory epithelium to: (A) control (C) and 0.1 mmol l–1 L-arginine (Arg); (B) putrescine, (C) cadaverine and (D) spermine (inset shows an example of spermine decreasing background neural activity). (E) The chart indicates the median (filled circle) and range (horizontal bar) of estimated electrophysiological thresholds (mol l–1) to putrescine (Put; N=5 fish), cadaverine (Cad; N=5) and spermine (Spe; N=2). Odorant concentrations (mol l–1) in A–D are listed below each trace.

View larger version (18K): [in a new window]   Fig. 5. Representative recordings from electro-olfactogram (EOG) cross-adaptation experiments recorded prior to (i), during (ii) and after (iii) adaptation to (A) 1 mmol l–1 glutaric acid (a deaminated analog of glutamate; pKa=4.34 and 5.22) and (B) 1 mmol l–1 L-glutamate (a negatively charged amino acid). The adapting solution is underlined. C, CFTW control; Glt, glutaric acid; Glu, L-glutamate; Arg, 0.1 mmol l–1 L-arginine; Put, 10 µmol l–1 putrescine; Cad, 10 µmol l–1 cadaverine; Spe, 3 µmol l–1 spermine.

View larger version (22K): [in a new window]   Fig. 6. Representative recordings of electro-olfactogram (EOG) cross-adaptation experiments prior to (i), during (ii) and after (iii) adaptation to: (A) a mixture of 0.1 mmol l–1 L-amino acids (AA; alanine, arginine, glutamate and methionine), (B) a mixture of 0.3 µmol l–1 bile salts (BS; sodium taurocholate and taurolithocholate), (C) 40 µmol l–1 ATP (compounds containing phosphate groups result in positive EOG deflections in goldfish), and (D) a mixture of polyamines (PA; 10 µmol l–1 putrescine and cadaverine and 3 µmol l–1 spermine). The adapting solution is underlined. The concentrations of the test solutions varied: in A, PA = 20 µmol l–1 putrescine, cadaverine and 2 µmol l–1 spermine) and BS = 10 µmol l–1 TCA and TCLA; in B, AA = 10 µmol l–1 alanine, arginine, glutamate and methionine and PA = 3 µmol l–1 putrescine, cadaverine and 1 µmol l–1 spermine; in C, AA = 100 µmol l–1 arginine, methionine, alanine and glutamate and PA = 10 µmol l–1 putrescine, cadaverine and 2 µmol l–1 spermine; in D, AA = 100 µmol l–1 alanine, arginine, glutamate and methionine and BS = 5 µmol l–1 TCA and TCLA; ATP = 40 µmol l–1. Upward deflections from the baseline are negative. Responses that are positive (downward) deflections were offset to enable comparison. Scale bars, 0.5 mV (vertical); 30 s (horizontal).

View larger version (26K): [in a new window]   Fig. 7. Results of cross-adaptation experiments utilizing mixtures of odorants. Adaptation to (A) L-amino acids (alanine, arginine, glutamate and methionine; 10–500 µmol l–1), (B) bile salts (sodium taurocholate and taurolithocholate; 0.3–10 µmol l–1), (C) ATP (30–70 µmol l–1) and (D) polyamines (1–40 µmol l–1 putrescine, cadaverine and spermine). The adapting solution in A–D is underlined. Bars indicate the percentage of unadapted response (mean ± S.D.). Numbers associated with each bar indicate the number of fish tested for each odorant category. Responses to test stimuli (A–D) are all significantly greater than the response to the adapting solution (one-way ANOVA, Tukey's post hoc test, P<0.05).

View larger version (13K): [in a new window]   Fig. 8. Results of cross-adaptation experiments to (A) putrescine (Put; 9–50 µmol l–1), (B) cadaverine (Cad; 20–40 µmol l–1) or (C) spermine (Spe; 2–7 µmol l–1). The adapting solution in A–C is underlined. Bars indicate percentage of unadapted response (mean ± S.D.). Numbers associated with each bar indicate the number of fish tested for each odorant. L-arginine (Arg; 0.1–1 mmol l–1); L-lysine, (Lys; 0.1–1 mmol l–1); L-methionine (Met; 0.1–0.5 mmol l–1); L-ornithine (Orn; 0.1–1 mmol l–1); amylamine (Amy; 0.03–0.5 mmol l–1); butylamine (But; 0.05–0.5 mmol l–1). Response magnitudes to test stimuli (A–C) are significantly greater than the response magnitude to the adapting solution (one-way ANOVA; Tukey's post hoc test, P<0.05).

View larger version (12K): [in a new window]   Fig. 9. The effects of forskolin and 1,9-dideoxyforskolin on odorant-evoked responses. (A) Representative electro-olfactogram (EOG) recordings to a mixture of 100 µmol l–1 L-amino acids (AA; alanine, arginine, glutamate and methionine), polyamines (PA; 20 µmol l–1 putrescine, cadaverine and 2 µmol l–1 spermine) and 10 µmol l–1 bile salts (BS; sodium taurocholate and taurolithocholate) prior to (i), during (ii) and after (iii) adaptation to 7 µmol l–1 forskolin (For). DS, dimethyl sulfoxide; C, CFTW control). (B) Representative EOG recordings to 100 µmol l–1 AA, 1 µmol l–1 BS and PA (10 µmol l–1 putrescine, cadaverine and 1 µmol l–1 spermine) during adaptation to 20 µmol l–1 1,9-dideoxyforskolin (De). (C) Percentage of unadapted response (mean ± S.D.) to mixtures of L-amino acids (50–500 µmol l–1), polyamines (1–20 µmol l–1), bile salts (10–50 µmol l–1), forskolin control and ATP (30–40 µmol l–1) during adaptation to forskolin (5–20 µmol l–1). Numbers associated with each bar indicate the number of fish tested for each odorant. The adapting solution is underlined. X, Y and Z designate statistical significance across groups (one-way ANOVA; Tukey's post hoc test, P<0.05).

View larger version (28K): [in a new window]   Fig. 10. The effects of the potent inhibitor of agonist-induced phospholipase C (PLC) activity, U-73122 (U7P), and its weaker analog, U-73343 (U7W) on odorant-evoked responses. (A,C) Representative electro-olfactogram (EOG) recordings to a mixture of 100 µmol l–1 L-amino acids (AA; alanine, arginine, glutamate and methionine), polyamines (PA; 10 µmol l–1 putrescine and cadaverine and 2 µmol l–1 spermine) and 10 µmol l–1 bile salts (BS; sodium taurocholate and taurolithocholate) prior to (i), during (ii) and after (iii) adaptation to 1 µmol l–1 U-73122 (U7P) and 1 µmol l–1 U-73343 (U7W), respectively. DS, dimethyl sulfoxide; C, CFTW control. The EOG response to ATP has been offset to allow for comparison. (B,D) Percentage of unadapted response (mean ± S.D.) to the adapting controls (underlined), (U7P in B; U7W in D), L-amino acids, polyamines, bile salts and ATP during adaptation to U7P (in B) and U7W (in D). Data in B and D were compiled from three fish. The adapting solution is underlined. X, Y and Z designate statistical significance across groups (one-way ANOVA; Tukey's post hoc test, P<0.05).

View larger version (19K): [in a new window]   Fig. 11. (A) Results of Behavior Experiment 1. The median (and 75% quartile) number of snapping and biting behaviors exhibited by groups of goldfish in a 4 min period before odor was added (`Pre') and then while odor was present (`During') in Experiment 1. Significant differences between the Pre- and test periods are noted. **P<0.01. (B) Results of Behavior Experiment 2. The median percentage time (75% quartile) spent by goldfish in the area of the maze to which test odors were added. Time spent during the 12 min period prior to odor addition (`Pre') and then after it had been added and was still present (`During') are shown for each stimulus. Significant differences between Pre- and test periods are noted. *P<0.05, **P<0.01.

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