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First published online May 1, 2006
Journal of Experimental Biology 209, 1914-1927 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02206

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Vomeronasal sensory neurons from Sternotherus odoratus (stinkpot/musk turtle) respond to chemosignals via the phospholipase C system

Jessica H. Brann^1,* and Debra A. Fadool^1,2,

¹ The Florida State University, Department of Biological Science, Program in Neuroscience, Biomedical Research Facility, Tallahassee, FL 32306, USA
² The Florida State University, Department of Biological Science, Program in Molecular Biophysics, Biomedical Research Facility, Tallahassee, FL 32306, USA

View larger version (15K): [in a new window]   Fig. 1. Plastron length and mass of Sternotherus odoratus, by year. (A) Adult female (filled circles) versus adult male (open squares) plastron length, S. odoratus versus collection year (asterisk denotes significantly different between sexes in years shown; mean ± s.e.m.; two-way ANOVA, SNK pairwise multiple comparison between sex and year, P0.05). (B) Mass of adult female (filled circles) and male (open squares) Sternotherus odoratus versus collection year (not significantly different; mean ± s.e.m.; two-way ANOVA, P0.05). N values are shown beside symbols.

View larger version (10K): [in a new window]   Fig. 2. Representative examples of chemosignal-activated current obtained with chemical stimulation of vomeronasal sensory neurons (VSNs). (A) A male vomeronasal sensory neuron (VSN) when stimulated with catfish extract exhibited an outward current, as defined by the polarity of the response obtained at holding potential Vh=–60 mV. The black bar above the trace denotes the time (700 ms) that the VSN was presented with chemosignal delivered via a pressurized glass pipette approximately two cell-widths away from the VSN. (B) As in A but for a female VSN stimulated with female musk. Current record is representative of an inward current, as defined by the polarity of the response obtained at Vh=–60 mV.

View larger version (31K): [in a new window]   Fig. 3. The percentage of cells responding with a given polarity to a particular chemosignal is different between male and female vomeronasal sensory neurons (VSNs). (A) Histogram plot of the three different response categories of VSNs, separated on the basis of sex; male, gray; female, black. (B–F) Histogram plots of the percentage of female and male VSNs responding with an outward (black bars) or inward (white bars) current to (B) male musk, (C) female musk, (D) male urine, (E) female urine, (F) catfish extract. *Significantly different response frequency of inward and outward current across sex by 2 analysis, P0.05; NS, not significantly different. N values are shown beside bars.

View larger version (24K): [in a new window]   Fig. 4. Ruthenium Red (RR), an antagonist of the IP3R, fails to block chemosignal-activated currents in S. odoratus vomeronasal sensory neurons (VSNs) while an inhibitor of phospholipase C (U73122) does block chemosignal-activated currents. (A) A representative example of the response of a female VSN to catfish extract at 0 min (top trace) and 6 min later (bottom trace) without pharmacological perturbation. The chemosignal presentation is shown as black bar above the response. This particular cell was used primarily for reversal potential analysis, hence the VSN had been exposed to the chemical signal twelve times within the time span described. (B) Histogram plot of the normalized current magnitude (normalized to first exposure to chemosignal; t0) over time. Cells were sampled at varying time points (tn), such that not every cell was sampled for each of the time points. The amplitude of the chemosignal-activated current was not altered over 12 min (one-way repeated measures ANOVA, P0.05). (C) A representative example of the response of a female VSN to male urine at 0 min (top trace) and 6 min (bottom trace) following bath application of 1 mmol 1–1 RR. (D) Histogram plot of the normalized current magnitude (normalized to t0) over time. The chemosignal-activated current is not altered over 10 min (not significantly different by treatment or time, two-way repeated measures ANOVA, P0.05) in response to RR treatment. The normal chemosignal response over time is denoted by the broken line. (E) A representative example of the response of a female VSN to catfish extract at 0 min (top trace) and 8 min (bottom trace) following bath application of 50 µmol l–1 U73122. (F) As in D but for U73122 treatment. Asterisks denote significant difference between treatments, two-way repeated measures ANOVA, followed by SNK pairwise multiple comparison between treatment and time, P0.05). N values are shown beside bars. (A,C,E) Broken line denotes baseline current.

View larger version (26K): [in a new window]   Fig. 5. Dialysis of the second messengers cAMP, IP3, OAG, arachidonic acid (AA), and SAG in isolated S. odoratus vomeronasal sensory neurons (VSNs). Analogues were made as described (see Materials and methods) and back-filled in the recording pipette to allow diffusion upon attaining the whole-cell configuration. VSNs were voltage-clamped at Vh=–60 mV. (A) A representative example of a VSN that had not been dialyzed with a second messenger analogue in order to illustrate stability of the patch. (B) 0.5 or 1 µmol l–1 cyclic adenosine monophosphate (cAMP) (N=27). (C) 240 µmol l–1 inositol 1,4,5-trisphosphate (IP3) (N=20). (D) 125 µmol l–1 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol (DAG), applied to either the bath surrounding the VSN or within the recording pipette (N=15). Trace shown is an example of pipette perfusion. (E) 10 µmol l–1 AA, a polyunsaturated fatty acid (PUFA) derivative of DAG (N=11). (F) 10 mmol l–1 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), a natural analogue of DAG (N=6).

View larger version (34K): [in a new window]   Fig. 6. Analysis of chemosignal-activated reversal potentials supports a cationic receptor potential in vomeronasal sensory neurons (VSNs) of S. odoratus. (A,B) Response of a female neuron to (A) catfish extract (1:10 dilution), (B) female musk (1:100 dilution). Broken horizontal lines indicate baseline currents. (C,D) Plotted current–voltage relation for the families of currents evoked in A and B, respectively. (E,F) Scatter plot of estimated reversal potentials derived in C and D for chemosignal-evoked outward (E) and inward (F) currents, respectively. For each chemosignal, the vertical line depicts the mean reversal potential if more than one sample was obtained. The average reversal potential for the outward current was –28.2±2.37 mV (N=30) and that for the inward current was –5.7±7.79 mV (N=10).

View larger version (10K): [in a new window]   Fig. 7. Vomeronasal sensory neurons (VSNs) from S. odoratus exhibit a chemosignal-evoked conductance decrease linked with outward currents. A VSN from a female S. odoratus was held at –60 mV (Vh) with five hyperpolarizing steps to –90 mV (Vc) injected prior (black), during (gray) and following (black) chemosignal stimulation with catfish extract. Inward chemosignal-evoked conductance changes were not tested.

View larger version (21K): [in a new window]   Fig. 8. A peptide directed against the interaction domain between TRPC2 and IP3R3 blocks the chemosignal-activated currents in S. odoratus vomeronasal sensory neurons (VSNs) when included in the patch pipette in the whole-cell configuration. (A) Amino acid sequence of the synthesized peptide (derived from mTRPC2, amino acids 905–934) (Tang et al., 2001). (B) Histogram plot of the chemosignal-activated current over 10 min when 10 µmol l–1 peptide is included in the recording pipette. The normal chemosignal-activated current over time is denoted by a broken line (see Fig. 4). Current obtained for each cell at varying time points (tn) was normalized to its first exposure to chemosignal (t0) Asterisks denote significant differences between control and peptide, two-way repeated measures ANOVA, followed by SNK pairwise multiple comparison between treatment and time, P0.05). (C) Representative example of peptide treatment. While in the whole-cell configuration, the VSN was presented with a chemosignal, and a baseline (immediately after the whole-cell configuration was obtained) recording was made (top). The same chemosignal-activated current is shown 10 min after peptide infusion (bottom). Broken line denotes baseline current.