Patch-clamp analysis of voltage-activated and chemically activated currents in the vomeronasal organ of Sternotherus odoratus (stinkpot/musk turtle)
D. A. Fadool1,*,
M. Wachowiak2 and
J. H. Brann1
1 The Florida State University, Program in Neuroscience and Molecular Biophysics, Biomedical Research Facility, Tallahassee, FL 32306, USA and
2 Yale University School of Medicine, Department of Physiology, New Haven, CT 06520, USA
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Fig. 1. Visualization of the vomeronasal neural epithelium (VNE) of the musk turtle Sternotherus odoratus. (A) Coronal cryosection (10 µm thick) of the vomeronasal organ labeled with rhodamine-conjugated dextran to visualize the vomeronasal (VN) sensory neurons. Dextran was introduced into the left vomeronasal organ orifice and permitted to migrate for approximately 2 weeks. Note the absence of labeling on the contralateral VNE. Scale bar, 80 µm. (B) Higher magnification of the section shown in A. The dendrites of the microvillar layer (M) and more basally positioned somata (S) are visible. Scale bar, 40 µm. (C) Cryosection (10 µm thick) of the dextran labeling contained in axon terminals of the VN neurons at the level of the glomeruli of the accessory olfactory bulb. Scale bar, 40 µm. (D) The same type of section and orientation as in B counterstained with Masson’s Trichrome. Scale bar, 40 µm. (E,F) Isolated VN neurons from a female (E) and a male (F) turtle. Scale bars, 10 µm. Ca, cartilage; S, somata; M, microvilli; Ax, axon; De, dendrite.
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Fig. 4. K+ current block by tetraethylammonium (TEA+) is dissimilar between male and female vomeronasal (VN) neurons. Dose/response curves for TEA+ block of K+ currents in female (A,B) and male (C,D) VN neurons. Whole-cell currents were elicited using the voltage stimulation protocol described in Fig. 3, and neurons were pretreated with 20 nmol l–1 tetrodotoxin (TTX) to isolate predominantly outward currents. The initial current at +40 mV depolarization was normalized to that after a 5 min incubation with bath-applied TEA+ at various concentrations. Peak transient current was measured 50 ms after the beginning of the voltage stimulation, and peak sustained current was measured at the end of the 400 ms duration of the voltage stimulation. The smooth curves are fits to a single-exponential decay, where y=yo+Ae–(x–x0)t, with a half-maximal inhibition concentration (Ki) for the transient K+ current of 2.4 µmol l–1 and 2.0 µmol l–1 for male and female neurons, respectively. The sustained K+ current had a Ki of 4.8 µmol l–1 and 1.7 µmol l–1 for male and female neurons, respectively. Values are means ± S.E.M. (sample sizes are given on the figure).
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Fig. 5. Vomeronasal (VN) neurons are chemically activated by multiple natural compounds. (A) Whole-cell, voltage-clamp recording from a female VN neuron in response to stimulating the cell (black bar) with a 1:100 dilution of female musk, a 1:100 dilution of male musk, a 1:10 dilution of female urine, a 1:10 dilution of male urine and turtle Ringer alone. Holding potential, Vh, was –60 mV for all traces. Chemical stimulation lasted 700 ms. (B) Whole-cell, voltage-clamp recording from a male VN neuron. This neuron displays the typical outward current observed in both genders in response to urine. Cat, catfish extract. (C) Histogram of the percentage of 90 neurons displaying chemically evoked currents in response to at least one of five natural stimuli tested per neuron as in A or B. The filled area of the column on the far right shows the percentage of neurons responding to only one of the five natural chemicals, and the hatched area of the column shows the percentage of neurons responding to two or more of the five test chemicals. Sample sizes are given on the figure.
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Fig. 6. Vomeronasal (VN) neurons elicit both inward and outward currents in response to chemical stimulation. (A) Plot of the distribution of the peak current magnitude in response to exposure to five natural stimuli. The most frequent response magnitudes were between –15 and +15 pA. Data were fitted with a bin width of 10 pA and with a normal distribution (solid line) to approximate the mean frequency. Zero current responses are not plotted. (B) Plot of the percentage of VN neurons that respond to the stimuli with inward and outward currents. Urine was tested at a dilution of 1:10, musk at a dilution of 1:300 and catfish extract at a dilution of 1:100. The number of neurons tested is given in parentheses. Cat, catfish extract; M Musk, male musk; F Musk, female musk; M Urine, male urine; F Urine, female urine.
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Fig. 7. The breadth of responsiveness of vomeronasal (VN) neurons indicates a degree of diversity on the basis of entropy calculations. (A) The response profiles of 10 VN neurons to five chemicals. Each horizontal line depicts the responses of a different neuron; the height of the columns on each line indicates the amplitude and the direction of the columns indicates the polarity of the chemically evoked current (pA). The outward currents are denoted as positive (upward columns) and the inward currents are denoted as negative (downward columns). The calculated H metric for a given neuron is presented to the left of each response profile. Breadth of responsiveness is minimal (H=0) when a VN neuron responds to only one of five chemicals (narrow selectivity) and it is maximal (H=1.0) when there is an equal response to each of the five chemicals (no selectivity or low diversity in response). (B) Histogram of the breadth of responsiveness of 36 neurons. The arrow denotes the mean H value (0.11±0.02, mean ± S.E.M.) (see Materials and methods for calculation). Cat, catfish extract; M Musk, male musk; F Musk, female musk; M Urine, male urine; F Urine, female urine.
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© The Company of Biologists Ltd 2001
=0.05. (C,D) As in A and B but for total neurite process length. Data were less well fitted by a normal distribution and not significantly different between female and male VN neurons (Student’s t-test, 
) and the peak transient (
) and sustained (
) components of the outward current. (C,D) As in A and B but for a female VN neuron. Note that the outward currents of the female inactivated more rapidly than those of the male (see Results for calculations). The insets in A and C are the same recording on an expanded time scale to allow visualization of the rapid inward current and kinetics of activation. (E) Time to half-activation of the outward voltage-activated current measured from male (
; N=19) VN neurons. Note that the vertical axis is on a logarithmic scale. Values are means ± S.E.M.