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Responses of Xenopus laevis water nose to water-soluble and volatile odorants.
Iida A
,
Kashiwayanagi M
.
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Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents, and inward currents in response to water-soluble and volatile odorants from receptor neurons in the lateral diverticulum (water nose) of the olfactory sensory epithelium of Xenopus laevis. The resting membrane potential was -46.5 +/- 1.2 mV (mean +/- SEM, n = 68), and a current injection of 1-3 pA induced overshooting action potentials. Under voltage-clamp conditions, a voltage-dependent Na+ inward current, a sustained outward K+ current, and a Ca2+-activated K+ current were identified. Application of an amino acid cocktail induced inward currents in 32 of 238 olfactory neurons in the lateral diverticulum under voltage-clamp conditions. Application of volatile odorant cocktails also induced current responses in 23 of 238 olfactory neurons. These results suggest that the olfactory neurons respond to both water-soluble and volatile odorants. The application of alanine or arginine induced inward currents in a dose-dependent manner. More than 50% of the single olfactory neurons responded to multiple types of amino acids, including acidic, neutral, and basic amino acids applied at 100 microM or 1 mM. These results suggest that olfactory neurons in the lateral diverticulum have receptors for amino acids and volatile odorants.
Figure 1. Xenopus laevis olfactory receptor neurons in the sensory epithelium. Light microscopic photomicrographs of slices of olfactory epithelia in the lateral diverticulum (A) and in the medial diverticulum (B) after the frog was cultured in 0.1% methylene blue water for 6 h. (C) A schema of the olfactory organ of Xenopus laevis. (D) An olfactory receptor neuron in the lateral diverticulum dialyzed with 1% Lucifer yellow. The knoblike structure, long dendrite, axon, and soma are visible.
Figure 2. Current-clamp recordings from olfactory receptor neurons in the lateral diverticulum of Xenopus laevis. Current-clamp recordings showing the voltage responses to positive current injections from a conditioning potential of â60 mV (A). Above a certain threshold, repetitive action potential spikes were observed. The number next to the action potentials indicates the amplitude of currents injected. These traces were recorded from the same cell. (B) Spike frequency of olfactory receptor neurons as a function of current injection for six neurons that fired repeatedly. (C) An action potential recorded in response to a positive current pulse (40 pA) from the conditioning potential (â60 mV). This action potential response was reversibly blocked by 1 μM TTX.
Figure 3. (A) The currents seen under voltage-clamp conditions in response to negative and positive voltage pulses from â70 mV. (B) Currentâvoltage relations. The peak of the transient inward current (â¢) and the outward current (â). Isolated Na+ currents from olfactory neurons in the lateral diverticulum. The voltage-activated Na+ current is blocked after the substitution of Na+ with choline in the external solution (C) or the addition of 1 μM TTX to the external solution (D). The internal solution contained (mM): 115 CsCl, 2 MgCl2, 2 EGTA, 10 HEPES-NaOH, pH 7.4. The external EGTA solution contained (mM): 116 NaCl, 4 KCl, 1 MgCl2, 1 EGTA, 10 glucose, 10 HEPES-NaOH, pH 7.4. Isolated K+ currents in olfactory receptor neurons in the lateral diverticulum of Xenopus laevis, and the effect of the application of TEA and the elimination of Ca2+ (EâH). (E) The K+ current responses to positive voltage pulses from a holding potential of â70 mV. This current was reversibly blocked by 25 mM TEA (F and G). The external solution contained (mM): 116 N-methyl-d-glucamine, 1 EGTA, 2 CoCl2, 10 glucose, 10 HEPES-KOH, pH 7.4. (H) The outward K+ current is partially attenuated by the elimination of Ca2+ from the external solution. The Ca2+-free solution contained (mM): 116 NaCl, 4 KCl, 1 MgCl2, 1 EGTA, 10 glucose, 10 HEPES-KOH, pH 7.4.
Figure 4. Inward currents in response to the amino acid cocktail (A and B). Inward currents in response to volatile odorant cocktail I (C) and cocktail II (D). All responses were recorded from different neurons (Aâ¼D). (E) Inward currents in response to volatile odorant cocktail I, volatile odorant cocktail II, and the amino acid cocktail, respectively. The same inward current in response to the amino acid cocktail was shown with a short time scale in inset. All responses were recorded from the same neuron (E). The holding potential was â70 mV.
Figure 5. Response profile of single olfactory neurons to various odorants as obtained by the whole-cell clamp technique. The â and à indicate an inward current and no current, respectively, upon the application of odorants.
Figure 6. Inward currents in response to alanine of varying concentrations (A). Responses from different neurons were recorded. Mean magnitude of inward currents in response to alanine (B) and arginine (C) of varying concentrations. Each point is the mean ± SEM of data obtained from n preparations that responded to any of the amino acids applied. The holding potential was â70 mV.
Figure 7. Inward currents in response to 100 μM alanine, 100 μM arginine, and 100 μM glutamic acid (A), and those to 1 mM alanine, 1 mM arginine, and 1 mM glutamic acid (B). All responses to each of the three traces were recorded from the same neurons. The holding potential was â70 mV. (C) Response profile of single olfactory neurons to various odorants of 1 mM as obtained by the whole-cell clamp technique. The â and à indicate an inward current and no current, respectively, upon the application of odorants.
Figure 8. Inward currents in response to 1 and 10 μM lilial, and 1 mM alanine (A). All responses to each of the four traces were recorded from the same neurons. The holding potential was â70 mV. Mean magnitude of inward currents to lilial (B) and citralva (C) of varying concentrations. Each point is the mean ± SEM of data obtained from n preparations that responded to the odorants applied. The holding potential was â70 mV.
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