Dopamine reduces odor- and elevated-K(+)-induced calcium responses in mouse olfactory receptor neurons in situ

Abstract

Although D2 dopamine receptors have been localized to olfactory receptor neurons (ORNs) and dopamine has been shown to modulate voltage-gated ion channels in ORNs, dopaminergic modulation of either odor responses or excitability in mammalian ORNs has not previously been demonstrated. We found that <50 microM dopamine reversibly suppresses odor-induced Ca2+ transients in ORNs. Confocal laser imaging of 300-microm-thick slices of neonatal mouse olfactory epithelium loaded with the Ca(2+)-indicator dye fluo-4 AM revealed that dopaminergic suppression of odor responses could be blocked by the D2 dopamine receptor antagonist sulpiride (<500 microM). The dopamine-induced suppression of odor responses was completely reversed by 100 microM nifedipine, suggesting that D2 receptor activation leads to an inhibition of L-type Ca2+ channels in ORNs. In addition, dopamine reversibly reduced ORN excitability as evidenced by reduced amplitude and frequency of Ca2+ transients in response to elevated K(+), which activates voltage-gated Ca2+ channels in ORNs. As with the suppression of odor responses, the effects of dopamine on ORN excitability were blocked by the D2 dopamine receptor antagonist sulpiride (<500 microM). The observation of dopaminergic modulation of odor-induced Ca2+ transients in ORNs adds to the growing body of work showing that olfactory receptor neurons can be modulated at the periphery. Dopamine concentrations in nasal mucus increase in response to noxious stimuli, and thus D2 receptor-mediated suppression of voltage-gated Ca2+ channels may be a novel neuroprotective mechanism for ORNs.

FIG. 1

Confocal imaging of odor responses in olfactory epithelium slices. A: representative olfactory epithelium slice (300 µm) made from mouse (postnatal day 0–5). Scale bar = 500 µm. B–D: confocal images from a fluo-4 AM-loaded mouse olfactory epithelium (OE) slice before (B), during (C), and after (D) application of odors (10 µM n-amyl acetate +10 µM r-carvone). Odor-responsive olfactory receptor neurons (ORNs) are outlined in white. Note that in this focal plane, some ORNs have only the soma outlined. The arrowhead indicates the cell used for odor transients shown in E. Scale bars = 10 µm. E: time course of odor- and high-K⁺-evoked Ca²⁺ transients elicited from one ORN in the slice shown in B–D. Black triangles correspond to the time of loop injection of the odors high K⁺ or Ringer.

FIG. 2

Dopamine reduces odor-evoked calcium transients. Fluo-4 AM loaded olfactory epithelium slices were superfused with multiple applications of 10 µM n-amyl acetate +10 µM r-carvone (odor) in the absence (A and B) or presence (C and D) of 50 µM dopamine (DA). A and C: representative odor-evoked Ca²⁺ transients in the absence (A) and presence (C) of 50 µM dopamine. - - -, linear regressions of the peak amplitudes from the 1st and 3rd Ca²⁺ transients in this and all subsequent figures. B and D: average peak Ca²⁺ transient amplitudes are shown (mean ± SE), as are the predicted peak amplitudes for the 2nd application (■). B: n = 48 cells from 11 slices; D: n = 21 cells/3 slices. *P < 0.05, paired Student’s t-test on predicted vs. observed values.

FIG. 3

D2 dopamine receptors mediate dopamine-evoked reduction in odor response. A, C, and E: representative odor responses in the presence of 50–500 µM D2 dopamine receptor antagonist sulpiride, or dopamine (DA; 50 µM) + sulpiride. ORNs were exposed to dopamine and sulpiride for 5 min prior to odor superfusion. B, D, and F: average peak Ca²⁺ transient amplitudes are shown (means ± SE) as are the predicted peak amplitudes for the 2nd application (■). n = 20 cells/4 slices (B), 30 cells/7 slices (D), and 20 cells/4 slices (F). B: sulpiride (500 µM) did not significantly affect odor responses: P > 0.05, paired Student’s t-test performed on predicted vs. observed values. D: 50 µM dopamine significantly reduced odor responses even in the presence of 50 µM sulpiride: *P < 0.05, paired Student’s t-test as in B. F: 500 µM sulpiride blocked the suppressive effect of dopamine: P > 0.05, paired Student’s t-test as in B.

FIG. 4

High-K⁺ Ringer-evoked calcium transients are reduced in the presence of dopamine. A and C: representative high-K⁺-evoked Ca²⁺ transients elicited by multiple applications of 100 mM K⁺ Ringer in the absence (A) or presence (C) of 50 µM dopamine. B and D: average peak Ca²⁺ transient amplitudes are shown (means ± SE) as are the predicted peak amplitudes for the 2nd application (■). n = 12 cells/2 slices and 21 cells/4 slices, respectively. *P < 0.05, paired Student’s t-test on predicted vs. observed values.

FIG. 5

D2 dopamine receptor activation reduces high-K⁺-evoked Ca²⁺ transients. A and C: representative 100 mM K⁺-evoked Ca²⁺ transients in the presence of 500 µM sulpiride or dopamine + sulpiride. ORNs were exposed to dopamine and sulpiride for 5 min prior to high-K⁺ superfusion. - - -, linear regressions on the peak amplitudes of the 1st and 3rd calcium transients. B and D: average peak Ca²⁺ transient amplitudes are shown (means ± SE) as are the predicted peak amplitudes for the 2nd application (■). n = 19 cells/3 slices and 10 cells/2 slices, respectively. *P < 0.05, paired Student’s t-test on predicted vs. observed values. Sulpiride prevented both basal and exogenous dopamine reductions in high-K⁺-induced Ca²⁺ transients.

FIG. 6

Dopamine decreases the probability of obtaining a high-K⁺ response. A: representative Ca²⁺ responses to 50 mM K⁺ (transients not shown), 50 mM K⁺ + dopamine (0–500 µM), and a 3rd 50 mM K⁺ “recovery” were measured from fluo-4 AM loaded mouse olfactory epithelial slices using confocal microscopy. Note that the first high-K⁺-evoked Ca²⁺ transient is not shown. B: summary of the frequency of response to high K⁺ in the presence of dopamine. Note that the probability of obtaining a response decreases in the presence of increasing dopamine concentrations. The number of ORNs = 47, 65, and 48, respectively. C: the ratios of Ca²⁺ transient amplitudes from the 2nd high-K⁺ + dopamine (0 or 500 µM) response, over the 3rd high-K⁺ “recovery” response were binned at 0.5 intervals and plotted as a frequency histogram. Gaussian fits center over a ratio of 1 for the 0 µM dopamine control group, whereas ratios in the 500 µM dopamine group are shifted toward 0.

FIG. 7

Nifedipine block of Ca²⁺ channels eliminates the suppressive effect of dopamine on odor-induced Ca²⁺ transients. A: representative odor responses in the presence and absence of Ringer or 100 µM nifedipine (100 nif). B: averaged Ca²⁺ transient amplitudes show that 100 µM nifedipine significantly inhibits observed odor responses compared with predicted (■; *P = 0.01, paired Student’s t-test). C and E: representative odor responses during constant bath application of 50 or 100 µM nifedipine in the presence and absence of 50 µM dopamine (DA). D and F: averaged Ca²⁺ transient amplitudes show that in the presence of 50 µM nifedipine dopamine suppresses odor responses (P = 0.01, paired Student’s t-test) but that in 100 µM nifedipine, dopamine does not suppress odor responses (P = 0.63, paired Student’s t-test). G: averaged percentage reduction of the observed odor responses compared with control (mean ± SE; *P < 0.03, paired Student’s t-test).