Chloride accumulation in mammalian olfactory sensory neurons

Abstract

The generation of an excitatory receptor current in mammalian olfactory sensory neurons (OSNs) involves the sequential activation of two distinct types of ion channels: cAMP-gated Ca(2+)-permeable cation channels and Ca(2+)-gated Cl(-) channels, which conduct a depolarizing Cl(-) efflux. This unusual transduction mechanism requires an outward-directed driving force for Cl(-), established by active accumulation of Cl(-) within the lumen of the sensory cilia. We used two-photon fluorescence lifetime imaging microscopy of the Cl(-)-sensitive dye 6-methoxy-quinolyl acetoethyl ester to measure the intracellular Cl(-) concentration in dendritic knobs of OSNs from mice and rats. We found a uniform intracellular Cl(-) concentration in the range of 40-50 mm, which is indicative of active Cl(-) accumulation. Functional assays and PCR experiments revealed that NKCC1-mediated Cl(-) uptake through the apical membrane counteracts Cl(-) depletion in the sensory cilia, and thus maintains the responsiveness of OSNs to odor stimulation. To permit Cl(-) accumulation, OSNs avoid the "chloride switch": they do not express KCC2, the main Cl(-) extrusion cotransporter operating in neurons of the adult CNS. Cl(-) accumulation provides OSNs with the driving force for the depolarizing Cl(-) current that is the basis of the low-noise receptor current in these neurons.

Figure 1.

Olfactory signal transduction. A, Schematic drawing of the olfactory neuroepithelium. The epithelial surface is covered with mucus (MU) that forms the environment for the chemosensory cilia (CI) of OSNs. Epithelial supporting cells (SC) form the apical surface and maintain a regular pattern of dendritic knobs (DK), the apical endings of OSN dendrites. Basal cells (BC) are nondifferentiated neurons that continuously replace OSNs. The mucus layer is primarily supplied by Bowman's glands (BG). B, The current model of excitatory components in olfactory signal transduction. Odorants bind to olfactory receptor proteins (R), which induce activation of type III adenylyl cyclase (AC) through the G-protein G_olf. cAMP opens cyclic nucleotide-gated ion channel, leading to Ca²⁺ influx and activation of Ca²⁺-gated Cl^- channels.

Figure 3.

Determination of [Cl^-]_i in dendritic knobs. A, Dependence of fluorescence lifetimes in mouse olfactory epithelium on [Cl^-]_i. B, Determination of the Stern-Volmer constant (13 m^-1) for MQAE in the tissue. Lifetime at 0 mm Cl^- (τ₀) was calculated from the curve in A. The data were obtained from a tissue preparation in which [Cl^-]_i of OSNs and supporting cells was set to the indicated values using Cl^- ionophores and the standard solutions listed in Table 1. C, [Cl^-]_i in dendritic knobs of rat (left) and mouse (right) olfactory epithelium at an extracellular [Cl^-] of 50 mm, corresponding to the mucosal [Cl^-] in vivo. Mean values are 54 ± 4 mm in rat (96 knobs) and 37 ± 6 mm in mouse (406 knobs). Scale bar, 10 μm.

Figure 2.

2P-FLIM of the olfactory epithelium. A, 2P-FLIM recordings from MQAE-loaded olfactory epithelium yield different fluorescence lifetimes in dendritic knobs and supporting cells. The lower value measured in knobs indicates a higher intracellular Cl^- concentration compared with supporting cells. Np indicates the normalized number of photons analyzed for fluorescence lifetime. B, Fluorescence intensity image (left) and fluorescence lifetime image (right) of the same area of mouse olfactory epithelium loaded with MQAE. The outlines of supporting cells (SC) can be seen in both images. Dendritic knobs (DK), however, can be more easily discerned in the false-color 2P-FLIM representation where warmer colors indicate higher levels of [Cl^-]_i. In the top right corner, the epithelial surface retreats below the focal plane, and only the protruding dendritic knobs remain visible. Scale bar, 10 μm.

Figure 4.

The site of Cl^- uptake. A, Optical sectioning by 2P-FLIM extending from the surface of rat olfactory epithelium 10 μm deep into the tissue, recorded with either 50 mm (left stack) or 150 mm (right stack) extracellular Cl^-. The white lines follow dendrites through the 2 μm intervals. An increase in [Cl^-]_i occurs near the apical end of the dendrites in 150 mm extracellular Cl^-. B, Axial [Cl^-]_i profiles in dendrites near the apical surface. With 50 mm extracellular Cl^-, [Cl^-]_i was near 55 mm and no Cl^- gradient was established (blue line). At 150 mm extracellular Cl^- (red line), a standing [Cl^-]_i gradient formed between the knobs (70 mm) and the proximal dendrite (55 mm), suggesting that Cl^- enters OSNs via the cilia. Means ± SD (7-14 knobs per point).

Figure 5.

Cl^- uptake mechanisms in mouse OSNs. A, 2P-FLIM images of the epithelial surface during transient exposure to 50 mm extracellular Cl^-. [Cl^-]_i in dendritic knobs declined when extracellular Cl^- was reduced from 150 to 50 mm and recovered after high Cl^- was restored (a-c). Bumetanide (BU) (50 μm) prevented recovery (d-f) as did exposure to Na⁺-free solution (g-j). Scale bar, 10 μm. B, Quantitative analysis of [Cl^-]_i of the experiment shown in A. The light gray area indicates the time interval when extracellular Cl^- was reduced from 150 to 50 mm. Reuptake of Cl^- into the dendritic knobs was suppressed by bumetanide and by exposure to Na⁺-free solution. Na⁺-dependent, bumetanide-sensitive Cl^- uptake represents evidence for the activity of a NKCC-type Cl^- transporter. Mean ± SEM of five to eight knobs. C, Detection of Cl^- transporter mRNA in rat olfactory epithelium. RT-PCR experiments yielded signals for KCC1, NKCC1, and NCC in olfactory epithelium cDNA (OE). No expression of KCC2 or NKCC2 was detected. Positive controls for KCC2 [rat hippocampus (HC)] and for NKCC2 [rat kidney (K)] are shown on the right.

Figure 6.

Impaired Cl^- homeostasis in isolated OSNs. A, 2P-FLIM images from isolated rat OSNs loaded with MQAE, sorted by the [Cl^-]_i in their dendritic knobs. [Cl^-]_i ranges from 20 to 60 mm. The extracellular Cl^- concentration was 150 mm. Scale bar, 10 μm. B, Distribution of [Cl^-]_i values in dendritic knobs of isolated OSNs (blue histogram; 30 ± 8 mm; 100 cells) and in knobs in intact epithelium (red histogram; 62 ± 6 mm; 39 cells) in the presence of 150 mm extracellular Cl^-. Most isolated OSNs showed a reduced ability to accumulate Cl^-.