Olfactory and solitary chemosensory cells: two different chemosensory systems in the nasal cavity of the American alligator, Alligator mississippiensis

Abstract

Background: The nasal cavity of all vertebrates houses multiple chemosensors, either innervated by the Ist (olfactory) or the Vth (trigeminal) cranial nerve. Various types of receptor cells are present, either segregated in different compartments (e.g. in rodents) or mingled in one epithelium (e.g. fish). In addition, solitary chemosensory cells have been reported for several species. Alligators which seek their prey both above and under water have only one nasal compartment. Information about their olfactory epithelium is limited. Since alligators seem to detect both volatile and water-soluble odour cues, I tested whether different sensory cell types are present in the olfactory epithelium.

Results: Electron microscopy and immunocytochemistry were used to examine the sensory epithelium of the nasal cavity of the American alligator. Almost the entire nasal cavity is lined with olfactory (sensory) epithelium. Two types of olfactory sensory neurons are present. Both types bear cilia as well as microvilli at their apical endings and express the typical markers for olfactory neurons. The density of these olfactory neurons varies along the nasal cavity. In addition, solitary chemosensory cells innervated by trigeminal nerve fibres, are intermingled with olfactory sensory neurons. Solitary chemosensory cells express components of the PLC-transduction cascade found in solitary chemosensory cells in rodents.

Conclusion: The nasal cavity of the American alligator contains two different chemosensory systems incorporated in the same sensory epithelium: the olfactory system proper and solitary chemosensory cells. The olfactory system contains two morphological distinct types of ciliated olfactory receptor neurons.

Figure 1

Macroscopic view of split head of a juvenile alligator. A Lateral view. B Right half of the head showing the incurrent naris and the length of the nasal cavity. * A piece of the septum occludes the nasal cavity. OB – olfactory bulb, VB – vestibule. C Same view as in B depicting the 5 zones that were examined at the light microscopic and the electron microscopic level.

Figure 2

A – D Histological staining nuclear red/light green/orange G. Note that A – D are depicted at the same magnification and that all zones contain OSNs (based on results from semi- and ultrathin sections). A Olfactory epithelium in zone 1 close to the naris. B Ventrolateral olfactory epithelium in zone 2. The height of the epithelium is variable. Bowman glands (bg) are present. bv – blood vessel. C Olfactory epithelium in zone 4 (anterior turbinate). bv – blood vessel. D Olfactory epithelium in zone 4 (anterior turbinate) in a different region than shown in C. bg – Bowman gland. E – F Semithin (1 μm) sections stained with toluidin blue. Both E and F depict zone 3. Bowman glands are numerous (bg). Note in F how few OSNs are present (arrows). Sc shows a nucleus in the layer of nuclei of the supporting cells. osn shows the layer of nuclei of the olfactory neurons and bc the layer of basal cells. bg – Bowman gland, bv – blood vessel, ms – muscle.

Figure 3

TEM micrographs of alligator nasal epithelia. A Low magnification of olfactory epithelium. Long slim supporting cells are filled with electron-lucent vesicles (vs). Note the area at the distal portion of the cell (asterisk) where vesicles are mostly absent. The surface of the supporting cells is covered with small microvilli. OSNs are rare (arrows). n – nucleus. B Higher magnification of the two different OSNs seen in A. One OSN (I) has few mitochondria. The cytoplasm is electron-lucent and contains microtubules. The other OSN (II) has abundant mitochondria and an electron-denser cytoplasm. Both cell types bear cilia and microvilli on their olfactory knobs. sc – supporting cell. C Apical ending of an OSN (type II) filled with mitochondria. The olfactory knob seems to bear only microvilli. However, serial sections reveal that cilia are present as seen in D. D The same type II OSN as in C showing microvilli and cilia.

Figure 4

TEM micrographs of alligator nasal epithelia. A OSN (type I) with few mitochondria. The long arrow points to a centriole deep in the cytoplasm of the OSN. Short arrow – basal body of cilia. sc – supporting cell. B Basal portion of the olfactory epithelium. The basal lamina (arrowheads) follows the undulating shape of a basal cell (bc). Axons (ax) aggregate above the basal lamina into fila olfactoria that will penetrate the basal lamina in a different location. C Solitary chemosensory cell (SCC) in the olfactory epithelium. The cytoplasm is filled with small vesicles. The surrounding supporting cells (sc) constrict the apical ending of the SCC into a "neck" (small arrows). Large arrow – OSN. D Nonsensory epithelium in the most ventral part of the nasal cavity. Among ciliated nonsensory cells (arrowheads), a cell with small microvilli-like protrusions contains vesicles of various sizes (compare Fig. 5E). Debris-containing vesicles (arrow) suggest a phagocytotic function. E Another type of nonsensory epithelium with cells that bear no cilia but only small microvilli-like protrusions. The cytoplasm is also filled with vesicles of various sizes as seen in D, but cell debris never occurred within these cells. n – nucleus; vs – vesicles.

Figure 5

SEM micrographs of alligator nasal epithelia. A Fracture of olfactory epithelium in zone 5. With 70 μm this is one of the thinner regions of epithelium on the caudal turbinate. B Overview of nasal epithelium in zone 1 which TEM preparations proved to be olfactory. Arrowheads depict openings of Bowman glands. C Fracture showing the apical portion of olfactory epithelium in zone 4. The olfactory knob of an OSN bears cilia and microvilli whereas the apical surfaces of the supporting cells (SC) are covered by microvilli. D Overview of olfactory epithelium in zone 5 showing the "mixed" olfactory knobs bearing cilia and microvilli (*). Note that the cilia do not taper at their ends. The short microvilli of supporting cells are visible between the olfactory knobs. E Overview of nasal epithelium in zone 4. A dense population of ciliated nonsensory cells (compare Fig. 4D) covers the more ventrally located surface. Only occasionally, non-ciliated cells are interspersed (*). F Overview of olfactory epithelium in zone 5. Between the cilia of OSNs and supporting cells (distinguishable by their short microvilli), one thick dome-shaped apical ending indicates the presence of a SCC (arrow).

Figure 6

Cell counting of sensory cells. The average number of OSNs counted in the 5 different zones of the nasal cavity increases steadily from anterior towards the turbinates.

Figure 7

Cryosections (16 μm) of various zones immunoreacted for PGP9.5 and PLCβ2. A In zone 2 (anterior nasal cavity) few OSNs show PGP9.5-like immunoreactivity. B In zone 4 (anterior turbinate) considerably more OSNs are labelled with PGP9.5. Also, two larger apical endings (arrow) are darkly labelled (see also paragraph solitary chemosensory cells). C In the most ventral nonsensory portion of zone 3 no labelled cells occur. The apical fringe of nonsensory cilia is labelled non-specifically as is typical for nonsensory cilia. D A control section from zone 4 (anterior turbinate) where the primary antibody had been omitted shows no label at all. E PLCβ2 labels thick apical endings of few cells (arrows) in zone 2 (anterior nasal cavity) and also in F Zone 4 (anterior turbinate). Size and scarceness of the label indicates that these are SCCs.

Figure 8

Immunocytochemical experiments with fluorescent secondary antibodies. The insets in A – C show higher magnifications of the apical and inset D the basal epithelia. A Gα_olf labels a thick fringe of olfactory cilia in zone 5 (caudal turbinate). B In zone 2 (anterior nasal cavity) considerably fewer cilia are Gα_olf-positive. C The YOL 1/34 antibody, a marker for microtubules, shows a fair amount of olfactory cilia in zone 3 (between the anterior turbinate and the anterior nasal cavity). D Substance P-positive fibers are present beneath the olfactory epithelium in zone 1 (most anterior nasal cavity). Some fibers reach into the epithelium where they may innervate SCCs.