Neurotrophin and Trk neurotrophin receptors in the inner ear of Salmo salar and Salmo trutta

Abstract

Neurotrophins (NTs) and their signal transducing Trk receptors play a critical role in the development and maintenance of specific neuronal populations in the nervous system of higher vertebrates. They are responsible for the innervation of the inner ear cochlear and vestibular sensory epithelia. Neurotrophins and Trks are also present in teleosts but their distribution in the inner ear is unknown. Thus, in the present study, we used Western-blot analysis and immunohistochemistry to investigate the expression and cell localization of both NTs and Trk receptors in the inner ear of alevins of Salmo salar and Salmo trutta. Western-blot analysis revealed the occurrence of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), but not nerve growth factor (NGF), as well as all three Trk receptors, i.e. TrkA, TrkB and TrkC, the estimated molecular weights of which were similar to those expected for mammals. Specific immunoreactivity for neurotrophins was detected mainly in the sensory epithelia. In particular, BDNF immunoreactivity was found in the maculae of the utricle and saccule, whereas NT-3 immunoreactivity was present in the sensory epithelium of the cristae ampullaris. As a rule the sensory epithelia of the inner ear lacked immunoreactivity for Trks, thus excluding possible mechanisms of autocrinia and/or paracrinia. By contrast, overlapping subpopulations of neurons in the statoacoustic ganglion expressed TrkA (about 15%), TrkB (about 65%) and TrkC (about 45%). The present results demonstrate that, as in mammals and birds, the inner ear of teleosts expresses the components of the neurotrophin-Trk system, but their roles remain to be elucidated.

Fig. 1

Transverse (a) and horizontal (b) sections of the head of Salmo salar and Salmo trutta through the inner ear (asterisks). Arrows indicate maculae and cristae ampullaris. Both the maculae (c) and the cristae ampullaris (d) consist of hair sensory cells, basal cells, supporting cells and mantle cells. Orientation coordinates: c, cephalic; d, dorsal; l, lateral; bs, brain stem; r, retina. Scale bars = 1 mm for (a) and (b); 40 µm for (c) and (d).

Fig. 2

Immunohistochemical localization of S100 protein (a, c, e) and neurofilament proteins (b, d, f) in the sensory epithelia of the inner ear of Salmo salar and Salmo trutta. S100 protein was used to identify hair sensory cells, and neurofilament proteins to label the axons supplying them. All the sensory hair cells of the cristae ampullaris displayed cytoplasmic immunoreactivity for S100 protein whereas in the maculae only two-thirds of hair sensory cells were inmunoreactive. S100 protein immunoreactivity also labelled Schwann cells in nerve bundles. Axons supplying sensory epithelia displayed neurofilament protein immunoreactivity. Arrows in (f) indicate nerve fibres in the macula of the utriclulus in Salmo trutta. c, cupula. Scale bar = 40 µm.

Fig. 3

(a) Western blot detection of BDNF and NT-3 in head homogenates of alevins of Salmo salar (lanes 1 and 2) and Salmo trutta (lanes 3 and 4). The anti-BDNF and anti-NT-3 antibodies used recognize protein bands of ∼13 and ∼14 kDa, respectively. (b) Western-blot detection of TrkA, TrkB and TrkC in head homogenates of alevins of Salmo salar (lanes 1 and 2) and Salmo trutta (lanes 3 and 4). The anti-TrkA antibody used recognizes a protein band of ∼140 kDa, whereas the antibodies against TrB and TrkC recognize proteins of ∼145 kDa. The antibodies used recognize sequences within the catalytic domain of TrkA, TrkB and TrkC, and therefore the proteins detected are mature full-length isoforms of these proteins.

Fig. 4

Immunohistochemical detection of neurotrophins and Trks in the maculae of the utriculus (a–c; Salmo salar) and sacculus (d–f; Salmo trutta). BDNF immunoreactivity was found in cells of the utriculus and sacculus sensory epithelia. A faint NT-3 immunoreactivity was found to be restricted to some sensory cells in the macula of the utriculus. No immunoreactivity for TrkA, TrkB or TrkC was detected in the maculae. Non-sensory epithelial cells covering the walls of both the utriculus and the sacculus displayed immunoreactivity for all Trk receptors and NT-3. Scale bars = 40 µm.

Fig. 5

Immunohistochemical detection of neurotrophins and Trks in the cristae ampullaris of the semicircular canals of Salmo salar (b, e, f) and Salmo trutta (a, c, d, g). BDNF immunoreactivity was found in few non-sensory epithelial cells (arrow in a) whereas NT-3 immunoreactivity was detected in most if not all hair sensory cells (hsc in b and c). TrkA immunoreactivity was absent from hair sensory cells, but was occasionally detected in isolated sensory cells (small arrow in d); some of them displayed a moderate cytoplasmic staining (Fig. 5d). TrkB immunoreactivity was only found in the apical segment of the mantle cells (small arrow in e), and TrkC immunoreactivity was observed in nerve fibre profiles (arrows in f and g). TrkA and TrkB immunoreactivity was also found in cubical epithelial cells placed at the basis of the cristae (large arrows in d and e). c, cupule. Scale bars = 40 µm (identical for a, d–f).

Fig. 6

Immunohistochemical localization of neurotrophins and Trk neurotrophin receptors in the statoacustic ganglion in approximate serial sections of the head of Salmo trutta (a–c) and Salmo salar (d–f). The typical bipolar morphology of the neurons in the cochleo-vestibular ganglion was evident in ganglia innunostained for TrkC (f). s, sacculus. Scale bar = 40 µm (identical for b–f).

Fig. 7

Percentage of neurons displaying immunoreactivity for neurotrophins (a) and Trk receptors (b) in the statoacoustic ganglion of Salmo salar and Salmo trutta. Measurements were made on five sections per animal (n = 5 Salmo salar; n = 5 Salmo trutta), 100 µm apart, per specimen and per antibody evaluating all nerve cell profiles present in the entire section.

Fig. 8

A theoretical model of the NT–Trk system in the inner ear of teleosts, based on the results of immunohistochemistry. The maculae of the utriculus and sacculus express BDNF and NT-3, whereas the sensory epithelia of the cristae ampullaris only express NT-3. Presumably these growth factors are convoyed via retrograde axonal transport to the statoacoustic ganglion. The division of the statoacoustic ganglion into utricular (UG) and saccular (SG) is artificial, and suggests that there is a predominance of TrkC-positive nerve fibres and neurons in the SG, whereas in the UG the non-TrkC-positive cells predominate. AC, anterior semicircular canal; HC, horizontal semicircular canal; PC, posterior semicircular canal; MU, maculeae of the utriculus; MS, maculae of the sacculus.