Regeneration of vestibular horizontal semicircular canal afferents in pigeons.

Spontaneous regeneration of vestibular and auditory receptors and their innervating afferents in birds, reptiles, and amphibians are well known. Here, we produced a complete vestibular receptor loss and epithelial denervation using an ototoxic agent (streptomycin), after which we quantitatively characterized the afferent innervation of the horizontal semicircular canals following completed regeneration. We found that calyx, dimorph, and bouton afferents all regenerate in a manner the recapitulates the epithelial topography of normal birds, but over a slow time course. Similar to previous findings in the vestibular otolith maculae, regeneration occurs according to a three-stage temporal sequence. Bouton afferents regenerate during the first month of regeneration, followed by calyceal-bearing afferents in the second and third months. Calyx afferents were the last to regenerate in the final stage of recovery after 3 mo. We also found that regenerated afferents exhibited terminal morphologies that are significantly smaller, less complex, and innervate fewer receptor cells over smaller epithelial areas than those that develop through normative morphogenesis. These structural fiber changes in afferent innervation correlate to alterations in gaze responses during regeneration, although the exact underlying mechanisms responsible for behavioral changes remain unknown. Plasticity in central vestibular neurons processing motion information seem to be required to explain the observed morphologic and response adaptations observed in regenerating vestibular systems.

Recovery of gaze stability during vestibular regeneration.

Many motion related behaviors, such as gaze stabilization, balance, orientation, and navigation largely depend on a properly functioning vestibular system. After vestibular insult, many of these responses are compromised but can return during the regeneration of vestibular receptors and afferents as is known to occur in birds, reptiles, and amphibians. Here we characterize gaze stability in pigeons to rotational motion during regeneration after complete bilateral vestibular loss via an ototoxic antibiotic. Immediate postlesion effects included severe head oscillations, postural ataxia, and total lack of gaze control. We found that these abnormal behaviors gradually subsided, and gaze stability slowly returned to normal function according to a temporal sequence that lasted several months. We also found that the dynamic recovery of gaze function during regeneration was not homogeneous for all types of motion. Instead high-frequency motion stability was first achieved, followed much later by slow movement stability. In addition, we found that initial gaze stability was established using almost exclusive head-response components with little eye-movement contribution. However, that trend reversed as recovery progressed so that when gaze stability was complete, the eye component had increased and the head response had decreased to levels significantly different from that observed in normal birds. This was true even though the head-fixed VOR response recovered normally. Recovery of gaze stability coincided well with the three stage temporal sequence of morphologic regeneration previously described by our laboratory.

Regeneration of vestibular otolith afferents after ototoxic damage.

Regeneration of receptor cells and subsequent functional recovery after damage in the auditory and vestibular systems of many vertebrates is well known. Spontaneous regeneration of mammalian hair cells does not occur. However, recent approaches provide hope for similar restoration of hearing and balance in humans after loss. Newly regenerated hair cells receive afferent terminal contacts, yet nothing is known about how reinnervation progresses or whether regenerated afferents finally develop normal termination fields. We hypothesized that neural regeneration in the vestibular otolith system would recapitulate the topographic phenotype of afferent innervation so characteristic of normal development. We used an ototoxic agent to produce complete vestibular receptor cell loss and epithelial denervation, and then quantitatively examined afferent regeneration at discrete periods up to 1 year in otolith maculas. Here, we report that bouton, dimorph, and calyx afferents all regenerate slowly at different time epochs, through a progressive temporal sequence. Furthermore, our data suggest that both the hair cells and their innervating afferents transdifferentiate from an early form into more advanced forms during regeneration. Finally, we show that regeneration remarkably recapitulates the topographic organization of afferent macular innervation, comparable with that developed through normative morphogenesis. However, we also show that regenerated terminal morphologies were significantly less complex than normal fibers. Whether these structural fiber changes lead to alterations in afferent responsiveness is unknown. If true, adaptive plasticity in the central neural processing of motion information would be necessitated, because it is known that many vestibular-related behaviors fully recover during regeneration.

Afferent innervation of the utricular macula in pigeons.

Biotinylated dextran amine (BDA) was used to retrogradely label afferents innervating the utricular macula in adult pigeons. The pigeon utriclar macula consists of a large rectangular-shaped neuroepithelium with a dorsally curved anterior edge and an extended medioposterior tail. The macula could be demarcated into several regions based on cytoarchitectural differences. The striola occupied 30% of the macula and contained a large density of type I hair cells with fewer type II hair cells. Medial and lateral extrastriola zones were located outside the striola and contained only type II hair cells. A six- to eight-cell-wide band of type II hair cells existed near the center of the striola. The reversal line marked by the morphological polarization of hair cells coursed throughout the epithelium, near the peripheral margin, and through the center of the type II band. Calyx afferents innervated type I hair cells with calyceal terminals that contained between 2 and 15 receptor cells. Calyx afferents were located only in the striola region, exclusive of the type II band, had small total fiber innervation areas and low innervation densities. Dimorph afferents innervated both type I and type II hair cells with calyceal and bouton terminals and were primarily located in the striola region. Dimorph afferents had smaller calyceal terminals with few type I hair cells, extended fiber branches with bouton terminals and larger innervation areas. Bouton afferents innervated only type II hair cells in the extrastriola and type II band regions. Bouton afferents innervating the type II band had smaller terminal fields with fewer bouton terminals and smaller innervation areas than fibers located in the extrastriolar zones. Bouton afferents had the most bouton terminals on the longest fibers, the largest innervation areas with the highest innervation densities of all afferents. Among all afferents, smaller terminal innervation fields were observed in the striola and large fields were located in the extrastriola. The cellular organization and innervation patterns of the utricular maculae in birds appear to represent an organ in adaptive evolution, different from that observed for amphibians or mammals.

Morphology of physiologically identified otolith-related vestibular neurons in cats.

The morphology of physiologically identified otolith nerve-activated vestibular neurons was investigated using intracellular injections of horseradish peroxidase (HRP). Eleven utricular, 11 saccular and three utricular/saccular nerve-activated vestibular neurons were labeled with HRP. All of these neurons except one were secondary neurons, the exception being a convergent neuron. The labeled neurons were pyramidal, elongated and ovoidal in shape. Most of the labeled cells were medium to large (mean diameter: > or =30 micro m). There was no apparent correlation between morphology and the different types of otolith nerve-activated vestibular neurons. Thus, it seems likely that the functional type of vestibular neurons cannot be presumed on the basis of their morphology alone.