Efferent control of the electrical and mechanical properties of hair cells in the bullfrog's sacculus.
ABSTRACT: Hair cells in the auditory, vestibular, and lateral-line systems respond to mechanical stimulation and transmit information to afferent nerve fibers. The sensitivity of mechanoelectrical transduction is modulated by the efferent pathway, whose activity usually reduces the responsiveness of hair cells. The basis of this effect remains unknown.We employed immunocytological, electrophysiological, and micromechanical approaches to characterize the anatomy of efferent innervation and the effect of efferent activity on the electrical and mechanical properties of hair cells in the bullfrog's sacculus. We found that efferent fibers form extensive synaptic terminals on all macular and extramacular hair cells. Macular hair cells expressing the Ca(2+)-buffering protein calretinin contain half as many synaptic ribbons and are innervated by twice as many efferent terminals as calretinin-negative hair cells. Efferent activity elicits inhibitory postsynaptic potentials in hair cells and thus inhibits their electrical resonance. In hair cells that exhibit spiking activity, efferent stimulation suppresses the generation of action potentials. Finally, efferent activity triggers a displacement of the hair bundle's resting position.The hair cells of the bullfrog's sacculus receive a rich efferent innervation with the heaviest projection to calretinin-containing cells. Stimulation of efferent axons desensitizes the hair cells and suppresses their spiking activity. Although efferent activation influences mechanoelectrical transduction, the mechanical effects on hair bundles are inconsistent.
Project description:Mechanoelectrical transduction by hair cells commences with hair-bundle deflection, which is postulated to tense filamentous tip links connected to transduction channels. Because direct mechanical stimulation of tip links has not been experimentally possible, this hypothesis has not been tested. We have engineered DNA tethers that link superparamagnetic beads to tip links and exert mechanical forces on the links when exposed to a magnetic-field gradient. By pulling directly on tip links of the bullfrog's sacculus we have evoked transduction currents from hair cells, confirming the hypothesis that tension in the tip links opens transduction channels. This demonstration of direct mechanical access to tip links additionally lays a foundation for experiments probing the mechanics of individual channels.
Project description:The internal ear's sensory receptor, or hair cell, responds when stimuli deflect its mechanoreceptive hair bundle. As a hair cell adapts to sustained stimulation, mechanical adjustments within the bundle reset its position of sensitivity. Because several lines of experimentation suggest that a form of myosin I mediates adaptation, we endeavored to clone cDNAs encoding this motor molecule. By using degenerate oligonucleotide primers based upon the deduced amino acid sequence for mammalian myosin I beta, we performed reverse transcription and polymerase chain reactions (PCRs) to produce a candidate cDNA from polyadenylylated mRNA isolated from the frog's brain. The resultant product was used to probe a cDNA library, from which were isolated clones encoding an approximately 119-kDa isozyme of myosin I beta. PCR amplification disclosed the presence of mRNA encoding the same isozyme in tissue from the bullfrog's sacculus, an organ of the internal ear. When expressed as a bacterial fusion protein, a domain from the tail region of this form of myosin I was recognized by monoclonal antibodies that react with myosin I in hair bundles. This cloned approximately 119-kDa isozyme of myosin I is accordingly a candidate to be the motor molecule responsible for the adaptation of mechanoelectrical transduction by hair cells.
Project description:Ca2+ signaling serves distinct purposes in different parts of a hair cell. The Ca2+ concentration in stereocilia regulates adaptation and, through rapid transduction-channel reclosure, underlies amplification of mechanical signals. In presynaptic active zones, Ca2+ mediates the exocytotic release of afferent neurotransmitter. At efferent synapses, Ca2+ activates the K+ channels that dominate the inhibitory postsynaptic potential. A copious supply of diffusible protein buffer isolates the three signals by restricting the spread of free Ca2+ and limiting the duration of its action. Using cDNA subtraction and a gene expression assay based on in situ hybridization, we detected abundant expression of mRNAs encoding the Ca2+ buffer parvalbumin 3 in bullfrog saccular and chicken cochlear hair cells. We cloned cDNAs encoding this protein from the corresponding inner-ear libraries and raised antisera against recombinant bullfrog parvalbumin 3. Immunohistochemical labeling indicated that parvalbumin 3 is a prominent Ca2+-binding protein in the compact, cylindrical hair cells of the bullfrog's sacculus, and occurs as well in the narrow, peanut-shaped hair cells of that organ. Using quantitative Western blot analysis, we ascertained that the concentration of parvalbumin 3 in saccular hair cells is approximately 3 mM. Parvalbumin 3 is therefore a significant mobile Ca2+ buffer, and perhaps the dominant buffer, in many types of hair cell. Moreover, parvalbumin 3 provides an early marker for developing hair cells in the frog, chicken, and zebrafish.
Project description:In the vestibular periphery of nearly every vertebrate, cholinergic vestibular efferent neurons give rise to numerous presynaptic varicosities that target hair cells and afferent processes in the sensory neuroepithelium. Although pharmacological studies have described the postsynaptic actions of vestibular efferent stimulation in several species, characterization of efferent innervation patterns and the relative distribution of efferent varicosities among hair cells and afferents are also integral to understanding how efferent synapses operate. Vestibular efferent markers, however, have not been well characterized in the turtle, one of the animal models used by our laboratory. Here we sought to identify reliable efferent neuronal markers in the vestibular periphery of turtle, to use these markers to understand how efferent synapses are organized, and to compare efferent neuronal labeling patterns in turtle with two other amniotes using some of the same markers. Efferent fibers and varicosities were visualized in the semicircular canal of red-eared turtles (Trachemys scripta elegans), zebra finches (Taeniopygia guttata), and mice (Mus musculus) utilizing fluorescent immunohistochemistry with antibodies against choline acetyltransferase (ChAT). Vestibular hair cells and afferents were counterstained using antibodies to myosin VIIa and calretinin. In all species, ChAT labeled a population of small diameter fibers giving rise to numerous spherical varicosities abutting type II hair cells and afferent processes. That these ChAT-positive varicosities represent presynaptic release sites were demonstrated by colabeling with antibodies against the synaptic vesicle proteins synapsin I, SV2, or syntaxin and the neuropeptide calcitonin gene-related peptide. Comparisons of efferent innervation patterns among the three species are discussed.
Project description:Although a hair bundle is normally deflected by mechanical stimuli, we found that irradiation of a hair cell from the bullfrog's sacculus with ultraviolet light causes rapid motion of the hair bundle toward its tall edge. This movement is associated with opening of mechanotransduction channels and disappears when tip links are disrupted. We localized the absorptive element responsible for the motion to the region directly below the hair bundle and measured an action spectrum similar to the absorption spectra of mitochondrial constituents. Temperature measurements revealed heating around the site of absorption; direct heating of the hair bundle confirmed that the response to light is mediated through heat. Although mechanical offsets of the hair bundle revealed that heat softens gating springs, it also acts directly to open transduction channels. This study identifies an unconventional method of hair-cell stimulation and clarifies the previously unexplained sensitivity of auditory organs to thermal stimulation.
Project description:The dazzling sensitivity and frequency selectivity of the vertebrate ear rely on mechanical amplification of the hair cells' responsiveness to small stimuli. As revealed by spontaneous oscillations and forms of mechanical excitability in response to force steps, the hair bundle that adorns each hair cell is both a mechanosensory antenna and a force generator that might participate in the amplificatory process. To study the various incarnations of active hair-bundle motility, we combined Ca(2+) iontophoresis with mechanical stimulation of single hair bundles from the bullfrog's sacculus. We identified three classes of active hair-bundle movements: a hair bundle could be quiescent but display nonmonotonic twitches in response to either excitatory or inhibitory force steps, or oscillate spontaneously. Extracellular Ca(2+) changes could affect the kinetics of motion and, when large enough, evoke transitions between the three classes of motility. We found that the Ca(2+)-dependent location of a bundle's operating point within its force-displacement relation controlled the type of movement observed. In response to an iontophoretic pulse of Ca(2+) or of a Ca(2+) chelator, a hair bundle displayed a movement whose polarity could be reversed by applying a static bias to the bundle's position at rest. Moreover, such polarity reversal was accompanied by a 10-fold change in the kinetics of the Ca(2+)-evoked hair-bundle movement. A unified theoretical description, in which mechanical activity stems solely from myosin-based adaptation, could account for the fast and slow manifestations of active hair-bundle motility observed in frog, as well as in auditory organs of the turtle and the rat.
Project description:In the adult auditory organ, mechanoelectrical transducer (MET) channels are essential for transducing acoustic stimuli into electrical signals. In the absence of incoming sound, a fraction of the MET channels on top of the sensory hair cells are open, resulting in a sustained depolarizing current. By genetically manipulating the in vivo expression of molecular components of the MET apparatus, we show that during pre-hearing stages the MET current is essential for establishing the electrophysiological properties of mature inner hair cells (IHCs). If the MET current is abolished in adult IHCs, they revert into cells showing electrical and morphological features characteristic of pre-hearing IHCs, including the re-establishment of cholinergic efferent innervation. The MET current is thus critical for the maintenance of the functional properties of adult IHCs, implying a degree of plasticity in the mature auditory system in response to the absence of normal transduction of acoustic signals.
Project description:The ear relies on nonlinear amplification to enhance its sensitivity and frequency selectivity to oscillatory mechanical stimuli. It has been suggested that this active process results from the operation of dynamical systems that operate in the vicinity of an oscillatory instability, a Hopf bifurcation. In the bullfrog's sacculus, a hair cell can display spontaneous oscillations of its mechanosensory hair bundle. The behavior of an oscillatory hair bundle resembles that of a critical oscillator. We present here a theoretical description of the effects of intrinsic noise on active hair-bundle motility. An oscillatory instability can result from the interplay between a region of negative stiffness in the bundle's force-displacement relation and the Ca(2+)-regulated activity of molecular motors. We calculate a state diagram that describes the possible dynamical states of the hair bundle in the absence of fluctuations. Taking into account thermal fluctuations, the stochastic nature of transduction channels' gating, and of the forces generated by molecular motors, we discuss conditions that yield a response function and spontaneous noisy movements of the hair bundle in quantitative agreement with previously published experiments. We find that the magnitude of the fluctuations resulting from the active processes that mediate mechanical amplification remains just below that of thermal fluctuations. Fluctuations destroy the phase coherence of spontaneous oscillations and restrict the bundle's sensitivity as well as frequency selectivity to small oscillatory stimuli. We show, however, that a hair bundle studied experimentally operates near an optimum of mechanosensitivity in our state diagram.
Project description:Although homomeric channels assembled from the alpha9 nicotinic acetylcholine receptor (nAChR) subunit are functional in vitro, electrophysiological, anatomical, and molecular data suggest that native cholinergic olivocochlear function is mediated via heteromeric nAChRs composed of both alpha9 and alpha10 subunits. To gain insight into alpha10 subunit function in vivo, we examined olivo cochlear innervation and function in alpha10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8-9 inner hair cells revealed ACh-gated currents in alpha10(+/+) and alpha10(+/-) mice, with no detectable responses to ACh in alpha10(-/-) mice. In contrast, a proportion of alpha10(-/-) outer hair cells showed small ACh-evoked currents. In alpha10(-/-) mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual alpha9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, alpha10(-/-) mice exhibit both an abnormal olivocochlear morphology and innervation to outer hair cells and a highly disorganized efferent innervation to the inner hair cell region. Our results demonstrate that alpha9(-/-) and alpha10(-/-) mice have overlapping but nonidentical phenotypes. Moreover, alpha10 nAChR subunits are required for normal olivocochlear activity because alpha9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in alpha10(-/-) mutant mice.
Project description:The dynamic adjustment of hearing sensitivity and frequency selectivity is mediated by the medial olivocochlear efferent reflex, which suppresses the gain of the 'cochlear amplifier' in each ear. Such efferent feedback is important for promoting discrimination of sounds in background noise, sound localization and protecting the cochleae from acoustic overstimulation. However, the sensory driver for the olivocochlear reflex is unknown. Here, we resolve this longstanding question using a mouse model null for the gene encoding the type III intermediate filament peripherin (Prph). Prph((-/-)) mice lacked type II spiral ganglion neuron innervation of the outer hair cells, whereas innervation of the inner hair cells by type I spiral ganglion neurons was normal. Compared with Prph((+/+)) controls, both contralateral and ipsilateral olivocochlear efferent-mediated suppression of the cochlear amplifier were absent in Prph((-/-)) mice, demonstrating that outer hair cells and their type II afferents constitute the sensory drive for the olivocochlear efferent reflex.