Lukas Rüttiger

Nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3 : In Vivo evidence

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat in the MJD1 gene resulting in an expanded polyglutamine repeat in the ataxin-3 protein. To study the course of the disease, we generated transgenic mice for SCA3 using full-length ataxin-3 constructs containing 15, 70, or 148 CAG repeats, respectively. Control mice (15 CAGs) were phenotypically normal and had no neuropathological findings. However, mice transgenic for ataxin-3 with expanded polyglutamine repeats were severely affected by a strong neurological phenotype with tremor, behavioral deficits, strongly reduced motor and exploratory activity, a hunchback, and premature death at 3 to 6 months of age. Neuropathological examination by immunohistochemical staining revealed ubiquitin- and ataxin-3-positive intranuclear inclusion bodies in a multitude of neurons. Directing ataxin-3 with 148 CAGs to the nucleus revealed an even more pronounced phenotype with more inclusions and earlier death, whereas mice transgenic with the same construct but attached to a nuclear export signal developed a milder phenotype with less inclusions. These studies indicate that nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3 in vivo.

Synaptotagmin IV determines the linear Ca2+ dependence of vesicle fusion at auditory ribbon synapses

Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca2+ dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca2+ sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca2+ dependence of immature spiking cells. However, the nature of the Ca2+ sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca2+ dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca2+ dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca2+ sensitivity of vesicle fusion at hair cell synapses.

A behavioral paradigm to judge acute sodium salicylate-induced sound experience in rats: a new approach for an animal model on tinnitus

Behavioral conditioning studies on rats have been proven to be a valid animal model for the evaluation of acute and chronic phantom auditory experience (tinnitus). We developed an animal model for short-term, acute induced phantom auditory sensations in rats. Rats were trained in a conditioning chamber to actively access a liquid feeder whenever a constant white noise sound was present. During silence, no reward was given. Fulfilling the demands of animal protection laws for maximal avoidance of pain and fear, punitive paradigms were maximally reduced. After 15-17 learning sessions, all animals performed more accesses to the reward feeder during periods of sound than during periods of silence. Tinnitus was induced by the administration of sodium salicylate (350 mg/kg body weight) given 3 h before testing. The feeder access activity of a rat treated with salicylate was significantly increased during periods of silence, indicating a phantom auditory experience. The presumptive auditory experience was estimated to be comparable to a white noise sound of about 30 dB SPL rms. The activity increase was less pronounced for lower doses of sodium salicylate (150 mg/kg body weight) and was not found in animals trained on a dark-light paradigm, as expected. As the learning sessions of the operant conditioning were performed without pharmacological treatment, unintentional drug effects, for example, on learning and motivation of a rat were minimized in this behavioral paradigm. Furthermore, the behavioral changes reported here were shown to be a specific drug effect evoking a phantom auditory experience of a rat and cannot be explained by unspecific drug effects on motor activity, motivation, learning or hearing loss. The conditional paradigm is discussed in the context of its potential as a model for testing drugs that may have a therapeutic value in tinnitus research.

Tinnitus behavior and hearing function correlate with the reciprocal expression patterns of BDNF and Arg3.1/arc in auditory neurons following acoustic trauma

The molecular changes following sensory trauma and the subsequent response of the CNS are poorly understood. We focused on finding a molecular tool for monitoring the features of excitability which occur following acoustic trauma to the auditory system. Of particular interest are genes that alter their expression pattern during activity-induced changes in synaptic efficacy and plasticity. The expression of brain-derived neurotrophic factor (BDNF), the activity-dependent cytoskeletal protein (Arg3.1/arc), and the immediate early gene c-Fos were monitored in the peripheral and central auditory system hours and days following a traumatic acoustic stimulus that induced not only hearing loss but also phantom auditory perception (tinnitus), as shown in rodent animal behavior models. A reciprocal responsiveness of activity-dependent genes became evident between the periphery and the primary auditory cortex (AI): as c-Fos and BDNF exon IV expression was increased in spiral ganglion neurons, Arg3.1/arc and (later on) BDNF exon IV expression was reduced in AI. In line with studies indicating increased spontaneous spike activity at the level of the inferior colliculus (IC), an increase in BDNF and GABA-positive neurons was seen in the IC. The data clearly indicate the usefulness of Arg3.1/arc and BDNF for monitoring trauma-induced activity changes and the associated putative plasticity responses in the auditory system.

Two classes of outer hair cells along the tonotopic axis of the cochlea

The molecular basis of high versus low frequency hearing loss and the differences in the sensitivity of outer hair cells depending on their cochlear localization are currently not understood. Here we demonstrate the existence of two different outer hair cell phenotypes along the cochlear axis. Outer hair cells in low frequency regions exhibit early sensitivity for loss of Ca(v)1.3 (alpha1 subunit 1.3 forming the class D L-type voltage-gated Ca(2+) channel), while high frequency regions display a progressive susceptibility for loss of the Ca(2+)-activated large conductance K(+) (BK) channel. Despite deafness, young Ca(v)1.3-deficient mice displayed distortion-product otoacoustic emissions (DPOAEs), indicating functional outer hair cells in the higher frequency range of the cochlea. Considering that DPOAEs are also found in the human deafness syndrome DFNB9 caused by mutations in the synaptic vesicle protein otoferlin, we tested the expression of otoferlin in outer hair cells. Surprisingly, otoferlin showed a distinct tonotopic expression pattern at both the mRNA and protein level. Otoferlin-expressing, Ca(v)1.3 deletion-sensitive outer hair cells in the low frequency range could be clearly separated from otoferlin-negative, BK deletion-sensitive outer hair cells in the high frequency range. In addition, BK deletion led to a higher noise vulnerability in low frequency regions, which are normally unaffected by the BK deletion alone, suggesting that BK currents are involved in survival mechanisms of outer hair cells under noise conditions. Our findings propose new mechanisms and candidate genes for explaining high and low frequency hearing loss.

The reduced cochlear output and the failure to adapt the central auditory response causes tinnitus in noise exposed rats.

Tinnitus is proposed to be caused by decreased central input from the cochlea, followed by increased spontaneous and evoked subcortical activity that is interpreted as compensation for increased responsiveness of central auditory circuits. We compared equally noise exposed rats separated into groups with and without tinnitus for differences in brain responsiveness relative to the degree of deafferentation in the periphery. We analyzed (1) the number of CtBP2/RIBEYE-positive particles in ribbon synapses of the inner hair cell (IHC) as a measure for deafferentation; (2) the fine structure of the amplitudes of auditory brainstem responses (ABR) reflecting differences in sound responses following decreased auditory nerve activity and (3) the expression of the activity-regulated gene Arc in the auditory cortex (AC) to identify long-lasting central activity following sensory deprivation. Following moderate trauma, 30% of animals exhibited tinnitus, similar to the tinnitus prevalence among hearing impaired humans. Although both tinnitus and no-tinnitus animals exhibited a reduced ABR wave I amplitude (generated by primary auditory nerve fibers), IHCs ribbon loss and high-frequency hearing impairment was more severe in tinnitus animals, associated with significantly reduced amplitudes of the more centrally generated wave IV and V and less intense staining of Arc mRNA and protein in the AC. The observed severe IHCs ribbon loss, the minimal restoration of ABR wave size, and reduced cortical Arc expression suggest that tinnitus is linked to a failure to adapt central circuits to reduced cochlear input.

Noise-Induced Inner Hair Cell Ribbon Loss Disturbs Central Arc Mobilization: A Novel Molecular Paradigm for Understanding Tinnitus

Increasing evidence shows that hearing loss is a risk factor for tinnitus and hyperacusis. Although both often coincide, a causal relationship between tinnitus and hyperacusis has not been shown. Currently, tinnitus and hyperacusis are assumed to be caused by elevated responsiveness in subcortical circuits. We examined both the impact of different degrees of cochlear damage and the influence of stress priming on tinnitus induction. We used (1) a behavioral animal model for tinnitus designed to minimize stress, (2) ribbon synapses in inner hair cells (IHCs) as a measure for deafferentation, (3) the integrity of auditory brainstem responses (ABR) to detect differences in stimulus-evoked neuronal activity, (4) the expression of the activity-regulated cytoskeletal protein, Arc, to identify long-lasting changes in network activity within the basolateral amygdala (BLA), hippocampal CA1, and auditory cortex (AC), and (5) stress priming to investigate the influence of corticosteroid on trauma-induced brain responses. We observed that IHC ribbon loss (deafferentation) leads to tinnitus when ABR functions remain reduced and Arc is not mobilized in the hippocampal CA1 and AC. If, however, ABR waves are functionally restored and Arc is mobilized, tinnitus does not occur. Both central response patterns were found to be independent of a profound threshold loss and could be shifted by the corticosterone level at the time of trauma. We, therefore, discuss the findings in the context of a history of stress that can trigger either an adaptive or nonadaptive brain response following injury.

Orientation sensitivity of ganglion cells in primate retina

The two-dimensional shape of the receptive field center of macaque retinal ganglion cells was determined by measuring responses to drifting sinusoidal gratings of different spatial frequency and orientation. The responses of most cells to high spatial frequencies depended on grating orientation, indicating that their centers were not circularly symmetric. In general, center shape was well described by an ellipse. The major axis of the ellipse tended to point towards the fovea or perpendicular to this. Parvocellular pathway cells had greater center ellipticity than magnocellular pathway cells; the median ratio of the major-to-minor axis was 1.72 and 1.38, respectively. Parvocellular pathway cells also had centers that were often bimodal in shape, suggesting that they received patchy cone/bipolar cell input. We conclude that most ganglion cells in primate retina have elongated receptive field centers and thus show orientation sensitivity.

cGMP-Prkg1 signaling and Pde5 inhibition shelter cochlear hair cells and hearing function

Noise-induced hearing loss (NIHL) is a global health hazard with considerable pathophysiological and social consequences that has no effective treatment. In the heart, lung and other organs, cyclic guanosine monophosphate (cGMP) facilitates protective processes in response to traumatic events. We therefore analyzed NIHL in mice with a genetic deletion of the gene encoding cGMP-dependent protein kinase type I (Prkg1) and found a greater vulnerability to and markedly less recovery from NIHL in these mice as compared to mice without the deletion. Prkg1 was expressed in the sensory cells and neurons of the inner ear of wild-type mice, and its expression partly overlapped with the expression profile of cGMP-hydrolyzing phosphodiesterase 5 (Pde5). Treatment of rats and wild-type mice with the Pde5 inhibitor vardenafil almost completely prevented NIHL and caused a Prkg1-dependent upregulation of poly (ADP-ribose) in hair cells and the spiral ganglion, suggesting an endogenous protective cGMP-Prkg1 signaling pathway that culminates in the activation of poly (ADP-ribose) polymerase. These data suggest vardenafil or related drugs as possible candidates for the treatment of NIHL.

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells.

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cells apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.