Marlies Knipper

Protein Kinase CK2 Is Coassembled with Small Conductance Ca2+-Activated K+ Channels and Regulates Channel Gating

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.

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.

Developmental Regulation of Nicotinic Synapses on Cochlear Inner Hair Cells

In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both α9 and α10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88-100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16-P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10-P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that α9 mRNA is expressed in adult IHCs but thatα10 mRNA disappears after the onset of hearing. In the present study, antibodies toα10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of α10, but not α9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.

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.

Thyroid hormone is a critical determinant for the regulation of the cochlear motor protein prestin

The most impressive property of outer hair cells (OHCs) is their ability to change their length at high acoustic frequencies, thus providing the exquisite sensitivity and frequency-resolving capacity of the mammalian hearing organ. Prestin, a protein related to a sulfate/anion transport protein, recently has been identified and proposed as the OHC motor molecule. Homology searches of 1.5 kb of genomic DNA 5′ of the coding region of the prestin gene allowed the identification of a thyroid hormone (TH) response element (TRE) in the first intron upstream of the prestin ATG codon. PrestinTRE bound TH receptors as a monomer or presumptive heterodimer and mediated a triiodothyronine-dependent transactivation of a heterologous promotor in response to triiodothyronine receptors α and β. Retinoid X receptor-α had an additive effect. Expression of prestin mRNA and prestin protein was reduced strongly in the absence of TH. Although prestin protein typically was redistributed to the lateral membrane before the onset of hearing, an immature pattern of prestin protein distribution across the entire OHC membrane was noted in hypothyroid rats. The data suggest TH as a first transcriptional regulator of the motor protein prestin and as a direct or indirect modulator of subcellular prestin distribution.

Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea, whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea, which may also be of relevance for treatment of hearing loss in humans.

Tonotopic Variation in the Calcium Dependence of Neurotransmitter Release and Vesicle Pool Replenishment at Mammalian Auditory Ribbon Synapses

The mammalian cochlea is specialized to recognize and process complex auditory signals with remarkable acuity and temporal precision over a wide frequency range. The quality of the information relayed to the auditory afferent fibers mainly depends on the transfer characteristics of inner hair cell (IHC) ribbon synapses. To investigate the biophysical properties of the synaptic machinery, we measured changes in membrane capacitance (ΔCm) in low-frequency (apical region, ∼300 Hz) and high-frequency (basal, ∼30 kHz) gerbil IHCs maintained in near physiological conditions (1.3 mm extracellular Ca2+ and body temperature). With maturation, the Ca2+ efficiency of exocytosis improved in both apical and basal IHCs and was more pronounced in the latter. Prehearing IHCs showed a similar Ca2+ cooperativity of exocytosis despite the smaller ΔCm in apical cells. After maturation, ΔCm in high-frequency IHCs increased linearly with the Ca2+ current, whereas, somewhat surprisingly, the relationship was significantly more nonlinear in low-frequency cells. This tonotopic difference seemed to be correlated with ribbon synapse morphology (spherical in apical and ellipsoid in basal IHCs) but not with the expression level of the proposed Ca2+ sensor otoferlin or the spatial coupling between Ca2+ channels and active zones. Repetitive stimulation of adult IHCs showed that vesicle pool refilling could become rate limiting for vesicle release, with high-frequency IHCs able to sustain greater release rates. Together, our findings provide the first evidence for a tonotopic difference in the properties of the synaptic machinery in mammalian IHCs, which could be essential for fine-tuning their receptor characteristics during sound stimulation.

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.

Position-dependent patterning of spontaneous action potentials in immature cochlear inner hair cells

Spontaneous action potential activity is crucial for mammalian sensory system development. In the auditory system, patterned firing activity has been observed in immature spiral ganglion and brain-stem neurons and is likely to depend on cochlear inner hair cell (IHC) action potentials. It remains uncertain whether spiking activity is intrinsic to developing IHCs and whether it shows patterning. We found that action potentials were intrinsically generated by immature IHCs of altricial rodents and that apical IHCs showed bursting activity as opposed to more sustained firing in basal cells. We show that the efferent neurotransmitter acetylcholine fine-tunes the IHCs resting membrane potential (Vm), and as such is crucial for the bursting pattern in apical cells. Endogenous extracellular ATP also contributes to the Vm of apical and basal IHCs by triggering small-conductance Ca2+-activated K+ (SK2) channels. We propose that the difference in firing pattern along the cochlea instructs the tonotopic differentiation of IHCs and auditory pathway.