BDNF-Live-Exon-Visualization (BLEV) Allows Differential Detection of BDNF Transcripts in vitro and in vivo.

Bdnf exon-IV and exon-VI transcripts are driven by neuronal activity and are involved in pathologies related to sleep, fear or memory disorders. However, how their differential transcription translates activity changes into long-lasting network changes is elusive. Aiming to trace specifically the network controlled by exon-IV and -VI derived BDNF during activity-dependent plasticity changes, we generated a transgenic reporter mouse for B DNF- l ive- e xon- v isualization (BLEV), in which expression of Bdnf exon-IV and -VI can be visualized by co-expression of CFP and YFP. CFP and YFP expression was differentially activated and targeted in cell lines, primary cultures and BLEV reporter mice without interfering with BDNF protein synthesis. CFP and YFP expression, moreover, overlapped with BDNF protein expression in defined hippocampal neuronal, glial and vascular locations in vivo. So far, activity-dependent BDNF cannot be explicitly monitored independent of basal BDNF levels. The BLEV reporter mouse therefore provides a new model, which can be used to test whether stimulus-induced activity-dependent changes in BDNF expression are instrumental for long-lasting plasticity modifications.

Visualizing BDNF Transcript Usage During Sound-Induced Memory Linked Plasticity.

Activity-dependent BDNF (brain-derived neurotrophic factor) expression is hypothesized to be a cue for the context-specificity of memory formation. So far, activity-dependent BDNF cannot be explicitly monitored independently of basal BDNF levels. We used the BLEV ( B DNF- live-exon- visualization) reporter mouse to specifically detect activity-dependent usage of Bdnf exon-IV and -VI promoters through bi-cistronic co-expression of CFP and YFP, respectively. Enriching acoustic stimuli led to improved peripheral and central auditory brainstem responses, increased Schaffer collateral LTP, and enhanced performance in the Morris water maze. Within the brainstem, neuronal activity was increased and accompanied by a trend for higher expression levels of Bdnf exon-IV-CFP and exon-VI-YFP transcripts. In the hippocampus BDNF transcripts were clearly increased parallel to changes in parvalbumin expression and were localized to specific neurons and capillaries. Severe acoustic trauma, in contrast, elevated neither Bdnf transcript levels, nor auditory responses, parvalbumin or LTP. Together, this suggests that critical sensory input is essential for recruitment of activity-dependent auditory-specific BDNF expression that may shape network adaptation.

Deletion of myosin VI causes slow retinal optic neuropathy and age-related macular degeneration (AMD)-relevant retinal phenotype.

The unconventional myosin VI, a member of the actin-based motor protein family of myosins, is expressed in the retina. Its deletion was previously shown to reduce amplitudes of the a- and b-waves of the electroretinogram. Analyzing wild-type and myosin VI-deficient Snell's Waltzer mice in more detail, the expression pattern of myosin VI in retinal pigment epithelium, outer limiting membrane, and outer plexiform layer could be linked with differential progressing ocular deficits. These encompassed reduced a-waves and b-waves and disturbed oscillatory potentials in the electroretinogram, photoreceptor cell death, retinal microglia infiltration, and formation of basal laminar deposits. A phenotype comprising features of glaucoma (neurodegeneration) and age-related macular degeneration could thus be uncovered that suggests dysfunction of myosin VI and its variable cargo adaptor proteins for membrane sorting and autophagy, as possible candidate mediators for both disease forms.

L-type CaV1.2 deletion in the cochlea but not in the brainstem reduces noise vulnerability: implication for CaV1.2-mediated control of cochlear BDNF expression.

Voltage-gated L-type Ca(2+) channels (L-VGCCs) like CaV1.2 are assumed to play a crucial role for controlling release of trophic peptides including brain-derived neurotrophic factor (BDNF). In the inner ear of the adult mouse, besides the well-described L-VGCC CaV1.3, CaV1.2 is also expressed. Due to lethality of constitutive CaV1.2 knock-out mice, the function of this ion channel as well as its putative relationship to BDNF in the auditory system is entirely elusive. We recently described that BDNF plays a differential role for inner hair cell (IHC) vesicles release in normal and traumatized condition. To elucidate a presumptive role of CaV1.2 during this process, two tissue-specific conditional mouse lines were generated. To distinguish the impact of CaV1.2 on the cochlea from that on feedback loops from higher auditory centers CaV1.2 was deleted, in one mouse line, under the Pax2 promoter (CaV1.2(Pax2)) leading to a deletion in the spiral ganglion neurons, dorsal cochlear nucleus, and inferior colliculus. In the second mouse line, the Egr2 promoter was used for deleting CaV1.2 (CaV1.2(Egr2)) in auditory brainstem nuclei. In both mouse lines, normal hearing threshold and equal number of IHC release sites were observed. We found a slight reduction of auditory brainstem response wave I amplitudes in the CaV1.2(Pax2) mice, but not in the CaV1.2(Egr2) mice. After noise exposure, CaV1.2(Pax2) mice had less-pronounced hearing loss that correlated with maintenance of ribbons in IHCs and less reduced activity in auditory nerve fibers, as well as in higher brain centers at supra-threshold sound stimulation. As reduced cochlear BDNF mRNA levels were found in CaV1.2(Pax2) mice, we suggest that a CaV1.2-dependent step may participate in triggering part of the beneficial and deteriorating effects of cochlear BDNF in intact systems and during noise exposure through a pathway that is independent of CaV1.2 function in efferent circuits.

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.

Lack of brain-derived neurotrophic factor hampers inner hair cell synapse physiology, but protects against noise-induced hearing loss.

The precision of sound information transmitted to the brain depends on the transfer characteristics of the inner hair cell (IHC) ribbon synapse and its multiple contacting auditory fibers. We found that brain derived neurotrophic factor (BDNF) differentially influences IHC characteristics in the intact and injured cochlea. Using conditional knock-out mice (BDNF(Pax2) KO) we found that resting membrane potentials, membrane capacitance and resting linear leak conductance of adult BDNF(Pax2) KO IHCs showed a normal maturation. Likewise, in BDNF(Pax2) KO membrane capacitance (ΔC(m)) as a function of inward calcium current (I(Ca)) follows the linear relationship typical for normal adult IHCs. In contrast the maximal ΔC(m), but not the maximal size of the calcium current, was significantly reduced by 45% in basal but not in apical cochlear turns in BDNF(Pax2) KO IHCs. Maximal ΔC(m) correlated with a loss of IHC ribbons in these cochlear turns and a reduced activity of the auditory nerve (auditory brainstem response wave I). Remarkably, a noise-induced loss of IHC ribbons, followed by reduced activity of the auditory nerve and reduced centrally generated wave II and III observed in control mice, was prevented in equally noise-exposed BDNF(Pax2) KO mice. Data suggest that BDNF expressed in the cochlea is essential for maintenance of adult IHC transmitter release sites and that BDNF upholds opposing afferents in high-frequency turns and scales them down following noise exposure.

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.

Midazolam reverses salicylate-induced changes in brain-derived neurotrophic factor and arg3.1 expression: implications for tinnitus perception and auditory plasticity.

Tinnitus is a phantom auditory perception, which can be induced via application of concentrated sodium salicylate, and is known to be associated with hearing loss and altered neuronal excitability in peripheral and central auditory neurons. The molecular features of this excitability, however, has been poorly characterized to date. Brain-derived neurotrophic factor (BDNF), the activity-dependent cytoskeletal protein (Arg3.1, also known as Arc), and c-Fos are known to be affected by changes in excitability and plasticity. Using reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry, the expression of these genes was monitored in the rat auditory system after local (cochlear) and systemic application of salicylate. Induction of tinnitus and hearing loss was verified in a behavioral model. Regardless of the mode of salicylate application, a common pattern became evident: 1) BDNF mRNA expression was increased in the spiral ganglion neurons of the cochlea; and 2) Arg3.1 expression was significantly reduced in the auditory cortex. Local application of the GABA(A) receptor modulator midazolam resulted in the reversal not only of salicylate-induced changes in cochlear BDNF expression, but also in cortical Arg3.1 expression, indicating that the tinnitus-associated changes in cochlear BDNF expression trigger the decline of cortical Arg3.1 expression. Furthermore, local midazolam application reduced tinnitus perception in the animal model. These findings support Arg3.1 and BDNF as markers for activity changes in the auditory system and suggest a role of GABAergic inhibition of cochlear neurons in the modulation of Arg3.1 plasticity changes in the auditory cortex and tinnitus perception.

Expression of glycine receptors and gephyrin in the rat cochlea.

The cochlear efferent feedback system exerts direct impact on cochlear nerve activity and balances interaural sensitivity. So far, acetylcholine, GABA and dopamine are known to be transmitters of the inhibitory efferent system. Despite the wealth of information about glycinergic neurotransmission in the central auditory system, the inhibitory glycine receptor (GlyR) has not yet been regarded as a target molecule of efferent transmission in the cochlea. Using RT-PCR, in situ hybridization and immunohistochemistry, we show that GlyRalpha3, GlyRbeta and gephyrin are expressed in the organ of Corti and spiral ganglion neurons. Furthermore, two alternative splice variants of GlyRalpha3, corresponding to the long (alpha3_L) and short (alpha3_K) human isoforms, could be distinguished. The localization of glycine receptors below inner hair cells and in outer hair cells of the adult cochlea suggests that these inhibitory receptors may serve as target molecules of the efferent olivocochlear bundle.

Salicylate alters the expression of calcium response transcription factor 1 in the cochlea: implications for brain-derived neurotrophic factor transcriptional regulation.

Brain-derived neurotrophic factor (BDNF) is a key neurotrophin whose expression is altered in response to neurological activity, influencing both short- and long-term synaptic changes. The BDNF gene consists of eight upstream exons (I-VII), each of which has a distinct promoter and can be independently spliced to the ninth coding exon (IX). We showed recently that the expression of BDNF exon IV in the cochlea is altered after exposure to salicylate, an ototoxic drug that in high doses is able to induce hearing loss and tinnitus. These changes were a crucial trigger for plasticity changes in the central auditory system. BDNF exon IV expression is regulated via interaction between calcium-response elements CaRE1, CaRE2, and CaRE3/Cre (CaREs) that are bound by the transcription factors CaRF1, upstream stimulatory factors 1 and 2 (USF1/2), and cAMP/Ca(2+) response element-binding protein (CREB), respectively. To determine whether the salicylate-induced changes in cochlear BDNF exon IV expression include a differential use of the CaRE binding proteins, we studied the level of the corresponding binding proteins in the spiral ganglion neurons before and after systemic application of concentrated salicylate using in situ hybridization and RT-PCR. BDNF exon IV and CaRF1 expression were up-regulated after application of salicylate, whereas USF1/2 and CREB mRNA expression remained unaffected. The changes in BDNF exon IV and CaRF1 expression were also dose-dependent. The data show Ca(2+) and CaRF1 as messengers of trauma (salicylate)-induced altered BDNF levels in the cochlea. Furthermore, they also provide the first evidence that a differential regulation of BDNF transcription factors might participate in BDNF-mediated plasticity changes.

Thyroid hormone receptor alpha1 is a critical regulator for the expression of ion channels during final differentiation of outer hair cells.

Cochlear outer hair cells (OHCs) terminally differentiate prior to the onset of hearing. During this time period, thyroid hormone (TH) dramatically influences inner ear development. It has been shown recently that TH enhances the expression of the motor protein prestin via liganded TH receptor beta (TRbeta) while in contrast the expression of the potassium channel KCNQ4 is repressed by unliganded TRalpha1. These different mechanisms of TH regulation by TRalpha1 or TRbeta prompted us to analyse other ion channels that are required for the final differentiation of OHCs. We analysed the onset of expression of the Ca(2+) channel Ca(V)1.3, and the K(+) channels SK2 and BK and correlated the results with the regulation via TRalpha1 or TRbeta. The data support the hypothesis that proteins expressed in rodents prior to or briefly after birth like Ca(V)1.3 and prestin are either independent of TH (e.g. Ca(V)1.3) or enhanced through TRbeta (e.g. prestin). In contrast, proteins expressed in rodents later than P6 like KCNQ4 ( approximately P6), SK2 ( approximately P9) and BK ( approximately P11) are repressed through TRalpha1. We hypothesise that the precise regulation of expression of the latter genes requires a critical local TH level to overcome the TRalpha1 repression.

BDNF mRNA expression and protein localization are changed in age-related hearing loss.

A decline in neuronal plasticity during the adult life span has been proposed to be associated with a reduced level of the effectors of plasticity responses (e.g., BDNF). Alteration of plasticity is also correlated with age-related hearing loss (presbycusis), but to date no detailed studies of BDNF expression have been performed in the young or aging mature cochlea. We have used rat and gerbil animal models for presbycusis, which displayed hearing loss in the final third of the animals' natural life span. We demonstrate for the first time a co-localization of BDNF protein, transcripts III and IV in cochlear neurons with a declining distribution towards low-frequency processing cochlear turns. BDNF protein was also found within the neuronal projections of the cochlea. A significant reduction of BDNF transcripts in high-frequency processing cochlear neurons was observed during aging, though this did not coincide with a major reduction of BDNF protein. In contrast, BDNF protein in peripheral and central projections was drastically reduced. Our results suggest that reduced BDNF protein levels in auditory nerves over age may be a crucial factor in the altered brainstem plasticity observed during presbycusis.

Thyroid hormone receptors TRalpha1 and TRbeta differentially regulate gene expression of Kcnq4 and prestin during final differentiation of outer hair cells.

Thyroid hormone (TH or T3) and TH-receptor beta (TRbeta) have been reported to be relevant for cochlear development and hearing function. Mutations in the TRbeta gene result in deafness associated with resistance to TH syndrome. The effect of TRalpha1 on neither hearing function nor cochlear T3 target genes has been described to date. It is also uncertain whether TRalpha1 and TRbeta can act simultaneously on different target genes within a single cell. We focused on two concomitantly expressed outer hair cell genes, the potassium channel Kcnq4 and the motor protein prestin Slc26a5. In outer hair cells, TH enhanced the expression of the prestin gene through TRbeta. Simultaneously Kcnq4 expression was activated in the same cells by derepression of TRalpha1 aporeceptors mediated by an identified THresponse element, which modulates KCNQ4 promoter activity. We show that T3 target genes can differ in their sensitivity to TH receptors having the ligand either bound (holoreceptors) or not bound (aporeceptors) within single cells, and suggest a role for TRalpha1 in final cell differentiation.

Deletion of the Ca2+-activated potassium (BK) alpha-subunit but not the BKbeta1-subunit leads to progressive hearing loss.

The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming alpha-subunit coexpressed with the auxiliary beta1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel alpha-(BKalpha-/-) and beta1-subunit (BKbeta1-/-) knockout mice, we demonstrate normal hearing function and cochlear structure of BKbeta1-/- mice. During the first 4 postnatal weeks also, BKalpha-/- mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKalpha-/- mice only from approximately 8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.

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.

Expression of prestin-homologous solute carrier (SLC26) in auditory organs of nonmammalian vertebrates and insects.

Prestin, the fifth member of the anion transporter family SLC26, is the outer hair cell molecular motor thought to be responsible for active mechanical amplification in the mammalian cochlea. Active amplification is present in a variety of other auditory systems, yet the prevailing view is that prestin is a motor molecule unique to mammalian ears. Here we identify prestin-related SLC26 proteins that are expressed in the auditory organs of nonmammalian vertebrates and insects. Sequence comparisons revealed the presence of SLC26 proteins in fish (Danio, GenBank accession no. AY278118, and Anguilla, GenBank accession no. BAC16761), mosquitoes (Anopheles, GenBank accession nos. EAA07232 and EAA07052), and flies (Drosophila, GenBank accession no. AAF49285). The fly and zebrafish homologues were cloned and, by using in situ hybridization, shown to be expressed in the auditory organs. In mosquitoes, in turn, the expression of prestin homologues was demonstrated for the auditory organ by using highly specific riboprobes against rat prestin. We conclude that prestin-related SLC26 proteins are widespread, possibly ancestral, constituents of auditory organs and are likely to serve salient roles in mammals and across taxa.

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. Prestin(TRE) bound TH receptors as a monomer or presumptive heterodimer and mediated a triiodothyronine-dependent transactivation of a heterologous promotor in response to triiodothyronine receptors alpha and beta. Retinoid X receptor-alpha 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.