Hyun-Soon Geisler

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.

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.

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 (BDNFPax2 KO) we found that resting membrane potentials, membrane capacitance and resting linear leak conductance of adult BDNFPax2 KO IHCs showed a normal maturation. Likewise, in BDNFPax2 KO membrane capacitance (ΔCm) as a function of inward calcium current (ICa) follows the linear relationship typical for normal adult IHCs. In contrast the maximal ΔCm, but not the maximal size of the calcium current, was significantly reduced by 45% in basal but not in apical cochlear turns in BDNFPax2 KO IHCs. Maximal ΔCm 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 BDNFPax2 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.

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 GABAA 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.

BDNF in Lower Brain Parts Modifies Auditory Fiber Activity to Gain Fidelity but Increases the Risk for Generation of Central Noise After Injury

For all sensory organs, the establishment of spatial and temporal cortical resolution is assumed to be initiated by the first sensory experience and a BDNF-dependent increase in intracortical inhibition. To address the potential of cortical BDNF for sound processing, we used mice with a conditional deletion of BDNF in which Cre expression was under the control of the Pax2 or TrkC promoter. BDNF deletion profiles between these mice differ in the organ of Corti (BDNFPax2-KO) versus the auditory cortex and hippocampus (BDNFTrkC-KO). We demonstrate that BDNFPax2-KO but not BDNFTrkC-KO mice exhibit reduced sound-evoked suprathreshold ABR waves at the level of the auditory nerve (wave I) and inferior colliculus (IC) (wave IV), indicating that BDNF in lower brain regions but not in the auditory cortex improves sound sensitivity during hearing onset. Extracellular recording of IC neurons of BDNFPax2 mutant mice revealed that the reduced sensitivity of auditory fibers in these mice went hand in hand with elevated thresholds, reduced dynamic range, prolonged latency, and increased inhibitory strength in IC neurons. Reduced parvalbumin-positive contacts were found in the ascending auditory circuit, including the auditory cortex and hippocampus of BDNFPax2-KO, but not of BDNFTrkC-KO mice. Also, BDNFPax2-WT but not BDNFPax2-KO mice did lose basal inhibitory strength in IC neurons after acoustic trauma. These findings suggest that BDNF in the lower parts of the auditory system drives auditory fidelity along the entire ascending pathway up to the cortex by increasing inhibitory strength in behaviorally relevant frequency regions. Fidelity and inhibitory strength can be lost following auditory nerve injury leading to diminished sensory outcome and increased central noise.

NO-Sensitive Guanylate Cyclase Isoforms NO-GC1 and NO-GC2 Contribute to Noise-Induced Inner Hair Cell Synaptopathy

Nitric oxide (NO) activates the NO-sensitive soluble guanylate cyclase (NO-GC, sGC) and triggers intracellular signaling pathways involving cGMP. For survival of cochlear hair cells and preservation of hearing, NO-mediated cascades have both protective and detrimental potential. Here we examine the cochlear function of mice lacking one of the two NO-sensitive guanylate cyclase isoforms [NO-GC1 knockout (KO) or NO-GC2 KO]. The deletion of NO-GC1 or NO-GC2 did not influence electromechanical outer hair cell (OHC) properties, as measured by distortion product otoacoustic emissions, neither before nor after noise exposure, nor were click- or noise-burst–evoked auditory brainstem response thresholds different from controls. Yet inner hair cell (IHC) ribbons and auditory nerve responses showed significantly less deterioration in NO-GC1 KO and NO-GC2 KO mice after noise exposure. Consistent with a selective role of NO-GC in IHCs, NO-GC β1 mRNA was found in isolated IHCs but not in OHCs. Using transgenic mice expressing the fluorescence resonance energy transfer–based cGMP biosensor cGi500, NO-induced elevation of cGMP was detected in real-time in IHCs but not in OHCs. Pharmacologic long-term treatment with a NO-GC stimulator altered auditory nerve responses but did not affect OHC function and hearing thresholds. Interestingly, NO-GC stimulation exacerbated the loss of auditory nerve response in aged animals but attenuated the loss in younger animals. We propose NO-GC2 and, to some degree, NO-GC1 as targets for early pharmacologic prevention of auditory fiber loss (synaptopathy). Both isoforms provide selective benefits for hearing function by maintaining the functional integrity of auditory nerve fibers in early life rather than at old age.

Detection of Excitatory and Inhibitory Synapses in the Auditory System Using Fluorescence Immunohistochemistry and High-Resolution Fluorescence Microscopy.

In sensory systems, a balanced excitatory and inhibitory circuit along the ascending pathway is not only important for the establishment of topographically ordered connections from the periphery to the cortex but also for temporal precision of signal processing. The accomplishment of spatial and temporal cortical resolution in the central nervous system is a process that is likely initiated by the first sensory experiences that drive a period of increased intracortical inhibition. In the auditory system, the time of first sensory experience is also the period in which a reorganization of cochlear efferent and afferent fibers occurs leading to the mature innervation of inner and outer hair cells. This mature hair cell innervation is the basis of accurate sound processing along the ascending pathway up to the auditory cortex. We describe here, a protocol for detecting excitatory and inhibitory marker proteins along the ascending auditory pathway, which could be a useful tool for detecting changes in auditory signal processing during various forms of hearing disorders. Our protocol uses fluorescence immunohistochemistry in combination with high-resolution fluorescence microscopy in cochlear and brain tissue.

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 BDNF-live-exon-visualization (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 (BDNF-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.