Pharmacological modulation of Kv3.1 mitigates auditory midbrain temporal processing deficits following auditory nerve damage

Higher stages of central auditory processing compensate for a loss of cochlear nerve synapses by increasing the gain on remaining afferent inputs, thereby restoring firing rate codes for rudimentary sound features. The benefits of this compensatory plasticity are limited, as the recovery of precise temporal coding is comparatively modest. We reasoned that persistent temporal coding deficits could be ameliorated through modulation of voltage-gated potassium (Kv) channels that regulate temporal firing patterns. Here, we characterize AUT00063, a pharmacological compound that modulates Kv3.1, a high-threshold channel expressed in fast-spiking neurons throughout the central auditory pathway. Patch clamp recordings from auditory brainstem neurons and in silico modeling revealed that application of AUT00063 reduced action potential timing variability and improved temporal coding precision. Systemic injections of AUT00063 in vivo improved auditory synchronization and supported more accurate decoding of temporal sound features in the inferior colliculus and auditory cortex in adult mice with a near-complete loss of auditory nerve afferent synapses in the contralateral ear. These findings suggest modulating Kv3.1 in central neurons could be a promising therapeutic approach to mitigate temporal processing deficits that commonly accompany aging, tinnitus, ototoxic drug exposure or noise damage

E-LEARNING U BANKARSTVU

With this paper research results are presented on a successful e-learning implementation in banking. Previous experience with e-learning, general satisfaction with e-learning, satisfaction with particular elements of e-learning, on-line support, e-learning acceptance by office employees, as well as demanding way of assuring regular office business, indicate the possibility of implementing e-learning not only as a training process, but as a regular business process as well.U ovom radu je opisano istraživanje uspješnosti primjene e-learninga u bankarstvu. Dosadašnje iskustvo s e-learningom, općenito zadovoljstvo e-learningom, zadovoljstvo elementima e-learninga, on-line podrškom, prihvatljivost prakticiranja e-learninga za zaposlenike poslovnica, kao i zahtjevnost osiguranja redovnog rada poslovnica, ukazuju na mogućnost implementiranja e-learninga ne samo kao obrazovnog već i svakodnevnog poslovnog proces

Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

Kv3 potassium currents mediate rapid repolarisation of action potentials (APs), supporting fast spikes and high repetition rates. Of the four Kv3 gene family members, Kv3.1 and Kv3.3 are highly expressed in the auditory brainstem and we exploited this to test for subunit-specific roles at the calyx of Held presynaptic terminal in the mouse. Deletion of Kv3.3 (but not Kv3.1) reduced presynaptic Kv3 channel immunolabelling, increased presynaptic AP duration and facilitated excitatory transmitter release; which in turn enhanced short-term depression during high-frequency transmission. The response to sound was delayed in the Kv3.3KO, with higher spontaneous and lower evoked firing, thereby reducing signal-to-noise ratio. Computational modelling showed that the enhanced EPSC and short-term depression in the Kv3.3KO reflected increased vesicle release probability and accelerated activity-dependent vesicle replenishment. We conclude that Kv3.3 mediates fast repolarisation for short precise APs, conserving transmission during sustained high-frequency activity at this glutamatergic excitatory synapse

The Binding and Mechanism of a Positive Allosteric Modulator of Kv3 Channels

Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. The cryo-EM structure of the Kv3.1/AUT5 complex at a resolution of 2.5 Å reveals four equivalent AUT5 binding sites at the extracellular inter-subunit interface between the voltage-sensing and pore domains of the channel’s tetrameric assembly. Furthermore, we show that the unique extracellular turret regions of Kv3.1 and Kv3.2 essentially govern the selective positive modulation by AUT5. High-resolution apo and bound structures of Kv3.1 demonstrate how AUT5 binding promotes turret rearrangements and interactions with the voltage-sensing domain to favor the open conformation

Acoustic Overexposure Increases the Expression of VGLUT-2 Mediated Projections from the Lateral Vestibular Nucleus to the Dorsal Cochlear Nucleus

The dorsal cochlear nucleus (DCN) is a first relay of the central auditory system as well as a site for integration of multimodal information. Vesicular glutamate transporters VGLUT-1 and VGLUT-2 selectively package glutamate into synaptic vesicles and are found to have different patterns of organization in the DCN. Whereas auditory nerve fibers predominantly co-label with VGLUT-1, somatosensory inputs predominantly co-label with VGLUT-2. Here, we used retrograde and anterograde transport of fluorescent conjugated dextran amine (DA) to demonstrate that the lateral vestibular nucleus (LVN) exhibits ipsilateral projections to both fusiform and deep layers of the rat DCN. Stimulating the LVN induced glutamatergic synaptic currents in fusiform cells and granule cell interneurones. We combined the dextran amine neuronal tracing method with immunohistochemistry and showed that labeled projections from the LVN are co-labeled with VGLUT-2 by contrast to VGLUT-1. Wistar rats were exposed to a loud single tone (15 kHz, 110 dB SPL) for 6 hours. Five days after acoustic overexposure, the level of expression of VGLUT-1 in the DCN was decreased whereas the level of expression of VGLUT-2 in the DCN was increased including terminals originating from the LVN. VGLUT-2 mediated projections from the LVN to the DCN are likely to play a role in the head position in response to sound. Amplification of VGLUT-2 expression after acoustic overexposure could be a compensatory mechanism from vestibular inputs in response to hearing loss and to a decrease of VGLUT-1 expression from auditory nerve fibers

Acoustic trauma slows AMPAR-mediated EPSCs in the auditory brainstem, reducing GluA4 subunit expression as a mechanism to rescue binaural function

Damaging levels of sound (acoustic trauma, AT) diminish peripheral synapses, but what is the impact on the central auditory pathway? Developmental maturation of synaptic function and hearing were characterized in the mouse lateral superior olive (LSO) from postnatal day 7 (P7) to P96 using voltage-clamp and auditory brainstem responses. IPSCs and EPSCs show rapid acceleration during development, so that decay kinetics converge to similar sub-millisecond time-constants (τ, 0.87 ± 0.11 and 0.77 ± 0.08 ms, respectively) in adult mice. This correlated with LSO mRNA levels for glycinergic and glutamatergic ionotropic receptor subunits, confirming a switch from Glyα2 to Glyα1 for IPSCs and increased expression of GluA3 and GluA4 subunits for EPSCs. The NMDA receptor (NMDAR)-EPSC decay τ accelerated from >40 ms in prehearing animals to 2.6 ± 0.4 ms in adults, as GluN2C expression increased. In vivo induction of AT at around P20 disrupted IPSC and EPSC integration in the LSO, so that 1 week later the AMPA receptor (AMPAR)-EPSC decay was slowed and mRNA for GluA1 increased while GluA4 decreased. In contrast, GlyR IPSC and NMDAR-EPSC decay times were unchanged. Computational modelling confirmed that matched IPSC and EPSC kinetics are required to generate mature interaural level difference functions, and that longer-lasting EPSCs compensate to maintain binaural function with raised auditory thresholds after AT. We conclude that LSO excitatory and inhibitory synaptic drive matures to identical time-courses, that AT changes synaptic AMPARs by expression of subunits with slow kinetics (which recover over 2 months) and that loud sounds reversibly modify excitatory synapses in the brain, changing synaptic function for several weeks after exposure

Exploring hearing loss and plastic adjustments in the dorsal cochlear nucleus

Acoustic over exposure (AOE) triggers hearing loss and tinnitus but cellular mechanisms underlying those auditory defects are still poorly understood. This thesis explores the changes of excitability produced by AOE in identified cells of the rat dorsal cochlear nucleus (DCN) within the auditory brainstem. A development of a method combining Golgi silver impregnation with Nissl staining allowed study of the morphology and the distribution of the main DCN neuronal subtypes within slices containing the DCN. Whole cell patch clamp recordings allowed characterisation of the distinctive electrophysiological properties of the main DCN neuronal subtypes. In vitro stimulations of auditory or multisensory synaptic inputs showed fundamental differences in terms of the principal neurones firing pattern and the role of inhibitory synaptic transmission on firing pattern. Wistar rats were exposed to loud (110 dB SPL) single tones (15 kHz) for a period of 4 hours (protocol of AOE). Non invasive auditory brainstem response recordings were performed after 3 to 4 days and showed a significant increase of the rat’s hearing threshold for frequencies above 8 kHz. Whole cell recordings performed at a similar time (3 to 4 days) after AOE, showed that AOE led to a change of the passive and the active properties of DCN interneurones and principal cells. AOE also decreased the general excitability of the cellular network and affected differently excitatory and inhibitory synaptic transmission onto principal neurones depending on whether multisensory or auditory synaptic inputs were stimulated. Computational modelling allowed simulation of the effects of AOE on principal cell firing patterns and elaboration of a general theory whereby AOE triggers shifts of hearing thresholds concomitant with plastic adjustments in the DCN network. In conclusion, an elevation of the hearing threshold accompanied by significant excitability changes within the central auditory system could represent fundamental steps towards the development of tinnitus

Exploring hearing loss and plastic adjustments in the dorsal cochlear nucleus

Acoustic over exposure (AOE) triggers hearing loss and tinnitus but cellular mechanisms underlying those auditory defects are still poorly understood. This thesis explores the changes of excitability produced by AOE in identified cells of the rat dorsal cochlear nucleus (DCN) within the auditory brainstem. A development of a method combining Golgi silver impregnation with Nissl staining allowed study of the morphology and the distribution of the main DCN neuronal subtypes within slices containing the DCN. Whole cell patch clamp recordings allowed characterisation of the distinctive electrophysiological properties of the main DCN neuronal subtypes. In vitro stimulations of auditory or multisensory synaptic inputs showed fundamental differences in terms of the principal neurones firing pattern and the role of inhibitory synaptic transmission on firing pattern. Wistar rats were exposed to loud (110 dB SPL) single tones (15 kHz) for a period of 4 hours (protocol of AOE). Non invasive auditory brainstem response recordings were performed after 3 to 4 days and showed a significant increase of the rat’s hearing threshold for frequencies above 8 kHz. Whole cell recordings performed at a similar time (3 to 4 days) after AOE, showed that AOE led to a change of the passive and the active properties of DCN interneurones and principal cells. AOE also decreased the general excitability of the cellular network and affected differently excitatory and inhibitory synaptic transmission onto principal neurones depending on whether multisensory or auditory synaptic inputs were stimulated. Computational modelling allowed simulation of the effects of AOE on principal cell firing patterns and elaboration of a general theory whereby AOE triggers shifts of hearing thresholds concomitant with plastic adjustments in the DCN network. In conclusion, an elevation of the hearing threshold accompanied by significant excitability changes within the central auditory system could represent fundamental steps towards the development of tinnitus.EThOS - Electronic Theses Online ServiceProject funded by GlaxoSmithKline and MedisearchGBUnited Kingdo

Kv3 channels contribute to the excitability of subpopulations of spinal cord neurons in Lamina VII

Autonomic parasympathetic preganglionic neurons (PGN) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be due to recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within lamina VII of the lumbosacral spinal cord, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphological analysis separated these neuronal classes into parasympathetic preganglionic populations (PGN) and a fast firing interneuronal population. Kv3 channels are voltage-gated potassium channels (Kv) that allow fast and precise firing of neurons. We found that blockade of Kv3 channels by tetraethylammonium (TEA) reduced neuronal firing frequency and isolated high-voltage-activated Kv currents in the fast-firing population but had no effect in PGN populations. Furthermore, Kv3 blockade potentiated the local and descending inhibitory inputs to PGN indicating that Kv3-expressing inhibitory neurons are synaptically connected to PGN. Taken together, our data reveal that Kv3 channels are crucial for fast and regulated neuronal output of a defined population that may be involved in intrinsic spinal bladder circuits that underpin recurrent inhibition of PGN. Neural circuits in the spinal cord and pons mediate the micturition reflex. The spinal cord drives bladder contraction during micturition through the activation of parasympathetic preganglionic neurons in lamina VII of the lumbosacral spinal cord. Despite the significant contribution of these neurons to a crucial physiological reflex, neurons in this region have been under-characterised. This study therefore elucidated and thoroughly characterised distinct neuronal populations in this lamina; we propose that these populations included a fast firing interneuron and subtypes of parasympathetic preganglionic neurons. Further investigation revealed the critical importance of Kv3 channels in the fast firing ability of the interneurons, as well as in synaptic release onto PGNs