K+ accumulation and clearance in the calyx synaptic cleft of type I mouse vestibular hair cells

Vestibular organs of Amniotes contain two types of sensory cells, named Type I and Type II hair cells. While Type II hair cells are contacted by several small bouton nerve terminals, Type I hair cells receive a giant terminal, called a calyx, which encloses their basolateral membrane almost completely. Both hair cell types release glutamate, which depolarizes the afferent terminal by binding to AMPA post-synaptic receptors. However, there is evidence that non-vesicular signal transmission also occurs at the Type I hair cell-calyx synapse, possibly involving direct depolarization of the calyx by K+ exiting the hair cell. To better investigate this aspect, we performed whole-cell patch-clamp recordings from mouse Type I hair cells or their associated calyx. We found that [K+] in the calyceal synaptic cleft is elevated at rest relative to the interstitial (extracellular) solution and can increase or decrease during hair cell depolarization or repolarization, respectively. The change in [K+] was primarily driven by GK,L, the low-voltage-activated, non-inactivating K+ conductance specifically expressed by Type I hair cells. Simple diffusion of K+ between the cleft and the extracellular compartment appeared substantially restricted by the calyx inner membrane, with the ion channels and active transporters playing a crucial role in regulating intercellular [K+]. Calyx recordings were consistent with K+ leaving the synaptic cleft through postsynaptic voltage-gated K+ channels involving KV1 and KV7 subunits. The above scenario is consistent with direct depolarization and hyperpolarization of the calyx membrane potential by intercellular K+

High-Performance Physical-Independent Address-Based Communication Interface for FPGA in Custom Scientific Equipment

Nowadays, in different scientific applications, custom processing systems are particularly suited for Field- Programmable Gate Arrays (FPGA), rather than for Application Specific Integrate Circuits (ASIC). This is mainly due to the added flexibility, simpler design and manufacturing process that FPGA solutions offer, fitting the needs of small-scale custom applications. While the intra-chip data-transfer between the IP-Cores (IPs) that compose the FPGA architecture is relatively easy to implement, the communication system with Temporal Computing (TC) devices is not trivial to build. This contribution focuses on this issue and presents our inter-chip communication system, that possesses the quality of not relying on any specific physical link feature, which allows the use of any type of connection between FPGA and TC devices, as long as it transmits ordered data. Encoding and communication errors are also automatically detected. The system is composed by a software part and a hardware one. The software part is developed in C++, with Python bindings, and provides the read and write methods, to be able to issue the relative commands to an internal standard bus of the FPGA. The hardware part is composed by the sub-modules Packet Transmission Engine (PTE) and Memory Management Engine (MME); the first one being responsible for packets’ data framing, integrity check and data multiplexing on the physical link, while the second one executing the read and write operations which were encoded within the packets

Acute effects of gentamicin on the ionic currents of semicircular canal hair cells of the frog

The effects of acute gentamicin application on hair cells isolated from the frog semicircular canals have been tested by using the patch clamp technique in the whole-cell configuration. Extracellular gentamicin (1mM) mostly affected the Ca2+ macrocurrent, ICa, and the Ca-dependent K+ current, IKCa. The drug, applied to the hair cell basolateral membrane through a fast perfusion system, produced a rapid and relevant decrease (~34%) of ICa amplitude, without apparently affecting its activation/deactivation kinetics. The IKCa component of the delayed IKD was similarly affected: peak and steady-state mean amplitudes were significantly reduced, by about 47 and 54%, respectively, whereas the time constant of the mono-exponential current rising phase did not change. The Ca2+ independent fraction of IKD, IKV, and the fast IA current were unaffected. Transduction channels (permeable to and blocked by gentamicin) are not available in the isolated hair cell, so the effect of intracellular gentamicin was tested by applying the drug through the patch pipette (1 mM in the pipette): again, it significantly reduced both ICa and IKD amplitude, without affecting currents kinetics. IA properties were also unaffected. The drug did not affect the onset and removal of IKD inactivation, although the changes were scaled to the reduced IKD amplitude. From these observations, it is expected that hair cells exposed to gentamicin ‘in vivo’ become unresponsive to physiological stimulation (block of the transduction channels) and transmitter release at the cytoneural junction be drastically depressed due to reduced Ca2+ inflow. In particular, functional impairment ensues much earlier than biochemical events that lead to hair cell apoptosis

Effects of the calyx on the apparent properties of vestibular type I hair cells K+ currents

Vestibular Type I hair cells are almost entirely enveloped by a single large afferent nerve terminal, called calyx, whose functional meaning is still enigmatic. Another defining property of Type I cells is the expression of a low-voltage-activated outward rectifying K+ current, named IK,L. By patch-clamp whole-cell recordings from in situ mouse vestibular Type I cells, we have found that IK,L activation can result in K+ accumulation around the cell, as inferred from the positive shift of K+ currents reversal potential (VrevK+), presumably due to the presence of a residual calyx [1]. This phenomenon accompanied to a slower IK,L deactivation during hyperpolarizing voltage steps. We have investigated this aspect in more detail, and found that IK,L deactivated with a slow complex time course, not consistent with the reported exponential decay [2]. Since most previous studies were done in isolated Type I cells, we repeated the experiments in enzimatically dissociated cells and found that both the shift of VrevK+ and the alteration of IK,L deactivation were absent or much less obvious. Our hypothesis is that most of the calyx survives in situ , but not after cell dissociation, which would represent a restriction to K+ diffusion, and a resistance (Re) to current flow between the ynaptic cleft and the bath. As a consequence, large ion currents would produce a significant voltage drop (Ve) across Re. Ve would be maximal at the peak amplitude of the instantaneous IK,L, and then decrease with IK,L deactivation. Thus, IK,L deactivation would be distorted by voltage-clamp failure due to Re, which we estimated in tens of MΩ, i.e. in the same magnitude order of the calyx input resistance (~90 MΩ; [1]). In conclusion, our data demonstrate for the first time that the calyx can significantly influence, via a purely electrical mechanism which adds to the effects of K+ accumulation in the cleft, the behavior of the currents generated by the hair cell membrane

Acute effects of gentamicin on the ionic currents of semicircular canal hair cells in the frog.

The effects of acute gentamicin application on hair cells isolated from the frog semicircular canals have been tested by using the patch-clamp technique in the whole-cell configuration. Extracellular gentamicin (1 mM) mostly affected the Ca(2+) macrocurrent, I(Ca), and the Ca-dependent K(+) current, I(KCa). The drug, applied to the hair cell basolateral membrane through a fast perfusion system, produced a rapid and relevant decrease ( 3c34%) of I(Ca) amplitude, without apparently affecting its activation-deactivation kinetics. The I(KCa) component of the delayed I(KD) was similarly affected: peak and steady-state mean amplitudes were significantly reduced, by about 47 and 54%, respectively, whereas the time constant of the mono-exponential current rising phase did not change. The Ca(2+) independent fraction of I(KD), I(KV), and the fast IA current were unaffected. Transduction channels (permeable to and blocked by gentamicin) are not available in the isolated hair cell, so the effect of intracellular gentamicin was tested by applying the drug through the patch pipette (1 mM in the pipette): again, it significantly reduced both I(Ca) and I(KD) amplitude, without affecting currents kinetics. IA properties were also unaffected. The drug did not affect the onset and removal of I(KD) inactivation, although the changes were scaled to the reduced I(KD) amplitude. From these observations, it is expected that hair cells exposed to gentamicin 'in vivo' become unresponsive to physiological stimulation (block of the transduction channels) and transmitter release at the cytoneural junction be drastically depressed due to reduced Ca(2+) inflow. In particular, functional impairment ensues much earlier than biochemical events that lead to hair cell apoptosis

Eps8 regulates K+ current expression in mouse cochlear inner but not outer hair cells nor in vestibular type I and type II hair cells

Eps8 is involved in modulating cell signaling and receptor trafficking, via its range of protein interactions. We recently showed that cochlear inner and outer hair cells of Eps8 knockout (KO) mice, which are born deaf, show shorter stereocilia than wild type (WT) mice [1]. Inner, but not outer, hair cells, moreover, showed an altered expression of the mature array of voltage-dependent K+ channels, despite the fact that they express similar conductances. Vestibular hair cells of Eps8 KO mice show shorter stereocilia than normal [2], too. However, it is not known if this is accompanied by some functional defect. We therefore patch-clamp whole-cell recorded the voltage-dependent K+ currents from vestibular Type I and Type II hair cells of Eps8 KO and WT mice at different postnatal developmental stages. We found that both vestibular hair cell types from KO mice showed a normal pattern of expression of K+ currents along with maturation up to the adult age. These results indicate that Eps8 is a specific regulator of K+ channel expression in mammalian cochlear inner hair cells. This notion appears particularly important in view of the recent discovery that a nonsense mutation in EPS8 is responsible for a non-syndromic form of human deafness [3]

Timescales and Mechanisms of Crystal-mush Rejuvenation and Melt Extraction Recorded in Permian Plutonic and Volcanic Rocks of the Sesia Magmatic System (Southern Alps, Italy)

Silicic calderas can evacuate 100 to >1000\u2009km3 of rhyolitic products in a matter of days to months, leading to questions on pre-eruptive melt generation and accumulation. Whereas silicic plutonic units may provide information on the igneous evolution of crystal-mush bodies, their connection with volcanic units remains enigmatic. In the Ivrea\u2013Verbano Zone of the southern Alps, the plumbing system of a Permian rhyolitic caldera is exposed to a depth of about 25\u2009km in tilted crustal blocks. The upper-crustal segment of this magmatic system (also known as the Sesia Magmatic System) is represented by the Valle Mosso pluton (VMP). The VMP is an 3c260\u2009km3 composite silicic intrusion ranging from quartz-monzonite to high-silica leucogranite ( 3c67\u201377\u2009wt% SiO2), which intrudes into roughly coeval rhyolitic products of the >15\u2009km diameter Sesia Caldera. In the caldera field, the emplacement of a large, crystal-rich rhyolite ignimbrite(s) (>400\u2009km3) is followed by eruption of minor volumes (1\u201310\u2009km3) of crystal-poor rhyolite. Here, we compare silicic plutonic and volcanic units of the Sesia Magmatic System through a combination of geochemical (X-ray fluorescence, inductively coupled plasma mass spectrometry and electron microprobe analyses) and petrological (rhyolite-MELTS, trace element and diffusion modeling) tools to explore their connection. Textural and compositional features shared by both VMP and crystal-rich ignimbrites imply thermal rejuvenation of crystal-mush as the mechanism to create large volumes of eruptible rhyolitic magma. Bulk-rock composition of crystal-rich rhyolite erupted during the caldera collapse overlaps that of the bulk VMP. Quartz and plagioclase from these two units show resorbed cores and inverse zoning, with Ti- and anorthite-rich rims, respectively. This indicates crystallization temperatures in rims >60\u2009\ub0C higher than in cores (780\u2013820 versus 3c720\u2009\ub0C), if temperature is the sole parameter responsible for zonation, suggesting heating and partial dissolution of the crystal-framework. Decrease in crystallinity associated with thermal energy input was calculated through rhyolite-MELTS and indicates lowering of the mush crystal fraction below the rheological lock-up threshold, which probably promoted eruptive activity. Also, after the climatic eruption, Si-rich melts in the Sesia Magmatic System were produced by extraction of interstitial melt from un-erupted, largely crystalline mush. Regarding both textures and chemical variations, we interpret the deep quartz-monzonite unit of the VMP as a compacted silicic cumulate. Fractionated melts extracted from this unit were emplaced as a leucogranite cupola atop the VMP, generating the final internal architecture of the silicic intrusion, or alternatively erupted as minor post-caldera, crystal-poor rhyolite. Ti-in-quartz diffusion profiles in thermally rejuvenated units of the Sesia Magmatic System demonstrate that the process of reheating, mobilization and eruption of crystal-mush took place rapidly (c. 101\u2013102\u2009years). A protracted cooling history is instead recorded in the diffusion timescales of quartz from the silicic cumulate units (c. 104\u2013106\u2009years). These longer timescales encompass the duration of evolved melt extraction from the cumulate residue. We argue that the VMP preserves a complex record of pre-eruptive processes, which span mechanisms and timescales universally identified in volcanic systems and are consistent with recently proposed numerical models

Intercellular K(+) accumulation depolarizes Type I vestibular hair cells and their associated afferent nerve calyx

Mammalian vestibular organs contain two types of sensory receptors, named Type I and Type II hair cells. While Type II hair cells are contacted by several small afferent nerve terminals, the basolateral surface of Type I hair cells is almost entirely enveloped by a single large afferent nerve terminal, called calyx. Moreover Type I, but not Type II hair cells, express a low-voltage-activated outward K(+) current, I(K,L), which is responsible for their much lower input resistance (Rm) at rest as compared to Type II hair cells. The functional meaning of I(K,L) and associated calyx is still enigmatic. By combining the patch-clamp whole-cell technique with the mouse whole crista preparation, we have recorded the current- and voltage responses of in situ hair cells. Outward K(+) current activation resulted in K(+) accumulation around Type I hair cells, since it induced a rightward shift of the K(+) reversal potential the magnitude of which depended on the amplitude and duration of K(+) current flow. Since this phenomenon was never observed for Type II hair cells, we ascribed it to the presence of a residual calyx limiting K(+) efflux from the synaptic cleft. Intercellular K(+) accumulation added a slow (τ>100ms) depolarizing component to the cell voltage response. In a few cases we were able to record from the calyx and found evidence for intercellular K(+) accumulation as well. The resulting depolarization could trigger a discharge of action potentials in the afferent nerve fiber. Present results support a model where pre- and postsynaptic depolarization produced by intercellular K(+) accumulation cooperates with neurotransmitter exocytosis in sustaining afferent transmission arising from Type I hair cells. While vesicular transmission together with the low Rm of Type I hair cells appears best suited for signaling fast head movements, depolarization produced by intercellular K(+) accumulation could enhance signal transmission during slow head movements