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+

Efferent Control of the Electrical and Mechanical Properties of Hair Cells in the Bullfrog's Sacculus

Background: Hair cells in the auditory, vestibular, and lateral-line systems respond to mechanical stimulation and transmit information to afferent nerve fibers. The sensitivity of mechanoelectrical transduction is modulated by the efferent pathway, whose activity usually reduces the responsiveness of hair cells. The basis of this effect remains unknown. Methodology and Principal Findings: We employed immunocytological, electrophysiological, and micromechanical approaches to characterize the anatomy of efferent innervation and the effect of efferent activity on the electrical and mechanical properties of hair cells in the bullfrog’s sacculus. We found that efferent fibers form extensive synaptic terminals on all macular and extramacular hair cells. Macular hair cells expressing the Ca 2+-buffering protein calretinin contain half as many synaptic ribbons and are innervated by twice as many efferent terminals as calretinin-negative hair cells. Efferent activity elicits inhibitory postsynaptic potentials in hair cells and thus inhibits their electrical resonance. In hair cells that exhibit spiking activity, efferent stimulation suppresses the generation of action potentials. Finally, efferent activity triggers a displacement of the hair bundle’s resting position. Conclusions and Significance: The hair cells of the bullfrog’s sacculus receive a rich efferent innervation with the heaviest projection to calretinin-containing cells. Stimulation of efferent axons desensitizes the hair cells and suppresses their spiking activity. Although efferent activation influences mechanoelectrical transduction, the mechanical effects on hair bundles ar

Intracellular recordings from "recurrent neurons" in the rat superior cervical ganglion

Intracellular recordings in the isolated superior cervical ganglion of the rat showed that electrical stimulation of the cervical sympathetic trunk elicited in a cluster of neurons localized in the caudal part of the ganglion synaptically driven action potentials, and propagated potentials having the features of typical antidromic spikes. The results demonstrate that these neurons, besides synapsing with common preganglionic fibres, project their axons to the cervical sympathetic trunk. The recurrent neurons showed a very low threshold to direct intracellular stimulation and a high input resistance, suggesting that they have a small size. Almost all recurrent neurons were activated synaptically also by stimulating the postganglionic trunks, indicating that they are innervated by collaterals of preganglionic through-fibres which are known to sustain a direct pathway between pre- and postganglionic nerves. Moreover, some recurrent neurons could also be activated antidromically following stimulation of the external carotid nerve, indicating that their axons divide into collaterals which project not only to the preganglionic trunk but also to a postganglionic nerve. The presence of recurrent neurons in the superior cervical ganglion of the rat provides further evidence for the concept that sympathetic ganglia consist of discrete cell subpopulations which are segregated in different regions and probably subserve different functions

Acetylcholine-induced back-firing in the preganglionic trunk of the rat superior cervical ganglion

ACh (5 X 10(-4) M), when applied to isolated ganglion preparations elicited an apparently antidromic discharge in the cervical sympathetic trunk. The intensity of this back-firing was found to be about 10 times lower than that of the postganglionic discharge evoked by ACh in the internal carotid nerve. Both responses, however, displayed a similar time course and consisted of an early and a late component. In the back-firing the early component died out in a few seconds, while the late one lasted for 20-30 s. The two components were cancelled by d-tubocurarine (5 X 10(-6) M) and atropine (10(-6) M), respectively, suggesting that both nicotinic and muscarinic cholinoceptive sites are involved. In chronically decentralized preparations ACh evoked a clear back-firing response not substantially different from that elicited in normal ganglia. Therefore it is likely that the back-firing phenomenon is not due to antidromic activation of preganglionic fibers. The back-firing observed in the rat superior cervical ganglion was interpreted as being due to activation of sympathetic neurons known to give rise to recurrent axons in the cervical sympathetic trunk

Effetto della stimolazione elettrica delle fibre nervose efferenti nel nervo ampollare del canale verticale posteriore della rana sui potenziali derivati da singoli assoni afferenti nello stesso nervo

I recettori labirintici della rana contraggono direttamente rapporti sinaptici sia con le terminazioni delle fibre nervose afferenti, che con terminazioni di fibre nervose efferenti. Dati presenti in letteratura, in strutture morfologicamente simili, indicano che il sistema efferente regola, attraverso un'azione inibitoria, l'attività afferente. Derivazioni endocellulari da singole fibre afferenti del nervo ampollare del canale verticale posteriore nella rana, ottenute con metodi convenzionali, ci hanno permesso di identificare sia potenziali propagati che EPSPs che si generano alla giunzione citoneurale. I meccanismi elementari della trasmissione sinaptica a livello delle fibre afferenti sono stati da noi esaminati in precedenza. Nella presente ricerca abbiamo studiato gli effetti della stimolazione delle fibre efferenti sugli eventi elettrici, sia propagati che sottoliminari, nelle fibre afferenti. A questo scopo, il ramo ampollare del canale verticale posteriore, isolato e separato dall’VIII nervo veniva aspirato in una pipetta che fungeva da elettrodo stimolante. La stimolazione provocava nelle unità afferenti impalate, accanto ai potenziali antidromici evocati dallo stimolo, una progressiva riduzione dell'ampiezza degli EPSPs e della frequenza dei potenziali propagati spontanei. In particolare aumentando la frequenza a 50 -100/sec, si poteva ottenere la completa scomparsa degli EPSPs. Il sistema efferente esercita quindi, anche a livello di questi recettori, un'azione inibitoria che si manifesta come una ridotta liberazione di mediatore chimico dal polo sinaptico delle cellule cigliate. Nelle nostre condizioni sperimentali non possiamo però escludere effetti collaterali dovuti all'invasione anttdromica delle fibre afferenti durante la stimolazione elettrica dell'intero nervo. Questi risultati verranno completati con esperimenti in cui si cercherà di eliminare l'effetto inibitorio, bloccando in modo selettivo le sinapsi efferenti

Preganglionic discharge induced by acetylcholine in the superior cervical ganglia of the rat

ACh (5.10(-4) M), when applied to isolated ganglion preparations elicited an apparently antidromic discharge in the cervical sympathetic trunk. The intensity of this back-firing was found to be about 10 times lower than that of the postganglionic discharge evoked by ACh in the internal carotid nerve. Both responses however displayed a similar time course consisting mainly of an early and a late component. In the back-firing the early component died out in few seconds, while the late one lasted 20-30 seconds. The two components were cancelled by d-tubocurarine (5.10(-6) M) and atropine (10(-6) M) respectively, suggesting that both nicotinic and muscarinic cholinoceptive sites are involved. In chronically decentralized preparations ACh evoked a clear back-firing response not substantially different from that elicited in normal ganglia. Therefore it is likely that the back-firing phenomenon is not due to antidromic activation of preganglionic fibers. The back-firing observed in the rat superior cervical ganglion was interpreted as being due to activation of sympathetic neurons, known to give rise to recurrent axons in the cervical sympathetic cord

Post-tetanic spontaneous spike activity in rat sympathetic neurons exposed to low potassium ion concentration

Preganglionic tetanic stimulation (30 sec at 50/sec) of rat superior cervical ganglia, performed in the presence of reduced external potassium concentration (0-1 mM), is followed by a long-lasting postganglionic afterdischarge which fails to appear if stimulation is repeated in normal (5.6 mM) postassium solution. Intracellular recordings revealed that tetanus is followed by 15-30 mV membrane hyperpolarization when the neuron is exposed to normal concentrations of potassium. Conversely, after the ganglion is soaked in low potassium, stimulation results in long-lasting depolarization of the nerve cell with the consequent appearance of spontaneous spikes. This effect is reversed on returing to normal external potassium. Spontaneous activity also occurs after antidromic activation of the cell. It is suggested that tetanus causes sodium loading of the neuron, which leads to stimulation of an electrogenic sodium pump. If potassium is available, the membrane will hyperpolarize, whereas depolarization and pacemaker activity ensues if external potassium is removed. The electrogenic sodium pump thus endows. the rat sympathetic neuron with a mechanism which enables it excitability to be controlled

Differential expression of potassium currents by hair cells in thin slices of frog crista ampullaris

1. Electrical responses in hair cells located in the peripheral regions and in the central region of the frog crista ampullaris were investigated in thin slice preparations by using the whole-cell configuration of the patch-clamp technique. 2. Hair cells from the peripheral regions exhibited mostly a club-like shape and had an average resting potential of -46 mV, whereas cells from the central region had mostly a cylindrical shape and a more negative resting potential (-57 mV). 3. Voltage-clamp recordings revealed that ionic conductances differed in the two epithelial regions. Cells from the peripheral regions exhibited a transient K+ current of A-type (IA) in conjunction with a slow rectifier outward K+ current (IK). Cells from the central region showed little or no IA and generated an IK together with an inward rectifier K+ current (IIR). In both regions, hair cells showed a rapidly activating Ca(2+)-dependent outward K+ current (IK(Ca)) that rapidly inactivated to reach a steady-state level during 150-ms test pulses. 4. IA activated close to -60 mV and was inhibited by 12 mM 4-aminopyridine (4-AP). The time course of this current showed time to peak values of 3-4 ms at 0 mV. Inactivation was fast and almost voltage-independent. The decay time constant was approximately 35 ms at 0 mV. 5. IK was recruited close to -60 mV and activated slowly, reaching peak values in approximately 100 ms at 0 mV. It showed no evidence of inactivation during 150-ms test pulses and it was insensitive to 4-AP. 6. IIR activated at membrane potentials more negative than -90 mV and was blocked by exposure to 6 mM Cs+ or to a K(+)-free medium. This current showed an outward relaxation at potentials more negative than -140 mV, an effect that disappeared after exposure to a Na(+)-free medium. 7. IK(Ca) was recruited close to -40 mV and was inhibited by exposure to a Ca(2+)-free external medium or to 0.5 mM Cd2+. The time to peak of this current was approximately 3 ms at 0 mV and inactivation was very fast and almost independent from the membrane potential. The decay time constant was approximately 4 ms at 0 mV. 8. IK and IA were prominent in hair cells from the peripheral regions, whereas IK accounted for most of the membrane conductance in cells from the central region. The contribution of IK(Ca) was comparable in cells from both epithelial regions

Inactivation of delayed rectifier K+ current in semicircular canal hair cells

Some properties of the inactivation process of delayed rectifier K+ current (Ik) were investigated in vestibular hair cells of the central region of frog crista ampullaris. These cells were chosen since they exhibited a very large Ik. Experiments were performed on thin slices of sensory epithelium using the whole-cell variant of the patch-clamp technique. Ik showed clear time-dependent inactivation over a period of some seconds, but the current did not completely inactivate even after 30 s depolarizing pulses. Another interesting finding was that inactivation could be well fitted by the sum of two exponentials: at 20 mV depolarization the fast time constant was 291.3 ms and the slow time constant was 2662.3 ms. In addition, an analysis of the steady-state inactivation process of Ik revealed that the inactivation curve was incomplete showing a non-inactivating current at potentials more positive than -50 mV. These results suggested that Ik in hair cells of frog crista ampullaris is composed of more than one component: by at least one inactivating and one non-inactivating component. The possible role of these components in hair cell excitability is discussed