KCNK5 channels mostly expressed in cochlear outer sulcus cells are indispensable for hearing

International audienceIn the cochlea, K þ is essential for mechano-electrical transduction. Here, we explore cochlear structure and function in mice lacking K þ channels of the two-pore domain family. A profound deafness associated with a decrease in endocochlear potential is found in adult Kcnk5 À / À mice. Hearing occurs around postnatal day 19 (P19), and completely disappears 2 days later. At P19, Kcnk5 À / À mice have a normal endolymphatic [K þ ] but a partly lowered endocochlear potential. Using Lac-Z as a gene reporter, KCNK5 is mainly found in outer sulcus Claudius', Boettcher's and root cells. Low levels of expression are also seen in the spiral ganglion, Reissner's membrane and stria vascularis. Essential channels (KCNJ10 and KCNQ1) contributing to K þ secretion in stria vascularis have normal expression in Kcnk5 À / À mice. Thus, KCNK5 channels are indispensable for the maintenance of hearing. Among several plausible mechanisms, we emphasize their role in K þ recycling along the outer sulcus lateral route

Effects of antidromic discharges in crayfish primary afferents.

Contrary to orthodromic spikes that are generated in sensory organs and conveyed to CNS, antidromic spikes are generated in the axon terminals of the sensory neurons within the CNS and are conveyed to the peripheral sensory organ. Antidromic discharges are observed in primary afferent neurons of both vertebrates and invertebrates and seem to be related to the rhythmic activity of central neural networks. In this study, we analyzed the effect of antidromic discharges on the sensory activity of a leg proprioceptor in in vitro preparations of the crayfish CNS. Intracellular microelectrodes were used both to record the orthodromic spikes and to elicit antidromic spikes by injecting squares pulses of depolarizing current at various frequencies. Experiments were performed on the three types of identified sensory afferents (tonic, phasotonic, and phasic). The main results showed a reduction of the firing frequency of the orthodromic activity in 82% of the tested afferents. In tonic afferents, during their occurrences and according to their frequency, antidromic spikes or bursts reduced or suppressed the orthodromic activity. Following their terminations, they also induced a silent period and a gradual recovery of the orthodromic activity, both of which increased as the duration and the frequency of the antidromic bursts increased. In phasotonic and phasic afferents, antidromic bursts reduced or suppressed the phasic responses as their frequency and durations increased. In phasotonic afferents, if elicited prior to the movements, long-duration bursts with increasing frequency reduced more rapidly the tonic background activity than the phasic one whereas short-duration bursts at high frequency produced strong decreases of both. The effect of antidromic bursts accumulated when they are repetitively elicited. Antidromic bursts induced a much larger decrease of the sensory activity than adaptation alone. The occurrences of antidromic spikes or bursts may have a functional role in modulating the incoming sensory messages during locomotion. The mechanisms by which antidromic spikes modulate the firing sensitivity of the primary afferents may well lie in modifications of the properties of either mecanotransduction and/or spike initiation

Antidromic modulation of a proprioceptor sensory discharge in crayfish.

In the proprioceptive neurons of the coxo-basal chortotonal organ, orthodromic spikes convey the sensory information from the cell somata (located peripherally) to the central output terminals. During fictive locomotion, presynaptic depolarizations of these central terminals elicit bursts of antidromic spikes that travel back to the periphery. To determine whether the antidromic spikes modified the orthodromic activity of the sensory neurons, single identified primary afferents of the proprioceptor were recorded intracellularly and stimulated in in vitro preparations of crayfish nervous system. Depolarizing current pulses were delivered in trains whose frequency and duration were controlled to reproduce bursts of antidromic spikes similar to those elicited during fictive locomotion. According to their frequencies, these antidromic bursts reduce or suppress the orthodromic discharges in both position- and movement-sensitive neurons. They induce both a long-lasting silence and a gradual recovery after their occurrences. Neither the collision between the afferent and the efferent messages nor the release of serotonin by the sensory neurons can explain these results. We therefore conclude that antidromic bursts produce a peripheral modulation of the orthodromic activity of the sensory neurons, modifying their sensitivity by mechanisms yet unknown

Antidromic modulation of a proprioceptor sensory discharge in crayfish.

In the proprioceptive neurons of the coxo-basal chortotonal organ, orthodromic spikes convey the sensory information from the cell somata (located peripherally) to the central output terminals. During fictive locomotion, presynaptic depolarizations of these central terminals elicit bursts of antidromic spikes that travel back to the periphery. To determine whether the antidromic spikes modified the orthodromic activity of the sensory neurons, single identified primary afferents of the proprioceptor were recorded intracellularly and stimulated in in vitro preparations of crayfish nervous system. Depolarizing current pulses were delivered in trains whose frequency and duration were controlled to reproduce bursts of antidromic spikes similar to those elicited during fictive locomotion. According to their frequencies, these antidromic bursts reduce or suppress the orthodromic discharges in both position- and movement-sensitive neurons. They induce both a long-lasting silence and a gradual recovery after their occurrences. Neither the collision between the afferent and the efferent messages nor the release of serotonin by the sensory neurons can explain these results. We therefore conclude that antidromic bursts produce a peripheral modulation of the orthodromic activity of the sensory neurons, modifying their sensitivity by mechanisms yet unknown

Presynaptic inhibition and antidromic discharges in crayfish primary afferents.

The mechanisms of presynaptic inhibition have been studied in sensory afferents of a stretch receptor in an in vitro preparation of the crayfish. Axon terminals of these sensory afferents display primary afferent depolarisations (PADs) mediated by the activation of GABA receptors that open chloride channels. Intracellular labeling of sensory axons by Lucifer yellow combined with GABA immunohistochemistry revealed the presence of close appositions between GABA-immunoreactive boutons and sensory axons close to their first branching point within the ganglion. Electrophysiological studies showed that GABA inputs mediating PADs appear to occur around the first axonal branching point, which corresponds to the area of transition between active and passive propagation of spikes. Moreover, this study demonstrated that whilst shunting appeared to be the sole mechanism involved during small amplitude PADs, sodium channel inactivation occurred with larger amplitude PADs. However, when the largest PADs (>25 mV) are produced, the threshold for spike generation is reached and antidromic action potentials are elicited. The mechanisms involved in the initiation of antidromic discharges were analyzed by combining electrophysiological and simulation studies. Three mechanisms act together to ensure that PAD-mediated spikes are not conveyed distally: 1) the lack of active propagation in distal regions of the sensory axons; 2) the inactivation of the sodium channels around the site where PADs are produced; and 3) a massive shunting through the opening of chloride channels associated with the activation of GABA receptors. The centrally generated spikes are, however, conveyed antidromically in the sensory nerve up to the proprioceptive organ, where they inhibit the activity of the sensory neurons for several hundreds of milliseconds

Inhibitory connections between antagonistic motor neurones of the crayfish walking legs.

The inhibitory relationship between two antagonistic groups of motor neurones (MNs) that control the second leg joint of the crayfish Procambarus clarkii, was investigated in an in vitro preparation of the ventral nerve cord. Paired intracellular recordings were used to test the hypothesis that reciprocal inhibitory connections between levator (Lev) and depressor (Dep) MNs are direct. The injection of depolarising current into a Lev MN induces a hyperpolarising response in the Dep MN. This inhibitory relationship does not require spikes in the presynaptic MN, because it persists when spikes are suppressed by the sodium channel blocker tetrodotoxin (TTX). This reciprocal inhibition is graded, and both the amplitude and the time constant of the hyperpolarising response increase with increasing amount of depolarising current injected into an antagonistic MN. Although this inhibition is slow (synaptic delay around 10 ms), it is probably supported by a direct glutamatergic synapse from the antagonistic glutamatergic MN because it persists in the presence of the gamma-amino-butyric acid (GABA) synthesis inhibitor 3-mercapto-propionic acid (3-MPA). This hypothesis is reinforced by the demonstration of close appositions between antagonistic MNs by using a confocal microscope, and by the presence of glutamate-immunoreactive synapses on the neurites of MNs labelled for electron microscopy by intracellular injection of horseradish peroxidase

Nasal trigeminal inputs release the A5 inhibition received by the respiratory rhythm generator of the mouse neonate.

Experiments were performed on neonatal mice to analyze why, in vitro, the respiratory rhythm generator (RRG) was silent and how it could be activated. We demonstrated that in vitro the RRG in intact brain stems is silenced by a powerful inhibition arising from the pontine A5 neurons through medullary alpha(2) adrenoceptors and that in vivo nasal trigeminal inputs facilitate the RRG as nasal continuous positive airway pressure increases the breathing frequency, whereas nasal occlusion and nasal afferent anesthesia depress it. Because nasal trigeminal afferents project to the A5 nuclei, we applied single trains of negative electric shocks to the trigeminal nerve in inactive ponto-medullary preparations. They induced rhythmic phrenic bursts during the stimulation and for 2-3 min afterward, whereas repetitive trains produced on-going rhythmic activity up to the end of the experiments. Electrolytic lesion or pharmacological inactivation of the ipsilateral A5 neurons altered both the phrenic burst frequency and occurrence after the stimulation. Extracellular unitary recordings and trans-neuronal tracing experiments with the rabies virus show that the medullary lateral reticular area contains respiratory-modulated neurons, not necessary for respiratory rhythmogenesis, but that may provide an excitatory pathway from the trigeminal inputs to the RRG as their electrolytic lesion suppresses any phrenic activity induced by the trigeminal nerve stimulation. The results lead to the hypothesis that the trigeminal afferents in the mouse neonate involve at least two pathways to activate the RRG, one that may act through the medullary lateral reticular area and one that releases the A5 inhibition received by the RRG

Release of glutamate by the embryonic spinal motoneurons of rat positively regulated by acetylcholine through the nicotinic and muscarinic receptors.

It has been shown that mature neurons in adult vertebrates can co-express glutamate and acetylcholine. Furthermore, interactions at the synaptic level have been demonstrated. In a previous study we found that also motoneurons at early embryonic stages, thus well prior to synapse formation, release acetylcholine, and that glutamate increases this release. We now report the existence of a glutamate release from embryonic motoneurons and the increase of glutamate release by acetylcholine. This effect is mediated by nicotinic and muscarinic cholinergic receptors present on embryonic motoneurons. Using conditions of partial or total depletion of calcium, we show that the glutamate release has two components: one is calcium-dependent and the other calcium-independent. Furthermore, we show that extracellular glutamate can be taken up by motoneurons, probably via the neuronal glutamate transporter EAAC1, which we find to be expressed at this stage. Monitoring of the glutamate release kinetics showed that extracellular glutamate concentration reached a steady-state level, strongly suggesting the establishment of equilibrium between glutamate release and uptake. Altogether, these results support the idea that glutamate can act as a neurotransmitter in embryonic motoneurons. We hypothesise that, glutamate acts as a regulator of motoneuron maturation and spinal cord development

Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies.

In vivo (plethysmography) and in vitro (en bloc preparations) experiments were performed from embryonic day 16 (E16) to postnatal day 9 (P9) in order to analyse the perinatal maturation of the respiratory rhythm-generator in mice. At E16, delivered foetuses did not ventilate and survive but at E18 they breathed at about 110 cycles/min with respiratory cycles of variable individual duration. From E18 to P0-P2, the respiratory cycles stabilised without changes in the breathing parameters. However, these increased several-fold during the next days. Hypoxia increased breathing frequency from E18-P5 and only significantly affected ventilation from P3 onwards. At E16, in vitro medullary preparations (pons resection) produced rhythmic phrenic bursts at a low frequency (about 5 cycles/min) with variable cycle duration. At E18, their frequency doubled but cycle duration remained variable. After birth, the frequency did not change although cycle duration stabilised. At E18 and P0-P2, the in vitro frequency decreased by around 50% under hypoxia, increased by 40-50% under noradrenaline or substance P and was permanently depressed by the pontine A5 areas. At E16 however, hypoxia had no effects, both noradrenaline and substance P drastically increased the frequency and area A5 inhibition was not expressed at this time. At E18 and P0-P2, electrical stimulation and electrolytic lesion of the rostral ventrolateral medulla affected the in vitro rhythm but failed to induce convincing effects at E16. Thus, a major maturational step in respiratory rhythmogenesis occurs between E16-E18, in agreement with the concept of multiple rhythmogenic mechanisms