RESUMO
BACKGROUND: Perineuronal nets (PNNs) are specialized aggregations of extracellular matrix (ECM) molecules surrounding specific neurons in the central nervous system (CNS). PNNs are supposed to control synaptic transmission and are frequently associated with neurons firing at high rates, including principal neurons of auditory brainstem nuclei. The origin of high-frequency activity of auditory brainstem neurons is the indefatigable sound-driven transmitter release of inner hair cells (IHCs) in the cochlea. RESULTS: Here, we show that synaptic poles of IHCs are ensheathed by basket-like ECM complexes formed by the same molecules that constitute PNNs of neurons in the CNS, including brevican, aggreccan, neurocan, hyaluronan, and proteoglycan link proteins 1 and 4 and tenascin-R. Genetic deletion of brevican, one of the main components, resulted in a massive degradation of ECM baskets at IHCs, a significant impairment in spatial coupling of pre- and postsynaptic elements and mild impairment of hearing. CONCLUSIONS: These ECM baskets potentially contribute to control of synaptic transmission at IHCs and might be functionally related to PNNs of neurons in the CNS.
Assuntos
Brevicam/genética , Orelha Interna/fisiologia , Matriz Extracelular/metabolismo , Transmissão Sináptica/fisiologia , Animais , Brevicam/metabolismo , Feminino , Masculino , Camundongos , Camundongos KnockoutRESUMO
The auxiliary subunit α2δ2 modulates the abundance and function of voltage-gated calcium channels. Here we show that α2δ2 mRNA is expressed in neonatal and mature hair cells. A functional α2δ2-null mouse, the ducky mouse (du), showed elevated auditory brainstem response click and frequency-dependent hearing thresholds. Otoacoustic emissions were not impaired pointing to normal outer hair cell function. Peak Ca2+ and Ba2+ currents of mature du/du inner hair cells (IHCs) were reduced by 30-40%, respectively, and gating properties, such as the voltage of half-maximum activation and voltage sensitivity, were altered, indicating that Cav1.3 channels normally coassemble with α2δ2 at IHC presynapses. The reduction of depolarization-evoked exocytosis in du/du IHCs reflected their reduced Ca2+ currents. Ca2+- and voltage-dependent K+ (BK) currents and the expression of the pore-forming BKα protein were normal. Cav1.3 and Cavß2 protein expression was unchanged in du/du IHCs, forming clusters at presynaptic ribbons. However, the close apposition of presynaptic Cav1.3 clusters with postsynaptic glutamate receptor GluA4 and PSD-95 clusters was significantly impaired in du/du mice. This implies that, in addition to controlling the expression and gating properties of Cav1.3 channels, the largely extracellularly localized α2δ2 subunit moreover plays a so far unknown role in mediating trans-synaptic alignment of presynaptic Ca2+ channels and postsynaptic AMPA receptors. SIGNIFICANCE STATEMENT: Inner hair cells possess calcium channels that are essential for transmitting sound information into synaptic transmitter release. Voltage-gated calcium channels can coassemble with auxiliary subunit α2δ isoforms 1-4. We found that hair cells of the mouse express the auxiliary subunit α2δ2, which is needed for normal hearing thresholds. Using a mouse model with a mutant, nonfunctional α2δ2 protein, we showed that the α2δ2 protein is necessary for normal calcium currents and exocytosis in inner hair cells. Unexpectedly, the α2δ2 protein is moreover required for the optimal spatial alignment of presynaptic calcium channels and postsynaptic glutamate receptor proteins across the synaptic cleft. This suggests that α2δ2 plays a novel role in organizing the synapse.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio/metabolismo , Células Ciliadas Auditivas Internas/fisiologia , Audição/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Sinalização do Cálcio/fisiologia , Feminino , Ativação do Canal Iônico/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The tectorial membrane (TM), a complex acellular structure that covers part of the organ of Corti and excites outer hair cells, is required for normal hearing. It consists of collagen fibrils and various glycoproteins, which are synthesized in embryonic and postnatal development by different cochlear cell types including the interdental cells (IDCs). At its modiolar side, the TM is fixed to the apical surfaces of IDCs, which form the covering epithelium of the spiral limbus. We performed confocal membrane imaging and Ca2+ imaging in IDCs of the developing mouse cochlea from birth to postnatal day 18 (P18). Using the fluorescent membrane markers FM 4-64 and CellMask™ Deep Red on explanted whole-mount cochlear epithelium, we identified the morphology of IDCs at different z-levels of the spiral limbus. Ca2+ imaging of Fluo-8 AM-loaded cochlear epithelia revealed spontaneous intracellular Ca2+ transients in IDCs at P0/1, P4/5, and P18. Their relative frequency was lowest on P0/1, increased by a factor of 12.5 on P4/5 and decreased to twice the initial value on P18. At all three ages, stimulation of IDCs with the trinucleotides ATP and UTP at 1 and 10 µM elicited Ca2+ transients of varying amplitude and shape. Before the onset of hearing, IDCs responded with robust Ca2+ oscillations. At P18, after the onset of hearing, ATP stimulation either caused Ca2+ oscillations or an initial Ca2+ peak followed by a plateau while the UTP response was unchanged from that at pre-hearing stage. Parameters of spontaneous and nucleotide-evoked Ca2+ transients such as amplitude, decay time and duration were markedly reduced during cochlear development, whereas the kinetics of the Ca2+ rise did not show relevant changes. Whether low-frequency spontaneous Ca2+ transients are necessary for the formation and maintenance of the tectorial membrane e.g. by regulating gene transcription needs to be elucidated in further studies.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Tectorial/metabolismo , Trifosfato de Adenosina/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Sinalização do Cálcio/efeitos dos fármacos , Feminino , Masculino , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Morfogênese , Membrana Tectorial/citologia , Membrana Tectorial/efeitos dos fármacos , Membrana Tectorial/crescimento & desenvolvimento , Fatores de Tempo , Uridina Trifosfato/farmacologiaRESUMO
Voltage-gated Ca2+ channels are composed of a pore-forming α1 subunit and auxiliary ß and α2δ subunits, which modulate Ca2+ current properties and channel trafficking. So far, the partial redundancy and specificity of α1 for α2δ subunits in the CNS have remained largely elusive. Mature spiral ganglion (SG) neurons express α2δ subunit isoforms 1, 2, and 3 and multiple Ca2+ channel subtypes. Differentiation and in vivo functions of their endbulb of Held synapses, which rely on presynaptic P/Q channels (Lin et al., 2011), require the α2δ3 subunit (Pirone et al., 2014). This led us to hypothesize that P/Q channels may preferentially co-assemble with α2δ3. Using a dissociated primary culture, we analyzed the effects of α2δ3 deletion on somatic Ca2+ currents (ICa ) of SG neurons isolated at postnatal day 20 (P20), when the cochlea is regarded to be mature. P/Q currents were the dominating steady-state Ca2+ currents (54% of total) followed by T-type, L-type, N-type, and R-type currents. Deletion of α2δ3 reduced P/Q- and R-type currents by 60 and 38%, respectively, whereas L-type, N-type, and T-type currents were not altered. A subset of ICa types was also analyzed in SG neurons isolated at P5, i.e., before the onset of hearing (P12). Both L-type and N-type current amplitudes of wildtype SG neurons were larger at P5 compared with P20. Deletion of α2δ3 reduced L-type and N-type currents by 23 and 44%, respectively. In contrast, small P/Q currents, which were just being up-regulated at P5, were unaffected by the lack of α2δ3. In summary, α2δ3 regulates amplitudes of L- and N-type currents of immature cultured SG neurons, whereas it regulates P/Q- and R-type currents at P20. Our data indicate a developmental switch from dominating somatic N- to P/Q-type currents in cultured SG neurons. A switch from N- to P/Q-type channels, which has been observed at several central synapses, may also occur at developing endbulbs of Held. In this case, reduction of both neonatal N- (P5) and more mature P/Q-type currents (around/after hearing onset) may contribute to the impaired morphology and function of endbulb synapses in α2δ3-deficient mice.
RESUMO
The Mongolian gerbil (Meriones unguiculatus) is one of the animal models in auditory research that has been used in several studies on age-related hearing loss. The standard laboratory strain is domesticated as it was bred in captivity for more than 70 years. We compared properties of distortion product otoacoustic emissions (DPOAEs) in domesticated gerbils with wild-type gerbils from F6-F7 generations of a strain originating from animals trapped in Central Asia in 1995. Up to an age of 9months, DPOAE thresholds were comparable between both strains and were below 10dB SPL for f2 frequencies between 4 and 44kHz. In older domesticated animals, the thresholds were increased by up to 12dB. Significant increases were found at stimulus frequencies of 2kHz, 12-20kHz, and 56-60kHz. The best frequency ratio f2/f1 to evoke maximum DPOAE amplitude was larger in domesticated animals at the age of 9 months or older. While these data show that there is a deterioration of cochlear sensitivity due to domestication, the magnitude of the described changes is small. Thus, the general suitability of domesticated gerbils for auditory research seems not to be affected.
Assuntos
Envelhecimento , Cóclea/fisiopatologia , Perda Auditiva Neurossensorial/fisiopatologia , Emissões Otoacústicas Espontâneas , Estimulação Acústica , Fatores Etários , Animais , Animais Domésticos , Animais Selvagens , Limiar Auditivo , Fenômenos Biomecânicos , Feminino , Gerbillinae , MasculinoRESUMO
Before the onset of hearing, which occurs around postnatal day 12 (P12) in mice, inner hair cells (IHCs) of the immature cochlea generate sound-independent Ca2+ action potentials (APs), which stimulate the auditory pathway and guide maturation of neuronal circuits. During these early postnatal days, intercellular propagating Ca2+ waves elicited by ATP-induced ATP release are found in inner supporting cells (ISCs). It is debated whether IHCs are able to fire Ca2+ APs independently or require a trigger by an ISC Ca2+ wave. To identify the Ca2+ transients of IHCs underlying Ca2+ APs and to analyze their dependence on ISC Ca2+ waves, we performed fast Ca2+ imaging of Fluo-8 AM-loaded organs of Corti at P4/P5. Fast Ca2+ transients (fCaTs) generated by IHCs were simultaneously imaged with Ca2+ waves in ISCs. ISC Ca2+ waves frequently evoked bursts consisting of >5 fCaTs in multiple adjacent IHCs. Although Ca2+ elevations of small amplitude appeared to be triggered by ISC Ca2+ waves in IHCs of Cav1.3 knockout mice we never observed fCaTs, indicating their requirement for Ca2+ influx through Cav1.3 channels. The Ca2+ wave-triggered Ca2+ upstroke in wildtype IHCs occurred 0.52 ± 0.27 s later than the rise of the Ca2+ signal in the adjacent ISCs. In comparison, superfusion of 1 µM ATP elicited bursts of fCaTs in IHCs starting 0.99 ± 0.34 s prior to Ca2+ elevations in adjacent ISCs. PPADS irreversibly abolished Ca2+ waves in ISCs and reversibly reduced fCaTs in IHCs indicating differential involvement of P2 receptors. IHC and ISC Ca2+ signals were however unaltered in P2X2R/P2X3R double knockout or in P2X7R knockout mice. Together, our data revealed a fairly similar occurrence of fCaTs within a burst (56.5%) compared with 43.5% as isolated single fCaTs or in groups of 2-5 fCaTs (minibursts). We provide evidence that IHCs autonomously generate single fCaTs and minibursts whereas bursts synchronized between neighboring IHCs were mostly triggered by ISC Ca2+ waves. Neonatal IHCs thus spontaneously generate electrical and Ca2+ activity, which is enhanced and largely synchronized by activity of ISCs of Kölliker's organ indicating two sources of spontaneous activity in the developing auditory system.
RESUMO
Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca(2+) action potentials before the onset of hearing. Although this firing activity is mainly sustained by a depolarizing L-type (Ca(V)1.3) Ca(2+) current (I(Ca)), IHCs also transiently express a large Na(+) current (I(Na)). We aimed to investigate the specific contribution of I(Na) to the action potentials, the nature of the channels carrying the current and whether the biophysical properties of I(Na) differ between low- and high-frequency IHCs. We show that I(Na) is highly temperature-dependent and activates at around -60 mV, close to the action potential threshold. Its size was larger in apical than in basal IHCs and between 5% and 20% should be available at around the resting membrane potential (-55 mV/-60 mV). However, in vivo the availability of I(Na) could potentially increase to >60% during inhibitory postsynaptic potential activity, which transiently hyperpolarize IHCs down to as far as -70 mV. When IHCs were held at -60 mV and I(Na) elicited using a simulated action potential as a voltage command, we found that I(Na) contributed to the subthreshold depolarization and upstroke of an action potential. We also found that I(Na) is likely to be carried by the TTX-sensitive channel subunits Na(V)1.1 and Na(V)1.6 in both apical and basal IHCs. The results provide insight into how the biophysical properties of I(Na) in mammalian cochlear IHCs could contribute to the spontaneous physiological activity during cochlear maturation in vivo.
Assuntos
Cóclea/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Ativação do Canal Iônico , Canais de Sódio/fisiologia , Potenciais de Ação , Animais , Imuno-Histoquímica , RatosRESUMO
Spontaneous action potential activity is crucial for mammalian sensory system development. In the auditory system, patterned firing activity has been observed in immature spiral ganglion and brain-stem neurons and is likely to depend on cochlear inner hair cell (IHC) action potentials. It remains uncertain whether spiking activity is intrinsic to developing IHCs and whether it shows patterning. We found that action potentials were intrinsically generated by immature IHCs of altricial rodents and that apical IHCs showed bursting activity as opposed to more sustained firing in basal cells. We show that the efferent neurotransmitter acetylcholine fine-tunes the IHC's resting membrane potential (V(m)), and as such is crucial for the bursting pattern in apical cells. Endogenous extracellular ATP also contributes to the V(m) of apical and basal IHCs by triggering small-conductance Ca(2+)-activated K(+) (SK2) channels. We propose that the difference in firing pattern along the cochlea instructs the tonotopic differentiation of IHCs and auditory pathway.