Spiking Pattern of the Mouse Developing Inner Hair Cells Is Mostly Invariant Along the Tonotopic Axis

During development, the sensory cells of the cochlea, the inner hair cells (IHCs), fire spontaneous calcium action potentials. This activity at the pre-hearing stage allows the IHCs to autonomously excite the auditory nerve fibers and hence, represents an efficient mechanism to shape the tonotopic organization along the ascending auditory pathway. Using calcium imaging, we show that the activity in the developing cochlea consists of calcium waves that propagate across the supporting and sensory cells. Both basal and apical IHCs were characterized by similar spontaneous calcium transients interspaced with silent periods, consistent with bursts of action potentials recorded in patch-clamp. In addition, adjacent auditory hair cells tend to have a synchronized [Ca2+]i activity, irrespective of their location along the base-to-apex gradient of the cochlea. Finally, we show that the mechanical ablation of the inner phalangeal cells (IPCs), a class of supporting cells, reduces the synchronized [Ca2+]i activity between neighboring sensory cells. These findings support the hypothesis that the tonotopic map refinement in higher auditory centers would depend on the synchronization of a discrete number of auditory sensory cells

Empreinte développementale des cellules sensorielles auditives

During development, the sensory cells of the cochlea, the inner hair cells (IHCs), fire spontaneous calcium action potentials. This spontaneous spiking activity at the pre-hearing stage allows the IHCs to automatically stimulate the auditory nerve fibers and hence, ensures the proper shaping of the tonotopic organization along the ascending auditory pathway. Spontaneous spiking patterns may depend on the IHCs position on the cochlea (the tonotopic axis). Those patterns may also rely on ATP secretion from neighboring supporting cells. In this study, we used calcium imaging in the immature neuro-sensory epithelium of the cochlea, the Kölliker´s organ, to gain insights in the IHCs spiking activity. After loading the Kölliker´s organ with the calcium dye fura-2 AM, propagation of spontaneous calcium waves was readily observed across supporting and sensory cells. Both basal and apical IHCs were characterized by similar spontaneous calcium transients interspaced with silent periods, reminiscent of bursts of action potential recorded in patch-clamp. In addition, neighboring cells show a strong degree of synchronous activity. Incubation with apyrase, which hydrolyzes ATP, prevents the spontaneous calcium increase that propagates across the supporting cells within the Kölliker's organ. However, it leaves the spontaneous calcium transients in IHCs mostly unaffected. All these results show that the tonotopic map refinement in higher auditory centers comes from a coordinated activity of neighboring sensory cells, whose activity seems to be independent of ATPLes cellules ciliées internes (CCI) sont les cellules sensorielles de l'organe de l'audition, elles transforment les ondes sonores en messages nerveux. Avant l’entrée en fonction de la cochlée, les CCI émettent spontanément des potentiels d’action (PA) calciques, ce qui active la voie auditive ascendante et assure le développement de l’axe tonotopique, à savoir la représentation du codage en fréquence, dans chaque relais de la voie auditive. Le profil et les mécanismes à l’origine des PA des CCI sont fortement débattus. Nous nous sommes donc attachés à étudier l’empreinte développementale des cellules sensorielles, c'est à dire déterminer le profil et les mécanismes à l’origine de leur activité.Après avoir incubé l’épithélium neuro-sensoriel avec la sonde calcique Fura2-AM, nous avons observé des vagues calciques se propageant le long des cellules de soutien et des cellules sensorielles. Plus précisément, l’activité des cellules ciliées se caractérisait par des élévations transitoires de calcium (pics calciques) à intervalles de temps réguliers. Nous avons ensuite démontré que les pics calciques des CCI correspondaient bien à des bouffées de PA en mesurant simultanément les oscillations calciques et l’émission de PA en patch-clamp. La fréquence, la durée et la distribution temporelle des pics calciques des CCI étaient en grande partie invariantes le long de l’axe base-apex de la cochlée. Enfin, les cellules voisines montraient une activité fortement synchrone à l’inverse des cellules spatialement éloignées. Ces résultats indiquent donc que l’activité des CCI est majoritairement identique le long de l’axe tonotopique de la cochlée.Nous nous sommes ensuite intéressés au mécanisme responsable de l’activité spontanée, la dépendance à l’ATP. L’incubation d’apyrase, une ecto-nucléotidase, entraine une diminution de l’activité des cellules de soutien, à savoir une réduction de l’aire et de la vitesse de propagation des vagues calciques. En revanche, l'activité des CCI n'est pas altérée par la déplétion d’ATP. Ces résultats suggèrent 2 mécanismes distincts, le premier ATP-dépendant et le second ATP-indépendant dans les cellules de soutien et sensorielles, respectivement.L’ensemble de ces résultats indique que la maturation des centres supérieurs serait déterminée par l’activation synchrone d’un nombre limité de cellules sensorielles

Developmental imprint of auditory sensory cells

Les cellules ciliées internes (CCI) sont les cellules sensorielles de l'organe de l'audition, elles transforment les ondes sonores en messages nerveux. Avant l’entrée en fonction de la cochlée, les CCI émettent spontanément des potentiels d’action (PA) calciques, ce qui active la voie auditive ascendante et assure le développement de l’axe tonotopique, à savoir la représentation du codage en fréquence, dans chaque relais de la voie auditive. Le profil et les mécanismes à l’origine des PA des CCI sont fortement débattus. Nous nous sommes donc attachés à étudier l’empreinte développementale des cellules sensorielles, c'est à dire déterminer le profil et les mécanismes à l’origine de leur activité.Après avoir incubé l’épithélium neuro-sensoriel avec la sonde calcique Fura2-AM, nous avons observé des vagues calciques se propageant le long des cellules de soutien et des cellules sensorielles. Plus précisément, l’activité des cellules ciliées se caractérisait par des élévations transitoires de calcium (pics calciques) à intervalles de temps réguliers. Nous avons ensuite démontré que les pics calciques des CCI correspondaient bien à des bouffées de PA en mesurant simultanément les oscillations calciques et l’émission de PA en patch-clamp. La fréquence, la durée et la distribution temporelle des pics calciques des CCI étaient en grande partie invariantes le long de l’axe base-apex de la cochlée. Enfin, les cellules voisines montraient une activité fortement synchrone à l’inverse des cellules spatialement éloignées. Ces résultats indiquent donc que l’activité des CCI est majoritairement identique le long de l’axe tonotopique de la cochlée.Nous nous sommes ensuite intéressés au mécanisme responsable de l’activité spontanée, la dépendance à l’ATP. L’incubation d’apyrase, une ecto-nucléotidase, entraine une diminution de l’activité des cellules de soutien, à savoir une réduction de l’aire et de la vitesse de propagation des vagues calciques. En revanche, l'activité des CCI n'est pas altérée par la déplétion d’ATP. Ces résultats suggèrent 2 mécanismes distincts, le premier ATP-dépendant et le second ATP-indépendant dans les cellules de soutien et sensorielles, respectivement.L’ensemble de ces résultats indique que la maturation des centres supérieurs serait déterminée par l’activation synchrone d’un nombre limité de cellules sensorielles.During development, the sensory cells of the cochlea, the inner hair cells (IHCs), fire spontaneous calcium action potentials. This spontaneous spiking activity at the pre-hearing stage allows the IHCs to automatically stimulate the auditory nerve fibers and hence, ensures the proper shaping of the tonotopic organization along the ascending auditory pathway. Spontaneous spiking patterns may depend on the IHCs position on the cochlea (the tonotopic axis). Those patterns may also rely on ATP secretion from neighboring supporting cells. In this study, we used calcium imaging in the immature neuro-sensory epithelium of the cochlea, the Kölliker´s organ, to gain insights in the IHCs spiking activity. After loading the Kölliker´s organ with the calcium dye fura-2 AM, propagation of spontaneous calcium waves was readily observed across supporting and sensory cells. Both basal and apical IHCs were characterized by similar spontaneous calcium transients interspaced with silent periods, reminiscent of bursts of action potential recorded in patch-clamp. In addition, neighboring cells show a strong degree of synchronous activity. Incubation with apyrase, which hydrolyzes ATP, prevents the spontaneous calcium increase that propagates across the supporting cells within the Kölliker's organ. However, it leaves the spontaneous calcium transients in IHCs mostly unaffected. All these results show that the tonotopic map refinement in higher auditory centers comes from a coordinated activity of neighboring sensory cells, whose activity seems to be independent of AT

Identification of cellular, functional and genetic causes of Hoxb1null sensorineural hearing loss

The assembly of central auditory subcircuits depends on the spatially and temporally ordered sequence of specification, migration and connectivity. Alterations of one of these events will lead to abnormal circuit formation and different types of deafness. Recessive mutations in the human homeobox HOXB1 gene cause sensorineural hearing impairments characterized by increased auditory threshold and loss of distortion product otoacoustic emissions, mainly due to abnormal cochlear activity of outer hair cells (OHCs) and absence of sound amplification Our studies in mouse show that Hoxb1 is essential for the specification of rhombomere 4 (r4)-derived auditory sensory and motor neurons contributing to the sound transmission pathway and to the establishment of sensorimotor reflex circuits. Hoxb1null mice have abnormal r4-derived sensory nuclei, lack inner ear efferent motor neurons and show degeneration of OHCs. This leads to defective cochlear amplification and altered auditory thresholds that well replicate the hearing loss observed in patients with mutations in the HOXB1 gene. However, the exact mechanisms leading to the auditory impairments described in mice and patients are not known. Hoxb1 is expressed in the medial olivocochlear (MOC) neurons that innervate the OHCs, but not in cochlear hair cells. In Hoxb1null mice, MOC neurons fail to be generated, but morphology of OHCs is not affected until P8, when MOC/OHC interactions normally occur; however, OHCs start to degenerate later when these interactions fail to be established, with consequent appearance of threshold impairments. Interesting, the persistence of few MOC neurons in Hoxb1flox b1r4-Cre mutants, in which Hoxb1 is inactivated only in r4, correlates well with less severe OHC morphology and threshold defects. To directly assess whether the degeneration of OHCs and the increased hearing threshold observed in Hoxb1null mutants depends on the absence of MOC innervation, or alternatively is due to affected sensory r4-derived components, we independently altered Hoxb1 function in r4 motor or sensory neurons. The OC efferent bundles were genetically deleted by using Hoxb1flox Nkx2.2-Cre mutants, in which Hoxb1 is exclusively inactivated in r4 motor neurons, whereas the sensory glutamatergic and GABAergic/glycinergic cochlear populations were affected in Hoxb1flox Atoh1-Cre and Hoxb1flox Ptf1a-Cre mice. By combining anatomical, electrophysiological and molecular analyses, we demonstrated a key role for MOC neurons on OHC survival and sound amplification as the major cause for the Hoxb1 phenotype. Overall, these data show that during a critical postnatal period interactions between MOC neurons and OHCs are crucial for establishing the correct sound amplification

Abnormal outer hair cell efferent innervation in Hoxb1-dependent sensorineural hearing loss.

Autosomal recessive mutation of HOXB1 and Hoxb1 causes sensorineural hearing loss in patients and mice, respectively, characterized by the presence of higher auditory thresholds; however, the origin of the defects along the auditory pathway is still unknown. In this study, we assessed whether the abnormal auditory threshold and malformation of the sensory auditory cells, the outer hair cells, described in Hoxb1null mutants depend on the absence of efferent motor innervation, or alternatively, is due to altered sensory auditory components. By using a whole series of conditional mutant mice, which inactivate Hoxb1 in either rhombomere 4-derived sensory cochlear neurons or efferent motor neurons, we found that the hearing phenotype is mainly reproduced when efferent motor neurons are specifically affected. Our data strongly suggest that the interactions between olivocochlear motor neurons and outer hair cells during a critical postnatal period are crucial for both hair cell survival and the establishment of the cochlear amplification of sound

Altered number of MOC/OHC interactions in mutant cochleae with high auditory thresholds.

The percentage of the presence (A) of MOC/OHC interactions was quantified relative to the control group (CKONkx2.2low vs Ctrl: 88.7 ± 7.7%; P = 0.5; CKOr4low vs Ctrl: 78.6± 8.6%; P = 0.2; CKONkx2.2high vs Ctrl: 0.7 ± 0.1%; PCKOr4high vs Ctrl: 0 ± 0%; PB) of MOC/OHC interactions was quantified relative to the Null group (CKONkx2.2low vs Null: 13.0 ± 6.8%; PCKOr4low vs Null: 21.5 ± 7.6%; PCKONkx2.2high vs Null: 100 ± 6.8%; P>0.9; CKOr4high vs Null: 100 ± 6.8%; P>0.9). Data were statistically analyzed with One-Way ANOVA followed by the Bonferroni test and results are shown as mean ± SEM.**** PS1 Data. (TIF)</p

Normal Auditory Brainstem Response in <i>DV-Cre</i> and <i>r4-Cre recombinase</i> lines.

Audiograms from r4-Cre, Nkx2.2-Cre, Atoh1-Cre, Ptf1a-Cre mice. Auditory thresholds in Cre recombinase lines are comparable to Ctrl mice. Mean threshold ± SEM: 33.9 ± 0.8 dB SPL, 38.3 ± 1.4 dB SPL, 35 ± 2.5 dB SPL, 34.6 ± 0.8 dB SPL, 32.9 ± 2.3 dB SPL in Ctrl, r4-Cre, Nkx2.2-Cre, Atoh1-Cre, Ptf1a-Cre mice respectively. P = 0.27, one-way analysis of variance. See also S1 Data. (TIF)</p

Altered medial olivocochlear neuron (MOC) innervation and outer hair cell (OHC) morphology in r4-motor conditional <i>Hoxb1</i> mutants.

Transmission electron microscopy (TEM) of MOC innervating OHC (A, C, E, G, I, K) and scanning electron microscopy (SEM) of OHCs (B, D, F, H, J, L) in the cochleae of the genotypes indicated to the left. MOC innervation and normal OHC morphology were observed in control (Ctrl) and conditional mice with low auditory thresholds (A, B, E, F, I, J). In mutant mice with altered high thresholds (C, D, G, H, K, L), no MOC efferent on OHCs and severe malformations of OHC arrangement and disarray of stereocilia were observed. Scale bars 1μm (A, C, E, G, I, K), 10μm (B, D, F, H, J, L). See also S2 Fig and S1 Data.</p

Numerical raw data related to Fig 1C–1F, Fig 2C–2H, Fig 5A–5E, Fig 6A–6G, S1 Fig, S2 Fig.

Numerical raw data related to Fig 1C–1F, Fig 2C–2H, Fig 5A–5E, Fig 6A–6G, S1 Fig, S2 Fig.</p

Conditional <i>Hoxb1</i> inactivation in r4-derived motor and sensory neurons and recorded audiograms.

(A) Schematics of half of rhombomere 4 (r4) in the developing hindbrain showing DV domain of the activity of the different Cre-recombinase lines used in this study. While the Nkx2.2-Cre line is expressed in ventral r4 from which MOC, LOC, and FMN efferent motor neurons originated, the Atoh1- and Ptf1-Cre lines are expressed in distinct dorsal domains from which the sensory glutamatergic or GABAergic/glycinergic populations, respectively, of the cochlear nucleus (CN) are generated. (B) The genetic strategy used to inactivate Hoxb1 in different domains. In the null group, Hoxb1 is inactivated from the onset of expression; in the conditional CKOr4 mice, Hoxb1 expression is exclusively abolished in r4 from E8.5 onwards. DV-restricted inactivation is obtained by using the CKOAtoh1 and CKOPtf1a mice for CN sensory components or the CKONkx2.2 mice for the motor efferent structures. (C-H) Audiograms from distinct mouse groups are indicated above each graph. Empty circles: mean ABR ± SEM, dashed lines: individual cochleae. n indicates the number of cochleae recorded. See also S1 Data.</p