Using Interteaching to Promote Online Learning Outcomes.

Due to the COVID-19 pandemic, educators have been forced to rapidly transition away from in-person learning environments to completely online formats. Many of these educators have had little or no training and experience teaching online, contributing to stress and anxiety. To compound this problem even further, there are a multitude of online learning technologies from which to choose that can be relatively costly and require an intensive production process. In an effort to provide immediate relief to those dealing with this problem, we detail how interteaching, an empirically supported behavioral teaching technique, can be used to cultivate an interactive online learning environment in either an asynchronous or synchronous format. Specifically, we describe some best practices and provide some examples on how to generate active student responding (ASR) as well as provide pinpointed performance-based feedback. We specifically reference the relatively easy-to-use online software Kaltura, but it is hoped that our suggestions inspire others to develop and use these strategies across a variety of platforms in effort to provide evidence-based quality education during this crisis.

Spray-Dried Nanoparticle-in-Microparticle Delivery Systems (NiMDS) for Gene Delivery, Comprising Polyethylenimine (PEI)-Based Nanoparticles in a Poly(Vinyl Alcohol) Matrix.

Nucleic acid-based therapies rely on efficient formulations for nucleic acid protection and delivery. As nonviral strategies, polymeric and lipid-based nanoparticles have been introduced; however, biological efficacy and biocompatibility as well as poor storage properties due to colloidal instability and their unavailability as ready-to-use systems are still major issues. Polyethylenimine is the most widely explored and promising candidate for gene delivery. Polyethylenimine-based polyplexes and their combination with liposomes, lipopolyplexes, are efficient for DNA or siRNA delivery in vitro and in vivo. In this study, a highly potent spray-dried nanoparticle-in-microparticle delivery system is presented for the encapsulation of polyethylenimine-based polyplexes and lipopolyplexes into poly(vinyl alcohol) microparticles, without requiring additional stabilizing agents. This easy-to-handle gene delivery device allows prolonged nanoparticle storage and protection at ambient temperature. Biological analyses reveal further advantages regarding profoundly reduced cytotoxicity and enhanced transfection efficacies of polyethylenimine-based nanoparticles from the nanoparticle-in-microparticle delivery system over their freshly prepared counterparts, as determined in various cell lines. Importantly, this nanoparticle-in-microparticle delivery system is demonstrated as ready-to-use dry powder to be an efficient device for the inhalative delivery of polyethylenimine-based lipopolyplexes in vivo, as shown by transgene expression in mice after only one administration.

Mononuclear-cell-derived microparticles attenuate endothelial inflammation by transfer of miR-142-3p in a CD39 dependent manner.

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p < 0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p < 0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p < 0.05) and endothelial apoptosis (p < 0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication-with implications in organ regeneration.

Allergen-Induced IL-6 Regulates IL-9/IL-17A Balance in CD4+ T Cells in Allergic Airway Inflammation.

IL-9-secreting Th9 cells have been considered to play a pivotal role in the pathogenesis of atopic diseases. To what extent IL-9-producing cells are induced or regulated by sensitization with naturally occurring allergens is not yet clear. Naturally occurring allergens are capable of inducing IL-6 production in dendritic cells (DCs). Whether allergen-induced IL-6 supports a Th9 subtype by increasing IL-9 production, as observed in in vitro studies, or rather favors Th17 differentiation is not finally resolved. Therefore, in the present study we have investigated the impact of IL-6 on the Th9/Th17 balance depending on the predominant cytokine milieu and, additionally, in vivo using a DC-driven murine asthma model. In vitro, IL-6 increases Th9 cells under strong IL-4 and TGF-ß activation, whereas under moderate IL-4 and TGF-ß activation the presence of IL-6 shifts naive CD4(+) cells to Th17 cells. To induce allergic airway inflammation, OVA-pulsed DCs from IL-6-deficient or wild-type donors were adoptively transferred into BALB/c mice. Recipients receiving IL-6-producing wild-type DCs showed a significant decrease of Th9- and IL-4-producing Th2 cells but an increase of Th17 cells in lung tissue in comparison with recipients sensitized with IL-6-deficient DCs. Our data suggest that the IL-6-mediated reduction of Th2-related IL-4 leads to a decline of the Th9 immune response and allows Th17 differentiation.

Restoration of auditory evoked responses by human ES-cell-derived otic progenitors.

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells, is responsible for a substantial proportion of patients with hearing impairment. Although the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss, as poor innervation limits the prospective performance of an implant. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair-cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory-evoked response thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

Correlation of cell-free DNA plasma concentration with severity of non-alcoholic fatty liver disease.

BACKGROUND: The assessment of fibrosis and inflammatory activity is essential to identify patients with non-alcoholic fatty liver disease (NAFLD) at risk for progressive disease. Serum markers and ultrasound-based methods can replace liver biopsy for fibrosis staging, whereas non-invasive characterization of inflammatory activity remains a clinical challenge. Cell-free DNA (cfDNA) is a novel non-invasive biomarker for assessing cellular inflammation and cell death, which has not been evaluated in NAFLD. METHODS: Patients and healthy controls from two previous studies were included. NAFLD disease activity and severity were non-invasively characterized by liver stiffness measurement (transient elastography, TE) including steatosis assessment with controlled attenuation parameter (CAP), single-proton magnetic resonance spectroscopy (1H-MRS) for determination of hepatic fat fraction, aminotransferases and serum ferritin. cfDNA levels (90 and 222 bp fragments) were analyzed using quantitative real-time PCR. RESULTS: Fifty-eight NAFLD patients (age 62 ± 11 years, BMI 28.2 ± 3.5 kg/m2) and 13 healthy controls (age 38 ± 12 years, BMI 22.4 ± 2.1 kg/m2) were included. 90 bp cfDNA levels were significantly higher in NAFLD patients compared to healthy controls: 3.7 (1.3-23.1) vs. 2.9 (1.4-4.1) ng/mL (p = 0.014). In the NAFLD cohort, circulating cfDNA correlated significantly with disease activity and severity, especially in patients with elevated liver stiffness (n = 13, 22%) compared to cases with TE values ≤7 kPa: cf90 bp 6.05 (2.41-23.13) vs. 3.16 (1.29-7.31) ng/mL (p < 0.001), and cf222 bp 14.41 (9.27-22.90) vs. 11.32 (6.05-18.28) ng/mL (p = 0.0041). CONCLUSIONS: Cell-free DNA plasma concentration correlates with established non-invasive markers of NAFLD activity and severity. Therefore, cfDNA should be further evaluated as biomarker for identifying patients at risk for progressive NAFLD.

Syndecan-1 regulates dendritic cell migration in cutaneous hypersensitivity to haptens.

In human dendritic cells (DCs), we previously demonstrated in vitro that syndecan-1 (SDC1) is downregulated during maturation correlating with enhanced motility. We investigated the effects of SDC1 on DC migration in vivo during TNCB(2,4,6-trinitro-1-chlorobenzene)-induced cutaneous hypersensitivity reaction (CHS) in mice. We show that DC in SDC1-deficient mice migrated faster and at a higher rate to lymph nodes draining the hapten-painted skin. Adoptive transfer of SDC1-deficient hapten- and fluorochrome-labelled DC into wild-type (WT) mice led to increased and faster migration of DC to paracortical lymph nodes, and to a stronger CHS compared to WT DC. In SDC1-/- mice, CCR7 remains longer on the DC surface within the first 15-minutes maturation (after LPS-induced maturation). In addition, a time-dependent upregulation of CCL2, CCL3, VCAM1 and talin was found during maturation in SDC1-/- DC. However, no difference in T-cell-stimulating capacity of SDC1-deficient DC was found compared to WT DC. Mechanistically, SDC1-deficient DC showed enhanced migration towards CCL21 and CCL19. This may result from functional overexpression of CCR7 in SDC1-/- DC. Increased and accelerated migration of otherwise functionally intact SDC1-deficient DC leads to an exacerbated CHS. Based on our results, we conclude that SDC1 on DC negatively regulates DC migration.

Spatial Surveillance of Childhood Lead Exposure in a Targeted Screening State: An Application of Generalized Additive Models in Denver, Colorado.

CONTEXT: The targeted nature of Colorado's childhood lead screening program presents several analytical issues that complicate routine epidemiologic surveillance. OBJECTIVES: To analyze spatial patterns of childhood lead exposure among children younger than 6 years, identifying areas of increased risk along with associated covariates. METHODS: We analyzed a spatial case-control data set of childhood lead poisoning using generalized additive models. Incident cases were represented by the residential locations of children younger than 6 years with confirmed elevated blood lead levels (EBLL) of 5 µg/dL or more recorded between calendar years 2010 and 2014, and controls were sampled from the population at risk. We modeled the effect of spatial location, adjusting for potential spatial confounders. We also adjusted for a number of covariates previously identified in the childhood lead poisoning literature to understand the ecologic-level drivers of spatial variation in risk. MAIN OUTCOME MEASURE(S): Crude and adjusted spatial odds ratios describing the relative frequency of lead poisoning among different locations in Denver, Colorado. RESULTS: We found evidence of statistically significant spatial clustering in incident cases of lead poisoning even after adjustment for age, sex, year, season, and spatially smoothed screening rate. Spatial confounder-adjusted odds ratios in the Denver study area ranged from 0.22 to 2.7. Adjusting for additional ecologic-level covariates effectively accounted for the observed spatial variation. We found that ecologic-level indicators of low socioeconomic status, Hispanic ethnicity, Asian race, and older housing age were all positively and significantly associated with an increased EBLL risk. CONCLUSION: Housing and socioeconomic factors continue to be the primary ecologic risk factors associated with childhood lead exposure and can be used to predict risk at a fine spatial resolution in the Denver study area. Our analysis demonstrates how other targeted screening states can be proactive about childhood lead surveillance within their major population centers and enhance the spatial specificity of lead mitigation efforts.

Prospective study of pregnancy and newborn outcomes in mothers with West nile illness during pregnancy.

BACKGROUND: A previous case report of West Nile virus (WNV) illness during pregnancy suggested that WNV could be a cause of congenital defects. We performed a prospective, longitudinal cohort study of pregnant women with WNV illness to increase our knowledge of the effects of WNV illness during pregnancy. METHODS: Participants were enrolled in 2005 to 2008 from pregnant women with serologically confirmed WNV illness reported to the Centers for Disease Control and Prevention. Comparison was made to WNV-uninfected women, matched on maternal age and enrollment month. Pregnancy and newborn data were collected; cord blood WNV serology was obtained. Pediatric exams and the Bayley Scales of Infant and Toddler Development-Third Edition (Bayley-III) were performed. RESULTS: Twenty-eight WNV-infected mothers and 25 WNV-uninfected mothers participated. Maternal demographics were similar except for a higher rate of planned pregnancies, education, and household income in the WNV-uninfected mothers. There were no differences in pregnancy and delivery characteristics except that infected mothers had a higher incidence of febrile illnesses and used more medications. Birth weight, length, head circumference, and rate of congenital malformations were similar in babies born to WNV-infected and -uninfected mothers. Follow-up physical exams were generally normal. The Bayley-III assessments, available for 17 children born to mothers with WNV illness, showed performance at or above age level across domains. CONCLUSION: The risk for adverse pregnancy and newborn outcomes in women experiencing WNV illness in pregnancy appears to be low, but future studies with larger numbers are needed to rule out a small risk. Birth Defects Research (Part A) 106:716-723, 2016. © 2016 Wiley Periodicals, Inc.

Presynaptic maturation in auditory hair cells requires a critical period of sensory-independent spiking activity.

The development of neural circuits relies on spontaneous electrical activity that occurs during immature stages of development. In the developing mammalian auditory system, spontaneous calcium action potentials are generated by inner hair cells (IHCs), which form the primary sensory synapse. It remains unknown whether this electrical activity is required for the functional maturation of the auditory system. We found that sensory-independent electrical activity controls synaptic maturation in IHCs. We used a mouse model in which the potassium channel SK2 is normally overexpressed, but can be modulated in vivo using doxycycline. SK2 overexpression affected the frequency and duration of spontaneous action potentials, which prevented the development of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their morphology or general cell development. By manipulating the in vivo expression of SK2 channels, we identified the "critical period" during which spiking activity influences IHC synaptic maturation. Here we provide direct evidence that IHC development depends upon a specific temporal pattern of calcium spikes before sound-driven neuronal activity.

Private well water in Colorado: collaboration, data use, and public health outreach.

As a result of participating in the Centers for Disease Control and Prevention's Private Well Initiative and Environmental Public Health Tracking Network (Tracking), the Colorado Department of Public Health and Environment was able to inventory private well water quality data, prioritize potential health concerns associated with drinking water from these wells, and create a Web portal for sharing public health information regarding private well water. The Colorado Department of Public Health and Environment collaborated with a local health department to pilot the project prior to a public implementation. Approximately 18 data sets were identified and inventoried. The Colorado Department of Public Health and Environment also participated in development and pilot testing of best practices for display of well water quality data with other Tracking states. Available data sets were compiled and summarized, and the data made available on the Colorado Tracking portal using geographic information system technology to support public health outreach regarding private wells.

Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells.

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.

HGF and SDF-1-mediated mobilization of CD133+ BMSC for hepatic regeneration following extensive liver resection.

BACKGROUND: The molecular mechanisms of haematopoietic stem cells (HSC) mobilization and homing to the liver after partial hepatectomy (PH) remain largely unexplored. METHODS: Functional liver volume loss and regain was determined by computerized tomography (CT) volumetry in 30 patients following PH. Peripheral HSC mobilization was investigated by fluorescence-activated cell sorting (FACS) analyses and cytokine enzyme-linked immunosorbent assay assays. Migration of purified HSC towards hepatic growth factor (HGF) and stroma-derived factor-1 (SDF-1) gradients was tested in vitro. Mice after 70% PH were examined for HSC mobilization by FACS and cytokine mRNA expression in the liver. FACS-sorted HSC were administered after PH and hepatocyte proliferation was evaluated by immunohistochemical staining for Ki67. RESULTS: Impaired liver function was noted after extended hepatic resection when compared to smaller resections. Patients with large liver resections were characterized by significantly higher levels of peripheral HSC which were positively correlated with the extent of resected liver volume and its regain after 3 weeks. Increased plasma levels of HGF, SDF-1 and insulin like growth factor (IGF-1) were evident within the first 6 hours post resection. Migration assays of human HSC in vitro showed a specific target-demonstrated migration towards recombinant HGF and SDF-1 gradients in a concentration and specific receptor (c-Met and CXCR4) dependent manner. The evaluation of peripheral human alpha foetoprotein expression demonstrated pronounced stemness following increased CD133(+) HSC in the course of liver regeneration following PH. Our human data were further validated in a murine model of PH and furthermore demonstrated increased hepatocyte proliferation subsequent to CD133(+) HSC treatment. CONCLUSION: HGF and SDF-1 are required for effective HSC mobilization and homing to the liver after hepatic resection. These findings have significant implications for potential therapeutic strategies targeting chemotactant modulation and stem cell mobilization for liver protection and regeneration.

miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells.

MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system.

Lack of brain-derived neurotrophic factor hampers inner hair cell synapse physiology, but protects against noise-induced hearing loss.

The precision of sound information transmitted to the brain depends on the transfer characteristics of the inner hair cell (IHC) ribbon synapse and its multiple contacting auditory fibers. We found that brain derived neurotrophic factor (BDNF) differentially influences IHC characteristics in the intact and injured cochlea. Using conditional knock-out mice (BDNF(Pax2) KO) we found that resting membrane potentials, membrane capacitance and resting linear leak conductance of adult BDNF(Pax2) KO IHCs showed a normal maturation. Likewise, in BDNF(Pax2) KO membrane capacitance (ΔC(m)) as a function of inward calcium current (I(Ca)) follows the linear relationship typical for normal adult IHCs. In contrast the maximal ΔC(m), but not the maximal size of the calcium current, was significantly reduced by 45% in basal but not in apical cochlear turns in BDNF(Pax2) KO IHCs. Maximal ΔC(m) correlated with a loss of IHC ribbons in these cochlear turns and a reduced activity of the auditory nerve (auditory brainstem response wave I). Remarkably, a noise-induced loss of IHC ribbons, followed by reduced activity of the auditory nerve and reduced centrally generated wave II and III observed in control mice, was prevented in equally noise-exposed BDNF(Pax2) KO mice. Data suggest that BDNF expressed in the cochlea is essential for maintenance of adult IHC transmitter release sites and that BDNF upholds opposing afferents in high-frequency turns and scales them down following noise exposure.

Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell.

Otoferlin has been proposed to be the Ca(2+) sensor in hair cell exocytosis, compensating for the classical synaptic fusion proteins synaptotagmin-1 and synaptotagmin-2. In the present study, yeast two-hybrid assays reveal myosin VI as a novel otoferlin binding partner. Co-immunoprecipitation assay and co-expression suggest an interaction of both proteins within the basolateral part of inner hair cells (IHCs). Comparison of otoferlin mutants and myosin VI mutant mice indicates non-complementary and complementary roles of myosin VI and otoferlin for synaptic maturation: (i) IHCs from otoferlin mutant mice exhibited a decoupling of CtBP2/RIBEYE and Ca(V)1.3 and severe reduction of exocytosis. (ii) Myosin VI mutant IHCs failed to transport BK channels to the membrane of the apical cell regions, and the exocytotic Ca(2+) efficiency did not mature. (iii) Otoferlin and myosin VI mutant IHCs showed a reduced basolateral synaptic surface area and altered active zone topography. Membrane infoldings in otoferlin mutant IHCs indicated disturbed transport of endocytotic membranes and link the above morphological changes to a complementary role of otoferlin and myosin VI in transport of intracellular compartments to the basolateral IHC membrane.

Deafness in TRbeta mutants is caused by malformation of the tectorial membrane.

Thyroid hormone receptor beta (TRbeta) dysfunction leads to deafness in humans and mice. Deafness in TRbeta(-/-) mutant mice has been attributed to TRbeta-mediated control of voltage- and Ca(2+)-activated K(+) (BK) channel expression in inner hair cells (IHCs). However, normal hearing in young constitutive BKalpha(-/-) mutants contradicts this hypothesis. Here, we show that mice with hair cell-specific deletion of TRbeta after postnatal day 11 (P11) have a delay in BKalpha expression but normal hearing, indicating that the origin of hearing loss in TRbeta(-/-) mutant mice manifested before P11. Analyzing the phenotype of IHCs in constitutive TRbeta(-/-) mice, we found normal Ca(2+) current amplitudes, exocytosis, and shape of compound action potential waveforms. In contrast, reduced distortion product otoacoustic emissions and cochlear microphonics associated with an abnormal structure of the tectorial membrane and enhanced tectorin levels suggest that disturbed mechanical performance is the primary cause of deafness resulting from TRbeta deficiency.

ECT for self-injury in an autistic boy.

OBJECTIVE: Self-injurious behavior presents a significant challenge in autism, and first-line psychopharmacological and behavioral interventions have limited efficacy in some patients. These intractable cases may be responsive to electroconvulsive therapy. CLINICAL PICTURE: This article presents an eight-year-old boy with autism, mental retardation, prominent mood lability and a five-year history of extreme self-injurious behavior towards his head, averaging 109 self-injurious attempts hourly. The patient was at high risk for serious head trauma, and required usage of bilateral arm restraints and protective equipment (i.e., padding on shoulders, arms, and legs). All areas of daily functioning were profoundly impacted by dangerous self-injury. TREATMENT: Fifteen bilateral ECT treatments resulted in excellent mood stabilization and reduction of self-injury to 19 attempts hourly, and maintenance ECT was pursued. The patient was able to return to developmentally-appropriate educational and social activities. CONCLUSION: ECT should be considered in the treatment algorithm of refractory cases of severe self-injury in autism.

Persistence of Ca(v)1.3 Ca2+ channels in mature outer hair cells supports outer hair cell afferent signaling.

Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca2+ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca2+ channel currents (charge carrier, 10 mM Ba2+) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from approximately 170 to approximately 50 pA at approximately the onset of hearing. Ba2+ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba2+ current amplitude of apical OHCs was 58 +/- 9 pA (n = 20, P19-P30) compared with that of the inner hair cells (IHCs) of 181 +/- 50 pA (n = 24, P17-P30). Properties of Ba2+ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by +9 mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Ca(v)1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Ca(v)1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Ca(v)1.3 and CtBP2/RIBEYE protein strongly suggests a role for Ca(v)1.3 channels in exocytosis of mature OHCs.

Thyroid hormone deficiency affects postnatal spiking activity and expression of Ca2+ and K+ channels in rodent inner hair cells.

Thyroid hormone (TH) is essential for the development of hearing. Lack of TH in a critical developmental period from embryonic day 17 to postnatal day 12 (P12) in rats and mice leads to morphological and functional deficits in the organ of Corti and the auditory pathway. We investigated the effects of TH on inner hair cells (IHCs) using patch-clamp recordings, capacitance measurements, and immunocytochemistry in hypothyroid rats and athyroid Pax8-/- mice. Spontaneous and evoked Ca2+ action potentials (APs) were present in control IHCs from P3-P11 rats and vanished in parallel with the expression of a rapidly activating Ca2+- and voltage-activated K+ (BK) conductance. IHCs of hypothyroid rats and athyroid Pax8-/- mice displayed APs until the end of the third postnatal week because of threefold elevated Ca2+ currents and missing expression of BK currents. After the fourth postnatal week, some IHCs showed BK currents whereas adjacent IHCs did not, demonstrated by electrophysiology and immunocytochemistry. To test whether the prolonged spiking activity during TH deficiency may be transmitted at IHC synapses, capacitance measurements were performed in parallel to analysis of otoferlin expression, a protein thought to play an essential role in exocytosis of IHCs. Strikingly, otoferlin was absent from IHCs of hypothyroid rats but not of Pax8-/- mice, although both cell types showed exocytosis with an efficiency typical for immature IHCs. These results demonstrate for the first time a TH-dependent control of IHC spiking activity before the onset of hearing attributable to effects of TH on Ca2+ and BK channels. Moreover, they question an indispensable role of otoferlin for exocytosis in IHCs.