A5 noradrenergic neurons and breathing control in neonate rats.

The pontine A5 noradrenergic group contributes to the maturation of the respiratory system before birth in rats. These neurons are connected to the neural network responsible for respiratory rhythmogenesis. In the present study, we investigated the participation of A5 noradrenergic neurons in neonates (P7-8 and P14-15) in the control of ventilation during hypoxia and hypercapnia in in vivo experiments using conjugated saporin anti-dopamine beta-hydroxylase (DßH-SAP) to specifically ablate noradrenergic neurons. Thus, DßH-SAP (420 ng/µL) or saporin (SAP, control) was injected into the A5 region of neonatal male Wistar rats. Hypoxia reduced respiratory variability in control animals; however, A5 lesion prevented this effect in P7-8 rats. Our data suggest that noradrenergic neurons of the A5 region in neonate rats do not participate in the control of ventilation under baseline and hypercapnic conditions, but exert an inhibitory modulation on breathing variability under hypoxic challenge in early life (P7-8).

Prenatal exposure to nicotine in mice is associated with alterations in development and cellular and synaptic effects of alcohol in a brainstem arousal nucleus.

Using drugs of abuse while pregnant has tremendous negative consequences for the offspring, including an enhanced risk for substance use disorder (SUD). This vulnerability suggests that gestational exposure to drugs alters the developmental trajectory of neurons important in SUD processes, which could lead to later life changes in responsiveness to motivationally salient stimuli. The laterodorsal tegmentum (LDT) gates the behaviorally relevant firing pattern signaling stimuli saliency in mesoaccumbal circuits. Accordingly, any alterations in LDT functionality could alter output, and play a role in negative outcomes on motivated behavior associated with early-life nicotine exposure. Therefore, we investigated whether prenatal exposure to nicotine (PNE), which is a known teratogen, altered responsiveness of LDT neurons to alcohol by conducting electrophysiology in brain slices. Alcohol induced an outward current in control LDT cells, which was not seen in PNE LDT neurons. The frequency of mEPSCs was significantly decreased by alcohol in LDT PNE cells and accompanied by a decrease in action potential frequency, which were actions not seen in controls. Changes in baseline activity of PNE LDT cells were also observed. In summary, PNE LDT neurons showed alterations in baseline activity and membrane and synaptic responses to postnatal exposures to alcohol. The differences in PNE baseline activity and alcohol responses likely lead to differential output from the LDT to mesoaccumbal targets that could play a role in biasing coding of relevant stimuli, which could participate in the enhanced proclivity for development of SUD in those exposed during gestation to nicotine.

Impact of Prematurity on the Tissue Properties of the Neonatal Brain Stem: A Quantitative MR Approach.

BACKGROUND AND PURPOSE: Preterm birth interferes with regular brain development. The aim of this study was to investigate the impact of prematurity on the physical tissue properties of the neonatal brain stem using a quantitative MR imaging approach. MATERIALS AND METHODS: A total of 55 neonates (extremely preterm [n = 30]: <28 + 0 weeks gestational age; preterm [n = 10]: 28 + 0-36 + 6 weeks gestational age; term [n = 15]: ≥37 + 0 weeks gestational age) were included in this retrospective study. In most cases, imaging was performed at approximately term-equivalent age using a standard MR protocol. MR data postprocessing software SyMRI was used to perform multidynamic multiecho sequence (acquisition time: 5 minutes, 24 seconds)-based MR postprocessing to determine T1 relaxation time, T2 relaxation time, and proton density. Mixed-model ANCOVA (covariate: gestational age at MR imaging) and the post hoc Bonferroni test were used to compare the groups. RESULTS: There were significant differences between premature and term infants for T1 relaxation time (midbrain: P < .001; pons: P < .001; basis pontis: P = .005; tegmentum pontis: P < .001; medulla oblongata: P < .001), T2 relaxation time (midbrain: P < .001; tegmentum pontis: P < .001), and proton density (tegmentum pontis: P = .004). The post hoc Bonferroni test revealed that T1 relaxation time/T2 relaxation time in the midbrain differed significantly between extremely preterm and preterm (T1 relaxation time: P < .001/T2 relaxation time: P = .02), extremely preterm and term (T1 relaxation time/T2 relaxation time: P < .001), and preterm and term infants (T1 relaxation time: P < .001/T2 relaxation time: P = .006). CONCLUSIONS: Quantitative MR parameters allow preterm and term neonates to be differentiated. T1 and T2 relaxation time metrics of the midbrain allow differentiation between the different stages of prematurity. SyMRI allows for a quantitative assessment of incomplete brain maturation by providing tissue-specific properties while not exceeding a clinically acceptable imaging time.

Normal human brainstem development in vivo: a quantitative fetal MRI study.

OBJECTIVES: To characterize spatiotemporal growth differences of prenatal brainstem substructures and cerebellum, using linear biometry and planimetry on fetal magnetic resonance imaging (MRI). METHODS: In this retrospective study, we included fetuses with normal brain and a precise midsagittal T2-weighted brain MRI sequence obtained between May 2003 and April 2019. The cross-sectional area, rostrocaudal diameter and anteroposterior diameter of the midbrain, pons (basis pontis and pontine tegmentum), medulla oblongata and cerebellar vermis, as well as the transverse cerebellar diameter, were quantified by a single observer. The diameters were also assessed by a second observer to test inter-rater variability. RESULTS: We included 161 fetuses with normal brain and a precise midsagittal MRI sequence, examined at a mean ± SD gestational age of 25.7 ± 5.4 (range, 14 + 0 to 39 + 2) weeks. All substructures of the fetal brainstem and the cerebellum could be measured consistently (mean ± SD interobserver intraclass correlation coefficient, 0.933 ± 0.065). We provide reference data for diameters and areas of the brainstem and cerebellum in the second and third trimesters. There was a significant quadratic relationship between vermian area and gestational age, and all other measured parameters showed a significant linear growth pattern within the observed period (P < 0.001). A significant change in the relative proportions of the brainstem substructures occurred between the beginning of the second trimester and the end of the third trimester, with an increase in the area of the pons (P < 0.001) and a decrease in that of the midbrain (P < 0.001), relative to the total brainstem area. CONCLUSIONS: The substructures of the fetal brainstem follow a distinct spatiotemporal growth pattern, characterized by a relative increase in the pons and decrease in the midbrain, between 15 and 40 weeks of gestation. Caution is needed when interpreting fetal brainstem appearance during the early second trimester, as the brainstem proportions differ significantly from the adult morphology. The reference data provided herein should help to increase diagnostic accuracy in detecting disorders of defective hindbrain segmentation. © 2020 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Auditory Brainstem Evoked Response Patterns in the Neonatal Intensive Care Unit.

OBJECTIVE: Delayed maturation of auditory brainstem pathway in neonates admitted to the neonatal intensive care unit (NICU) may lead to misdiagnosis of children with normal peripheral hearing and inappropriate use of amplification devices. The aim of this study is to determine the pattern of auditory brain stem response in neonates admitted to the NICU for proper hearing assessment in this high-risk population. STUDY DESIGN: This prospective study was conducted on 1,469 infants who were admitted to the NICU, of which 1,423 had one or more risk factors for permanent congenital hearing loss and were screened with automated auditory brain stem response (AABR). A total of 60 infants were referred for diagnostic ABR analysis after failure on AABR screening. The control group comprised 60 well-baby nursery neonates with no risk factors for PCHL. RESULTS: Mean values of absolute latencies of waves III and V; interpeak latencies I-III, III-V, and I-V; amplitude of waves I, and V; and I/V amplitude ratio at 90 dBnHL measured for the right and left ears at 1 and 3 months of age show significant difference in NICU neonates compared with controls (p < 0.05). All the diagnostic ABR measurements significantly improved at the age of 3 months (p < 0.001) except wave I absolute latency of both groups (p > 0.05). Significant correlations were found between ABR readings at the age of 1 and 3 months and the gestational age of the NICU neonates (p < 0.05). CONCLUSION: Diagnostic ABR findings in NICU neonates suggested delayed maturation of the auditory brainstem pathway with a great impact of gestational age on this maturation. Auditory maturational changes were observed at 3 months of age of patient and control groups.

Nitric oxide controls excitatory/inhibitory balance in the hypoglossal nucleus during early postnatal development.

Synaptic remodeling during early postnatal development lies behind neuronal networks refinement and nervous system maturation. In particular, the respiratory system is immature at birth and is subjected to significant postnatal development. In this context, the excitatory/inhibitory balance dramatically changes in the respiratory-related hypoglossal nucleus (HN) during the 3 perinatal weeks. Since, development abnormalities of hypoglossal motor neurons (HMNs) are associated with sudden infant death syndrome and obstructive sleep apnea, deciphering molecular partners behind synaptic remodeling in the HN is of basic and clinical relevance. Interestingly, a transient expression of the neuronal isoform of nitric oxide (NO) synthase (NOS) occurs in HMNs at neonatal stage that disappears before postnatal day 21 (P21). NO, in turn, is a determining factor for synaptic refinement in several physiopathological conditions. Here, intracerebroventricular chronic administration (P7-P21) of the broad spectrum NOS inhibitor L-NAME (N(ω)-nitro-L-arginine methyl ester) differentially affected excitatory and inhibitory rearrangement during this neonatal interval in the rat. Whilst L-NAME led to a reduction in the number of excitatory structures, inhibitory synaptic puncta were increased at P21 in comparison to administration of the inactive stereoisomer D-NAME. Finally, L-NAME decreased levels of the phosphorylated form of myosin light chain in the nucleus, which is known to regulate the actomyosin contraction apparatus. These outcomes indicate that physiologically synthesized NO modulates excitatory/inhibitory balance during early postnatal development by acting as an anti-synaptotrophic and/or synaptotoxic factor for inhibitory synapses, and as a synaptotrophin for excitatory ones. The mechanism of action could rely on the modulation of the actomyosin contraction apparatus.

Brainstem development requires galactosylceramidase and is critical for pathogenesis in a model of Krabbe disease.

Krabbe disease (KD) is caused by a deficiency of galactosylceramidase (GALC), which induces demyelination and neurodegeneration due to accumulation of cytotoxic psychosine. Hematopoietic stem cell transplantation (HSCT) improves clinical outcomes in KD patients only if delivered pre-symptomatically. Here, we hypothesize that the restricted temporal efficacy of HSCT reflects a requirement for GALC in early brain development. Using a novel Galc floxed allele, we induce ubiquitous GALC ablation (Galc-iKO) at various postnatal timepoints and identify a critical period of vulnerability to GALC ablation between P4-6 in mice. Early Galc-iKO induction causes a worse KD phenotype, higher psychosine levels in the rodent brainstem and spinal cord, and a significantly shorter life-span of the mice. Intriguingly, GALC expression peaks during this critical developmental period in mice. Further analysis of this mouse model reveals a cell autonomous role for GALC in the development and maturation of immature T-box-brain-1 positive brainstem neurons. These data identify a perinatal developmental period, in which neuronal GALC expression influences brainstem development that is critical for KD pathogenesis.

DARS2 is indispensable for Purkinje cell survival and protects against cerebellar ataxia.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation disorder (LBSL) arises from mutations in mitochondrial aspartyl-tRNA synthetase (DARS2) gene. The disease has a childhood or juvenile-onset and is clinically characterized by cerebellar ataxia, cognitive decline and distinct morphological abnormalities upon magnetic resonance imaging. We previously demonstrated that neurons and not adult myelin-producing cells are specifically sensitive to DARS2 loss, hence likely the primary culprit in LBSL disorder. We used conditional Purkinje cell (PCs)-specific Dars2 deletion to elucidate further the cell-type-specific contribution of this class of neurons to the cerebellar impairment observed in LBSL. We show that DARS2 depletion causes a severe mitochondrial dysfunction concomitant with a massive loss of PCs by the age of 15 weeks, thereby rapidly deteriorating motor skills. Our findings conclusively show that DARS2 is indispensable for PC survival and highlights the central role of neuroinflammation in DARS2-related PC degeneration.

Mechanical Ventilation Duration, Brainstem Development, and Neurodevelopment in Children Born Preterm: A Prospective Cohort Study.

OBJECTIVES: To determine, in children born preterm, the association of mechanical ventilation duration with brainstem development, white matter maturation, and neurodevelopmental outcomes at preschool age. STUDY DESIGN: This prospective cohort study included 144 neonates born at <30 weeks of gestation (75 male, mean gestational age 27.1 weeks, SD 1.6) with regional brainstem volumes automatically segmented on magnetic resonance imaging at term-equivalent age (TEA). The white matter maturation was assessed by diffusion tensor imaging and tract-based spatial statistics. Neurodevelopmental outcomes were assessed at 4.5 years of age using the Movement Assessment Battery for Children, 2nd Edition, and the Wechsler Primary and Preschool Scale of Intelligence, 4th Edition, full-scale IQ. The association between the duration of mechanical ventilation and brainstem development was validated in an independent cohort of children born very preterm. RESULTS: Each additional day of mechanical ventilation predicted lower motor scores (0.5-point decrease in the Movement Assessment Battery for Children, 2nd Edition, score by day of mechanical ventilation, 95% CI -0.6 to -0.3, P < .0001). Prolonged exposure to mechanical ventilation was associated with smaller pons and medulla volumes at TEA in 2 independent cohorts, along with widespread abnormalities in white matter maturation. Pons and medulla volumes at TEA predicted motor outcomes at 4.5 years of age. CONCLUSIONS: In neonates born very preterm, prolonged mechanical ventilation is associated with impaired brainstem development, abnormal white matter maturation, and lower motor scores at preschool age. Further research is needed to better understand the neural pathological mechanisms involved.

Altered local cerebellar and brainstem development in preterm infants.

BACKGROUND: Premature birth is associated with high prevalence of neurodevelopmental impairments in surviving infants. The putative role of cerebellar and brainstem dysfunction remains poorly understood, particularly in the absence of overt structural injury. METHOD: We compared in-utero versus ex-utero global, regional and local cerebellar and brainstem development in healthy fetuses (n â€‹= â€‹38) and prematurely born infants without evidence of structural brain injury on conventional MRI studies (n â€‹= â€‹74) that were performed at two time points: the first corresponding to the third trimester, either in utero or ex utero in the early postnatal period following preterm birth (30-40 weeks of gestation; 38 control fetuses; 52 premature infants) and the second at term equivalent age (37-46 weeks; 38 control infants; 58 premature infants). We compared 1) volumetric growth of 7 regions in the cerebellum (left and right hemispheres, left and right dentate nuclei, and the anterior, neo, and posterior vermis); 2) volumetric growth of 3 brainstem regions (midbrain, pons, and medulla); and 3) shape development in the cerebellum and brainstem using spherical harmonic description between the two groups. RESULTS: Both premature and control groups showed regional cerebellar differences in growth rates, with the left and right cerebellar hemispheres showing faster growth compared to the vermis. In the brainstem, the pons grew faster than the midbrain and medulla in both prematurely born infants and controls. Using shape analyses, premature infants had smaller left and right cerebellar hemispheres but larger regional vermis and paravermis compared to in-utero control fetuses. For the brainstem, premature infants showed impaired growth of the superior surface of the midbrain, anterior surface of the pons, and inferior aspects of the medulla compared to the control fetuses. At term-equivalent age, premature infants had smaller cerebellar hemispheres bilaterally, extending to the superior aspect of the left cerebellar hemisphere, and larger anterior vermis and posteroinferior cerebellar lobes than healthy newborns. For the brainstem, large differences between premature infants and healthy newborns were found in the anterior surface of the pons. CONCLUSION: This study analyzed both volumetric growth and shape development of the cerebellum and brainstem in premature infants compared to healthy fetuses using longitudinal MRI measurements. The findings in the present study suggested that preterm birth may alter global, regional and local development of the cerebellum and brainstem even in the absence of structural brain injury evident on conventional MRI.

Post-natal development of the envelope following response to amplitude modulated sounds in the bat Phyllostomus discolor.

Bats use a large repertoire of calls for social communication, which are often characterized by temporal amplitude and frequency modulations. As bats are considered to be among the few mammalian species capable of vocal learning, the perception of temporal sound modulations should be crucial for juvenile bats to develop social communication abilities. However, the post-natal development of auditory processing of temporal modulations has not been investigated in bats, so far. Here we use the minimally invasive technique of recording auditory brainstem responses to measure the envelope following response (EFR) to sinusoidally amplitude modulated noise (range of modulation frequencies: 11-130 Hz) in three juveniles (p8-p72) of the bat, Phyllostomus discolor. In two out of three animals, we show that although amplitude modulation processing is basically developed at p8, EFRs maturated further over a period of about two weeks until p33. Maturation of the EFR generally took longer for higher modulation frequencies (87-130 Hz) than for lower modulation frequencies (11-58 Hz).

Effects of inflammation on the developing respiratory system: Focus on hypoglossal (XII) neuron morphology, brainstem neurochemistry, and control of breathing.

Breathing is fundamental to life and any adverse change in respiratory function can endanger the health of an organism or even be fatal. Perinatal inflammation is known to adversely affect breathing in preterm babies, but lung infection/inflammation impacts all stages of life from birth to death. Little is known about the role of inflammation in respiratory control, neuronal morphology, or neural function during development. Animal models of inflammation can provide understanding and insight into respiratory development and how inflammatory processes alter developmental phenotype in addition to providing insight into new treatment modalities. In this review, we focus on recent work concerning the development of neurons, models of perinatal inflammation with an emphasis on two common LPS-based models, inflammation and its impact on development, and current and potential treatments for inflammation within the respiratory control circuitry of the mammalian brainstem. We have also discussed models of inflammation in adults and have specifically focused on hypoglossal motoneurons (XII) and neurons of the nucleus tractus solitarii (nTS) as these nuclei have been studied more extensively than other brainstem nuclei participating in breathing and airway control. Understanding the impact of inflammation on the developmental aspects of respiratory control and breathing pattern is critical to addressing problems of cardiorespiratory dysregulation in disease and this overview points out many gaps in our current knowledge.

An age- and sex-dependent role of catecholaminergic neurons in the control of breathing and hypoxic chemoreflex during postnatal development.

The respiratory system undergoes significant development during the postnatal phase. Maturation of brainstem catecholaminergic (CA) neurons is important for the control and modulation of respiratory rhythmogenesis, as well as for chemoreception in early life. We demonstrated an inhibitory role for CA neurons in CO2 chemosensitivity in neonatal and juvenile male and female rats, but information regarding their role in the hypoxic ventilatory response (HVR) is lacking. We evaluated the contribution of brainstem CA neurons in the HVR during postnatal (P) development (P7-8, P14-15 and P20-21) in male and female rats through chemical injury with conjugated saporin anti-dopamine beta-hydroxylase (DßH-SAP, 420 ng·µL-1) injected in the fourth ventricle. Ventilation (V̇E) and oxygen consumption were recorded one week after the lesion in unanesthetized rats during exposure to normoxia and hypoxia. Hypoxia reduced breathing variability in P7-8 control rats of both sexes. At P7-8, the HVR for lesioned males and females increased 27% and 24%, respectively. Additionally, the lesion reduced the normoxic breathing variability in both sexes at P7-8, but hypoxia partially reverted this effect. For P14-15, the increase in V̇E during hypoxia was 30% higher for male and 24% higher for female lesioned animals. A sex-specific difference was detected at P20-21, as lesioned males exhibited a 24% decrease in the HVR, while lesioned females experienced a 22% increase. Furthermore, the hypoxia-induced body temperature reduction was attenuated in P20-21 lesioned females. We conclude that brainstem CA neurons modulate the HRV during the postnatal phase, and possibly thermoregulation during hypoxia.

Presynaptic Mitochondria Volume and Abundance Increase during Development of a High-Fidelity Synapse.

The calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem undergoes developmental changes to support the high action-potential firing rates required for auditory information encoding. In addition, calyx terminals are morphologically diverse, which impacts vesicle release properties and synaptic plasticity. Mitochondria influence synaptic plasticity through calcium buffering and are crucial for providing the energy required for synaptic transmission. Therefore, it has been postulated that mitochondrial levels increase during development and contribute to the morphological-functional diversity in the mature calyx. However, the developmental profile of mitochondrial volumes and subsynaptic distribution at the calyx of Held remains unclear. To provide insight on this, we developed a helper-dependent adenoviral vector that expresses the genetically encoded peroxidase marker for mitochondria, mito-APEX2, at the mouse calyx of Held. We developed protocols to detect labeled mitochondria for use with serial block face scanning electron microscopy to carry out semiautomated segmentation of mitochondria, high-throughput whole-terminal reconstruction, and presynaptic ultrastructure in mice of either sex. Subsequently, we measured mitochondrial volumes and subsynaptic distributions at the immature postnatal day (P)7 and the mature (P21) calyx. We found an increase of mitochondria volumes in terminals and axons from P7 to P21 but did not observe differences between stalk and swelling subcompartments in the mature calyx. Based on these findings, we propose that mitochondrial volumes and synaptic localization developmentally increase to support high firing rates required in the initial stages of auditory information processing.SIGNIFICANCE STATEMENT Elucidating the developmental processes of auditory brainstem presynaptic terminals is critical to understanding auditory information encoding. Additionally, morphological-functional diversity at these terminals is proposed to enhance coding capacity. Mitochondria provide energy for synaptic transmission and can buffer calcium, impacting synaptic plasticity; however, their developmental profile to ultimately support the energetic demands of synapses following the onset of hearing remains unknown. Therefore, we created a helper-dependent adenoviral vector with the mitochondria-targeting peroxidase mito-APEX2 and expressed it at the mouse calyx of Held. Volumetric reconstructions of serial block face electron microscopy data of immature and mature labeled calyces reveal that mitochondrial volumes are increased to support high firing rates upon maturity.

Sustained envelope periodicity representations are associated with speech-in-noise performance in difficult listening conditions for younger and older adults.

Temporal modulations are an important part of speech signals. An accurate perception of these time-varying qualities of sound is necessary for successful communication. The current study investigates the relationship between sustained envelope encoding and speech-in-noise perception in a cohort of normal-hearing younger (ages 18-30 yr, n = 22) and older adults (ages 55-90+ yr, n = 35) using the subcortical auditory steady-state response (ASSR). ASSRs were measured in response to the envelope of 400-ms amplitude-modulated (AM) tones with 3,000-Hz carrier frequencies and 80-Hz modulation frequencies. AM tones had modulation depths of 0, -4, and -8 dB relative to m = 1 (m = 1, 0.631, and 0.398, respectively). The robustness, strength at modulation frequency, and synchrony of subcortical envelope encoding were quantified via time-domain correlations, spectral amplitude, and phase-locking value, respectively. Speech-in-noise ability was quantified via the QuickSIN test in the 0- and 5-dB signal-to-noise (SNR) conditions. All ASSR metrics increased with increasing modulation depth and there were no effects of age group. ASSR metrics in response to shallow modulation depths predicted 0-dB speech scores. Results demonstrate that sustained amplitude envelope processing in the brainstem relates to speech-in-noise abilities, but primarily in difficult listening conditions at low SNRs. These findings furthermore highlight the utility of shallow modulation depths for studying temporal processing. The absence of age effects in these data demonstrate that individual differences in the robustness, strength, and specificity of subcortical envelope processing, and not age, predict speech-in-noise performance in the most difficult listening conditions.NEW & NOTEWORTHY Failure to correctly understand speech in the presence of background noise is a significant problem for many normal-hearing adults and may impede healthy communication. The relationship between sustained envelope encoding in the brainstem and speech-in-noise perception remains to be clarified. The present study demonstrates that the strength, specificity, and robustness of the brainstem's representations of sustained stimulus periodicity relates to speech-in-noise perception in older and younger normal-hearing adults, but only in highly challenging listening environments.

Developmental expression patterns of erythropoietin and its receptor in mouse brainstem respiratory regions.

Erythropoietin (EPO) is a hypoxia-inducible hormone, classically known to enhance red blood cell production upon binding its receptor (EPOR) present on the surface of the erythroid progenitor cells. EPO and its receptor are also expressed in the central nervous system (CNS), exerting several non-hematopoietic actions. EPO also plays an important role in the control of breathing. In this review, we summarize the known physiological actions of EPO in the neural control of ventilation during postnatal development and at adulthood in rodents under normoxic and hypoxic conditions. Furthermore, we present the developmental expression patterns of EPO and EPORs in the brainstem, and with the use of in situ hybridization (ISH) and immunofluorescence techniques we provide original data showing that EPOR is abundantly present in specific brainstem nuclei associated with central chemosensitivity and control of ventilation in the ventrolateral medulla, mainly on somatostatin negative cells. Thus, we conclude that EPO signaling may act through glutamatergic neuron populations that are the primary source of rhythmic inspiratory excitatory drive. This work underlies the importance of EPO signaling in the central control of ventilation across development and adulthood and provides new insights on the expression of EPOR at the cellular level.

Newborn hearing screening at the Neonatal Intensive Care Unit and Auditory Brainstem Maturation in preterm infants.

OBJECTIVES: Aim of this study is to report and discuss the results of 4 years of Newborn hearing screening (NHS) program at the Neonatal Intensive Care Unit (NICU), particularly evaluating the clinical ABR results. METHODS: Retrospective study. NHS data from NICU newborns, admitted for ≥5 days, in the period from January 1st, 2013 and December 31st, 2016, were retrieved and analyzed. NHS results were classified as following: (i) "pass" when both ears for both the a-TEOAE (automated Transient-Evoked Otoacoustic Emissions) and the a-ABR (automated Auditory Brainstem Response) protocol resulted as "pass"; (ii) "fail" when one ear, at either one of the two performed tests resulted as "fail"; (iii) "missing" when the newborns were not tested with both protocols. All "fail" and "missing" newborns were retested (with both tests): in the case of a second "fail" result, a clinical ABR was performed within a period of 3 months. RESULTS: A total of 1191 newborns were screened. From those, 1044/1191 resulted as "pass", 108/1191 as "fail", and 39/1191 as "missing". During the re-testing of these 147 newborns, 43 were assigned as "missing", 63 were assigned as "pass" (showing bilaterally a wave V identifiable within 30 dB nHL) and 25 failed the retest and/or did not present an identifiable wave V within 30 dB nHL. Among the 147 retested infants, we identified a group of 16 subjects who resulted as NHS "refer" and who, during the audiological follow-up, showed either: (i) a unilateral or bilateral wave V identifiable over 30 dB nHL, at the first clinical ABR assessment; or (ii) a bilateral wave V identifiable within 30 dB nHL, in a following clinical ABR test during the first year of life. These 16 subjects were defined to have an 'Auditory Brainstem Maturation' issue. CONCLUSIONS: A possible "maturation" of the ABR response (and therefore of the auditory pathway) has been hypothesised in 16 out of 1191 infants (1.3%). A delay of the auditory pathway maturation in preterm babies compared to term newborns has already been suggested in the literature. A possible delay of the NHS retest could be considered, in selected cases, with significant savings in economic resources and parental anxiety.

NADH-linked mitochondrial respiration in the developing mouse brain is sex-, age- and tissue-dependent.

Mitochondria play a major role in the brain. Apart from energy production, mitochondria regulate key factors in the activation of cell signaling pathways such as survival, proliferation, and differentiation. While all these processes occur during the physiological development of the brain, it is surprising that the mitochondrial functions and functioning in the brain during the postnatal development remain poorly explored. In this work, we collected samples of brainstem and cortex of mice at postnatal ages 3 (P3), 21 (P21), and at adulthood (3 months old) and evaluated the mitochondrial oxygen consumption after complex I activation. To do so, we used our oxygraph-2 K system (OROBOROS) that measures the mitochondrial bioenergetics in saponin-permeabilized tissue punches of 2  mg weight. Furthermore, as sex dimorphism in the brain occurs since very early stages of development, we performed experiments in brain samples of male and female mice. Accordingly, the mitochondrial oxygen consumption rate (OCR) was evaluated under activation of complex I (NADH-linked respiration - mitochondrial state 3), and during the inhibition of the complex V (ATP synthase) with oligomycin (mitochondrial state 4). In following, the respiratory control ratio (RCR - state 3/state4) was calculated as an index of mitochondrial oxidative-phosphorylation coupling. Our results show that the activity of the mitochondrial complex I in the brain increases along with the postnatal development in a sex- and tissue-dependent manner, with males showing higher activity than females, and with brainstem tissue showing higher activity than cortex. Our data may contribute to a better understanding of the sex-dependent maturation of the cortex and the cardiorespiratory network located in the brainstem.

Role of BMP Signaling for the Formation of Auditory Brainstem Nuclei and Large Auditory Relay Synapses.

Large excitatory synapses are found at specific points in the neuronal circuits of the auditory brainstem, to enable fast information transfer and the preservation of acoustic timing information. The extracellular cues and signaling mechanisms that lead to the development of these specialized synaptic connections, exemplified by the calyx of Held in the medial nucleus of the trapezoid body (MNTB), are still largely unknown. Here, we investigate the role of BMP signaling for the early development of the ventral cochlear nucleus (VCN) and MNTB, and for the initial formation of the calyx of Held synaptic connection. We used conditional alleles of two BMP type-1 receptors in the background of a constitutive BMPR1b knock-out (KO), or else a conditional allele of SMAD4. The conditional alleles were recombined by the Krox20Cre mouse line that is active around mid-gestation in rhombomeres (r) 3 and 5 from which the VCN and MNTB are derived; alternatively, virus-mediated Cre-expression was performed early postnatally in the VCN. The data show that embryonic SMAD-dependent BMP-signaling in r3 and r5 contributes to the histogenesis of auditory brainstem nuclei. On the other hand, BMP-receptor signaling early postnatally in presynaptic neurons of the calyx of Held projection is necessary for correct axon branch retraction, which suggests a cell-autonomous role of presynaptic BMP-receptors in synapse elimination at the developing calyx of Held. Thus, our work dissects developmentally early and late roles of BMP-signaling for the formation of auditory brainstem nuclei, and the highly specialized synaptic connectivity in these structures.