Chronic maternal exposure to titanium dioxide nanoparticles alters breathing in newborn offspring.

BACKGROUND: Over the last two decades, nanotechnologies and the use of nanoparticles represent one of the greatest technological advances in many fields of human activity. Particles of titanium dioxide (TiO2) are one of the nanomaterials most frequently found in everyday consumer products. But, due in particular to their extremely small size, TiO2 nanoparticles (NPs) are prone to cross biological barriers and potentially lead to adverse health effects. The presence of TiO2 NPs found in human placentae and in the infant meconium has indicated unequivocally the capacity for a materno-fetal transfer of this nanomaterial. Although chronic exposure to TiO2 NPs during pregnancy is known to induce offspring cognitive deficits associated with neurotoxicity, the impact of a gestational exposure on a vital motor function such as respiration, whose functional emergence occurs during fetal development, remains unknown. RESULTS: Using in vivo whole-body plethysmographic recordings from neonatal mice, we show that a chronic exposure to TiO2 NPs during pregnancy alters the respiratory activity of offspring, characterized by an abnormally elevated rate of breathing. Correspondingly, using ex vivo electrophysiological recordings performed on isolated brainstem-spinal cord preparations of newborn mice and medullary slice preparations containing specific nuclei controlling breathing frequency, we show that the spontaneously generated respiratory-related rhythm is significantly and abnormally accelerated in animals prenatally exposed to TiO2 NPs. Moreover, such a chronic prenatal exposure was found to impair the capacity of respiratory neural circuitry to effectively adjust breathing rates in response to excitatory environmental stimuli such as an increase in ambient temperature. CONCLUSIONS: Our findings thus demonstrate that a maternal exposure to TiO2 NPs during pregnancy affects the normal development and operation of the respiratory centers in progeny.

Obstructive Apneas in a Mouse Model of Congenital Central Hypoventilation Syndrome.

Rationale: Congenital central hypoventilation syndrome (CCHS) is characterized by life-threatening sleep hypoventilation and is caused by PHOX2B gene mutations, most frequently the PHOX2B27Ala/+ mutation, with patients requiring lifelong ventilatory support. It is unclear whether obstructive apneas are part of the syndrome. Objectives: To determine if Phox2b27Ala/+ mice, which present the main symptoms of CCHS and die within hours after birth, also express obstructive apneas, and to investigate potential underlying mechanisms. Methods: Apneas were classified as central, obstructive, or mixed by using a novel system combining pneumotachography and laser detection of abdominal movement immediately after birth. Several respiratory nuclei involved in airway patency were examined by immunohistochemistry and electrophysiology in brainstem-spinal cord preparations. Measurements and Main Results: The median (interquartile range) of obstructive apnea frequency was 2.3 (1.5-3.3)/min in Phox2b27Ala/+ pups versus 0.6 (0.4-1.0)/min in wild types (P < 0.0001). Obstructive apnea duration was 2.7 seconds (2.3-3.9) in Phox2b27Ala/+ pups versus 1.7 seconds (1.1-1.9) in wild types (P < 0.0001). Central and mixed apneas presented similar significant differences. In Phox2b27Ala/+ preparations, the hypoglossal nucleus had fewer (P < 0.05) and smaller (P < 0.01) neurons, compared with wild-type preparations. Importantly, coordination of phrenic and hypoglossal motor activities was disrupted, as evidenced by the longer and variable delay of hypoglossal activity with respect to phrenic activity onset (P < 0.001). Conclusions: The Phox2b27Ala/+ mutation predisposed pups not only to hypoventilation and central apneas, but also to obstructive and mixed apneas, likely because of hypoglossal dysgenesis. These results thus demand attention toward obstructive events in infants with CCHS.

Functional organization of vestibulospinal inputs on thoracic motoneurons responsible for trunk postural control in Xenopus.

KEY POINTS: Vestibulospinal reflexes participate in postural control. How this is achieved has not been investigated fully. We combined electrophysiological, neuroanatomical and imaging techniques to decipher the vestibulospinal network controlling the activation of back and limb muscles responsible for postural adjustments. We describe two distinct pathways activating either thoracic postural motoneurons alone or thoracic and lumbar motoneurons together, with the latter co-ordinating specifically hindlimb extensors and postural back muscles. ABSTRACT: In vertebrates, trunk postural stabilization is known to rely mainly on direct vestibulospinal inputs on spinal axial motoneurons. However, a substantial role of central spinal commands ascending from lumbar segments is not excluded during active locomotion. In the adult Xenopus, a lumbar drive dramatically overwhelms the descending inputs onto thoracic postural motoneurons during swimming. Given that vestibulospinal fibres also project onto the lumbar segments that shelter the locomotor generators, we investigated whether such a lumbo-thoracic pathway may relay vestibular information and consequently, also be involved in the control of posture at rest. We show that thoracic postural motoneurons exhibit particular dendritic spatial organization allowing them to gather information from both sides of the cord. In response to passive head motion, these motoneurons display both early and delayed discharges, with the latter occurring in phase with ipsilateral hindlimb extensor bursts. We demonstrate that both vestibulospinal and lumbar ascending fibres converge onto postural motoneurons, and that thoracic motoneurons monosynaptically respond to the electrical stimulation of either pathway. Finally, we show that vestibulospinal fibres project to and activate lumbar interneurons with thoracic projections. Taken together, our results complete the scheme of the vestibulospinal control of posture by illustrating the existence of a novel, indirect pathway, which implicates lumbar interneurons relaying vestibular inputs to thoracic motoneurons, and participating in global body postural stabilization in the absence of active locomotion.

Modulation of respiratory network activity by forelimb and hindlimb locomotor generators.

Early at the onset of exercise, breathing rate accelerates in order to anticipate the increasing metabolic demand resulting from the extra effort produced. Accordingly, the respiratory neural networks are the target of various input signals originating either centrally or peripherally. For example, during locomotion, the activation of muscle sensory afferents is able to entrain and thereby increase the frequency of spontaneous respiratory rhythmogenesis. Moreover, the lumbar spinal networks engaged in generating hindlimb locomotor rhythms are also capable of activating the medullary respiratory generators through an ascending excitatory command. However, in the context of quadrupedal locomotion, the influence of other spinal cord regions, such as cervical and thoracic segments, remains unknown. Using isolated brainstem-spinal cord preparations from neonatal rats and mice, we show that cervicothoracic circuitry may also contribute to locomotion-induced acceleration of respiratory cycle frequency. As previously observed for the hindlimb CPGs, the pharmacological activation of forelimb locomotor networks produces episodes of fictive locomotion that in turn increase the ongoing respiratory rhythm. Thoracic neuronal circuitry may also participate indirectly in this modulation via the activation of both cervical and lumbar CPG neurons. Furthermore, using light stimulation of CHR2-expressing glutamatergic neurons, we found that the modulation of the respiratory rate during locomotion involves lumbar glutamatergic circuitry. Our results demonstrate that during locomotion, the respiratory rhythm-generating networks receive excitatory ascending inputs from the spinal circuits responsible for generating and coordinating fore- and hindlimb movements. This constitutes a distributed central mechanism that contributes to matching breathing rate to the speed of locomotion.

Brainstem Steering of Locomotor Activity in the Newborn Rat.

Control of locomotion relies on motor loops conveying modulatory signals between brainstem and spinal motor circuits. We investigated the steering control of the brainstem reticular formation over the spinal locomotor networks using isolated brainstem-spinal cord preparations of male and female neonatal rats. First, we performed patch-clamp recordings of identified reticulospinal cells during episodes of fictive locomotion. This revealed that a spinal ascending phasic modulation of reticulospinal cell activity is already present at birth. Half of the cells exhibited tonic firing during locomotion, while the other half emitted phasic discharges of action potentials phase locked to ongoing activity. We next showed that mimicking the phasic activity of reticulospinal neurons by applying patterned electrical stimulation bilaterally at the ventral caudal medulla level triggered fictive locomotion efficiently. Moreover, the brainstem stimuli-induced locomotor rhythm was entrained in a one-to-one coupling over a range of cycle periods (2-6 s). Additionally, we induced turning like motor outputs by either increasing or decreasing the relative duration of the stimulation trains on one side of the brainstem compared to the other. The ability of the patterned descending command to control the locomotor output depended on the functional integrity of ventral reticulospinal pathways and the involvement of local spinal central pattern generator circuitry. Altogether, this study provides a mechanism by which brainstem reticulospinal neurons relay steering and speed commands to the spinal locomotor networks.SIGNIFICANCE STATEMENT Locomotor function allows the survival of most animal species while sustaining the expression of fundamental behaviors. Locomotor activities adapt from moment to moment to behavioral and environmental changes. We show that the brainstem can control the spinal locomotor network outputs through phasic descending commands that alternate bilaterally. Manipulating the periodicity and/or the relative durations of the left and right descending commands at the brainstem level is efficient to set the locomotor speed and sustain directional changes.

Bimodal Respiratory-Locomotor Neurons in the Neonatal Rat Spinal Cord.

Neural networks that can generate rhythmic motor output in the absence of sensory feedback, commonly called central pattern generators (CPGs), are involved in many vital functions such as locomotion or respiration. In certain circumstances, these neural networks must interact to produce coordinated motor behavior adapted to environmental constraints and to satisfy the basic needs of an organism. In this context, we recently reported the existence of an ascending excitatory influence from lumbar locomotor CPG circuitry to the medullary respiratory networks that is able to depolarize neurons of the parafacial respiratory group during fictive locomotion and to subsequently induce an increased respiratory rhythmicity (Le Gal et al., 2014b). Here, using an isolated in vitro brainstem-spinal cord preparation from neonatal rat in which the respiratory and the locomotor networks remain intact, we show that during fictive locomotion induced either pharmacologically or by sacrocaudal afferent stimulation, the activity of both thoracolumbar expiratory motoneurons and interneurons is rhythmically modulated with the locomotor activity. Completely absent in spinal inspiratory cells, this rhythmic pattern is highly correlated with the hindlimb ipsilateral flexor activities. Furthermore, silencing brainstem neural circuits by pharmacological manipulation revealed that this locomotor-related drive to expiratory motoneurons is solely dependent on propriospinal pathways. Together these data provide the first evidence in the newborn rat spinal cord for the existence of bimodal respiratory-locomotor motoneurons and interneurons onto which both central efferent expiratory and locomotor drives converge, presumably facilitating the coordination between the rhythmogenic networks responsible for two different motor functions. Significance statement: In freely moving animals, distant regions of the brain and spinal cord controlling distinct motor acts must interact to produce the best adapted behavioral response to environmental constraints. In this context, it is well established that locomotion and respiration must to be tightly coordinated to reduce muscular interferences and facilitate breathing rate acceleration during exercise. Here, using electrophysiological recordings in an isolated in vitro brainstem-spinal cord preparation from neonatal rat, we report that the locomotor-related signal produced by the lumbar central pattern generator for locomotion selectively modulates the intracellular activity of spinal respiratory neurons engaged in expiration. Our results thus contribute to our understanding of the cellular bases for coordinating the rhythmic neural circuitry responsible for different behaviors.

Mechanisms Underlying Adaptation of Respiratory Network Activity to Modulatory Stimuli in the Mouse Embryo.

Breathing is a rhythmic behavior that requires organized contractions of respiratory effector muscles. This behavior must adapt to constantly changing conditions in order to ensure homeostasis, proper body oxygenation, and CO2/pH regulation. Respiratory rhythmogenesis is controlled by neural networks located in the brainstem. One area considered to be essential for generating the inspiratory phase of the respiratory rhythm is the preBötzinger complex (preBötC). Rhythmogenesis emerges from this network through the interplay between the activation of intrinsic cellular properties (pacemaker properties) and intercellular synaptic connections. Respiratory activity continuously changes under the impact of numerous modulatory substances depending on organismal needs and environmental conditions. The preBötC network has been shown to become active during the last third of gestation. But only little is known regarding the modulation of inspiratory rhythmicity at embryonic stages and even less on a possible role of pacemaker neurons in this functional flexibility during the prenatal period. By combining electrophysiology and calcium imaging performed on embryonic brainstem slice preparations, we provide evidence showing that embryonic inspiratory pacemaker neurons are already intrinsically sensitive to neuromodulation and external conditions (i.e., temperature) affecting respiratory network activity, suggesting a potential role of pacemaker neurons in mediating rhythm adaptation to modulatory stimuli in the embryo.

Activation of subthalamic alpha 2 noradrenergic receptors induces motor deficits as a consequence of neuronal burst firing.

The subthalamic nucleus (STN) plays a key role in the pathophysiology of Parkinson's disease. This was demonstrated by the fact that STN neurons express more bursts in animal models of the disease and by the ability of STN inactivation to alleviate motor deficits. However, the origin of the bursts and the causal link between STN bursts and motor deficits remain unknown. The present study aimed to investigate the role of noradrenergic receptor modulation on the firing activity of STN neurons and the impact of this modulation on locomotor activity in sham and 6-hydroxydopamine-lesioned rats. Using selective agonists and antagonists of α1- and α2-adrenergic receptors (AR), we show that local infusion of clonidine, an α2-AR agonist, induced a switch from tonic to bursty pattern without changing the firing rate. This change in the pattern was prevented by the local infusion of idazoxan, an α2-AR antagonist. Furthermore, clonidine injection into the STN reduced locomotor activity in sham and 6-hydroxydopamine-lesioned rats. In contrast, local injection of phenylephrine, an α1-AR agonist, increased the firing rate of STN neurons without changing the firing pattern. In parallel, phenylephrine did not change locomotor activity. This is the first evidence showing the implication of α1-ARs in the modulation of firing rate and α2-ARs in the modulation of the firing pattern of STN neurons. Furthermore, our data provide also evidence that activation of the STN α2-ARs plays a key role in the genesis of subthalamic burst activity, which may be at the origin of motor deficits.

Emerging dysfunctions consequent to combined monoaminergic depletions in Parkinsonism.

The loss of dopamine (DA) neurons has been the pathophysiological focus of the devastating conditions of Parkinson's disease, but depletion of DA alone in animal models has failed to simultaneously elicit both the motor and non-motor deficits of PD. The present study aimed to investigate, in rats, the respective role of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) depletions on motor and non-motor behaviors and on subthalamic (STN) neuronal activity. We show that NA or DA depletion significantly decreased locomotor activity and enhanced the proportion of bursty and irregular STN neurons. Anxiety-like states required DA depletion plus the depletion of 5-HT or NA. Anhedonia and "depressive-like" behavior emerged only from the combined depletion of all three monoamines, an effect paralleled by an increase in the firing rate and the proportion of bursty and irregular STN neurons. Here, we provide evidence for the exacerbation of behavioral deficits when NA and/or 5-HT depletions are combined with DA depletion, bringing new insight into the combined roles of the three monoamines in PD.

Perinatal development of central vestibular neurons in mice.

Central circuitry of the vestibular nuclei integrates sensory inputs in the adaptive control of motor behaviors such as posture, locomotion, and gaze stabilization. Thus far, such circuits have been mostly examined at mature stages, whereas their emergence and early development have remained poorly described. Here, we focused on the perinatal period of murine development, from embryonic day E14.5 to post-natal day P5, to investigate the ontogeny of two functionally distinct vestibular neuronal groups, neurons projecting to the spinal cord via the lateral vestibulospinal tract (LVST) and commissural neurons of the medial vestibular nucleus that cross the midline to the contralateral nucleus. Using transgenic mice and retrograde labeling, we found that network-constitutive GABAergic and glycinergic neurons are already established in the two vestibular groups at embryonic stages. Although incapable of repetitive firing at E14.5, neurons of both groups can generate spike trains from E15.5 onward and diverge into previously established A or B subtypes according to the absence (A) or presence (B) of a two-stage spike after hyperpolarization. Investigation of several voltage-dependent membrane properties indicated that solely LVST neurons undergo significant maturational changes in their electrophysiological characteristics during perinatal development. The proportions of A vs B subtypes also evolve in both groups, with type A neurons remaining predominant at all stages, and type B commissural neurons appearing only post-natally. Together, our results indicate that vestibular neurons acquire their distinct morpho-functional identities after E14.5 and that the early maturation of membrane properties does not emerge uniformly in the different functional subpopulations of vestibulo-motor pathways.

Microglia shape the embryonic development of mammalian respiratory networks.

Microglia, brain-resident macrophages, play key roles during prenatal development in defining neural circuitry function, including ensuring proper synaptic wiring and maintaining homeostasis. Mammalian breathing rhythmogenesis arises from interacting brainstem neural networks that are assembled during embryonic development, but the specific role of microglia in this process remains unknown. Here, we investigated the anatomical and functional consequences of respiratory circuit formation in the absence of microglia. We first established the normal distribution of microglia within the wild-type (WT, Spi1+/+ (Pu.1 WT)) mouse (Mus musculus) brainstem at embryonic ages when the respiratory networks are known to emerge (embryonic day (E) 14.5 for the parafacial respiratory group (epF) and E16.5 for the preBötzinger complex (preBötC)). In transgenic mice depleted of microglia (Spi1-/- (Pu.1 KO) mutant), we performed anatomical staining, calcium imaging, and electrophysiological recordings of neuronal activities in vitro to assess the status of these circuits at their respective times of functional emergence. Spontaneous respiratory-related activity recorded from reduced in vitro preparations showed an abnormally slow rhythm frequency expressed by the epF at E14.5, the preBötC at E16.5, and in the phrenic motor nerves from E16.5 onwards. These deficits were associated with a reduced number of active epF neurons, defects in commissural projections that couple the bilateral preBötC half-centers, and an accompanying decrease in their functional coordination. These abnormalities probably contribute to eventual neonatal death, since plethysmography revealed that E18.5 Spi1-/- embryos are unable to sustain breathing activity ex utero. Our results thus point to a crucial contribution of microglia in the proper establishment of the central respiratory command during embryonic development.

A Rodent Model of Mild Neonatal Hypoxic Ischemic Encephalopathy.

In the brain of full-term newborns, Hypoxic Ischemic Encephalopathy (HIE), a consequence of severe hypoxia and ischemia due to low cardiac output, is frequently observed and results in cerebral injuries with dramatic consequences for life. To investigate the physiopathology of HIE, several animal models have been developed, but none closely replicate human cases, mostly because they are based on a single carotid ligation protocol. In the present study we aimed to develop a novel and more accurate HIE model in juvenile (post-natal days (PND) 14-16) rats. For this, we induced a 9 min hypoxic cardiac arrest (CA) by stopping mechanical ventilation of intubated, ventilated and curarized rats followed by a cardiopulmonary resuscitation. To evaluate the consequences of the CA we performed radiological (cerebral MRI), behavioral (Open Field, Elevated Plus Maze, Fear Conditioning), and histological (Cresyl Violet and Fluoro-Jade B) testing on treated animals. We found that rats in the CA group developed an anxiolytic-like behavioral profile in adulthood without any locomotor impairment, nor memory deficits. However, MRI investigation performed early after CA failed to reveal any change in apparent diffusion coefficient (ADC) in brain tissue (including the hippocampus, striatum, and thalamus), suggesting no massive anatomical lesion had occurred. In contrast, signs of neurodegeneration were found in the Dentate Gyrus and the CA1 region of the hippocampus at day 1 post-CA, suggesting that the anxiolytic-like phenotype observed in adulthood could be related to an abnormal degeneration of this brain region beginning immediately after CA. Thus, our model, despite not representing a severe condition of HIE, nonetheless constitutes a potential model for studying mild, yet persistent and region-specific cerebral injury resulting from an acute oxygen deprivation.

Serotonergic modulation of sacral dorsal root stimulation-induced locomotor output in newborn rat.

Descending neuromodulators from the brainstem play a major role in the development and regulation of spinal sensorimotor functions. Here, the contribution of serotonergic signaling in the lumbar spinal cord was investigated in the context of the generation of locomotor activity. Experiments were performed on in vitro spinal cord preparations from newborn rats (0-5 days). Rhythmic locomotor episodes (fictive locomotion) triggered by tonic electrical stimulations (2Hz, 30s) of a single sacral dorsal root were recorded from bilateral flexor-dominated (L2) and extensor-dominated (L5) ventral roots. We found that the activity pattern induced by sacral stimulation evolves over the 5 post-natal (P) day period. Although alternating rhythmic flexor-like motor bursts were expressed at all ages, the locomotor pattern of extensor-like bursting was progressively lost from P1 to P5. At later stages, serotonin (5-HT) and quipazine (5-HT2A receptor agonist) at concentrations sub-threshold for direct locomotor network activation promoted sacral stimulation-induced fictive locomotion. The 5-HT2A receptor antagonist ketanserin could reverse the agonist's action but was ineffective when fictive locomotion was already expressed in the absence of 5-HT (mainly before P2). Although inhibiting 5-HT7 receptors with SB266990 did not affect locomotor pattern organization, activating 5-HT1A receptors with 8-OH-DPAT specifically deteriorated extensor phase motor burst activity. We conclude that during the first 5 post-natal days in rat, serotonergic signaling in the lumbar cord becomes increasingly critical for the expression of fictive locomotion. Our findings therefore further underline the importance of both descending serotonergic and sensory afferent pathways in shaping locomotor activity during postnatal development. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

Role of the K+-Cl- Cotransporter KCC2a Isoform in Mammalian Respiration at Birth.

In central respiratory circuitry, synaptic excitation is responsible for synchronizing neuronal activity in the different respiratory rhythm phases, whereas chloride-mediated inhibition is important for shaping the respiratory pattern itself. The potassium chloride cotransporter KCC2, which serves to maintain low intraneuronal Cl- concentration and thus render chloride-mediated synaptic signaling inhibitory, exists in two isoforms, KCC2a and KCC2b. KCC2 is essential for functional breathing motor control at birth, but the specific contribution of the KCC2a isoform remains unknown. Here, to address this issue, we investigated the respiratory phenotype of mice deficient for KCC2a. In vivo plethysmographic recordings revealed that KCC2a-deficient pups at P0 transiently express an abnormally low breathing rate and a high occurrence of apneas. Immunostainings confirmed that KCC2a is normally expressed in the brainstem neuronal groups involved in breathing (pre-Bötzinger complex, parafacial respiratory group, hypoglossus nucleus) and is absent in these regions in the KCC2a-/- mutant. However, in variously reduced in vitro medullary preparations, spontaneous rhythmic respiratory activity is similar to that expressed in wild-type preparations, as is hypoglossal motor output, and no respiratory pauses are detected, suggesting that the rhythm-generating networks are not intrinsically affected in mutants at P0. In contrast, inhibitory neuromodulatory influences exerted by the pons on respiratory rhythmogenesis are stronger in the mutant, thereby explaining the breathing anomalies observed in vivo. Thus, our results indicate that the KCC2a isoform is important for establishing proper breathing behavior at the time of birth, but by acting at sites that are extrinsic to the central respiratory networks themselves.

Functional limb muscle innervation prior to cholinergic transmitter specification during early metamorphosis in Xenopus.

In vertebrates, functional motoneurons are defined as differentiated neurons that are connected to a central premotor network and activate peripheral muscle using acetylcholine. Generally, motoneurons and muscles develop simultaneously during embryogenesis. However, during Xenopus metamorphosis, developing limb motoneurons must reach their target muscles through the already established larval cholinergic axial neuromuscular system. Here, we demonstrate that at metamorphosis onset, spinal neurons retrogradely labeled from the emerging hindlimbs initially express neither choline acetyltransferase nor vesicular acetylcholine transporter. Nevertheless, they are positive for the motoneuronal transcription factor Islet1/2 and exhibit intrinsic and axial locomotor-driven electrophysiological activity. Moreover, the early appendicular motoneurons activate developing limb muscles via nicotinic antagonist-resistant, glutamate antagonist-sensitive, neuromuscular synapses. Coincidently, the hindlimb muscles transiently express glutamate, but not nicotinic receptors. Subsequently, both pre- and postsynaptic neuromuscular partners switch definitively to typical cholinergic transmitter signaling. Thus, our results demonstrate a novel context-dependent re-specification of neurotransmitter phenotype during neuromuscular system development.

Acute exposure to zinc oxide nanoparticles critically disrupts operation of the respiratory neural network in neonatal rat.

Due to their extremely small size that gives them unique physicochemical properties, nanoparticles (NPs) are used in the production of everyday materials. However, NPs can accumulate in body organs and could cause various diseases. Moreover, NPs that cross biological membranes such as the blood-brain barrier can aggregate in the brain and potentially produce neuronal damage. Although studies have reported the effects of diverse NPs on the bioelectrical properties of individual neurons, their potential influences on the operation of whole neuronal networks have not been documented. Here, we aimed to evaluate the effects of an acute exposure to zinc oxide (ZnO) NPs on the central neural networks responsible for mammalian respiratory rhythm generation. Using an isolated ex vivo brainstem-spinal cord preparation from neonatal rat in which the circuitry for the central respiratory command remained intact, we show that ZnO NPs accelerate, then profoundly disrupt respiratory-related activity produced by the pre-Bötzinger complex (preBötC) responsible for inspiratory rhythm generation. Consequently, a sudden and definitive cessation of respiratory-related activity occurs in ZnO NPs-exposed preparations. Part of these effects is related to zinc ions released from NPs. Using brainstem slice preparations containing the preBötC network, whole-cell patch-clamp recordings revealed that ZnO NPs depolarize preBötC inspiratory neurons and affect their bioelectrical properties by reducing the amplitude of action potentials, thereby leading to a depression of intra-network activity and the ultimate termination of respiratory rhythmogenesis. These findings support the conclusion that ZnO NPs may have deleterious effects on the central respiratory centers of newborn mammals.

The embryonic development of hindbrain respiratory networks is unaffected by mutation of the planar polarity protein Scribble.

The central command for breathing arises mainly from two interconnected rhythmogenic hindbrain networks, the parafacial respiratory group (pFRG or epF at embryonic stages) and the preBötzinger complex (preBötC), which are comprised of a limited number of neurons located in confined regions of the ventral medulla. In rodents, both networks become active toward the end of gestation but little is known about the signaling pathways involved in their anatomical and functional establishment during embryogenesis. During embryonic development, epF and preBötC neurons migrate from their territories of origin to their final positions in ventral brainstem areas. Planar Cell Polarity (PCP) signaling, including the molecule Scrib, is known to control the developmental migration of several hindbrain neuronal groups. Accordingly, a homozygous mutation of Scrib leads to severe disruption of hindbrain anatomy and function. Here, we aimed to determine whether Scrib is also involved in the prenatal development of the hindbrain nuclei controlling breathing. We combined immunostaining, calcium imaging and electrophysiological recordings of neuronal activity in isolated in vitro preparations. In the Scrib mutant, despite severe neural tube defects, epF and preBötC neurons settled at their expected hindbrain positions. Furthermore, both networks remained capable of generating rhythmically organized, respiratory-related activities and exhibited normal sensitivity to pharmacological agents known to modify respiratory circuit function. Thus Scrib is not required for the proper migration of epF and preBötC neurons during mouse embryogenesis. Our findings thus further illustrate the robustness and specificity of the developmental processes involved in the establishment of hindbrain respiratory circuits.

Differential Alteration in Expression of Striatal GABAAR Subunits in Mouse Models of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive motor symptoms that are preceded by cognitive deficits and is considered as a disorder that primarily affects forebrain striatal neurons. To gain a better understanding of the molecular and cellular mechanisms associated with disease progression, we analyzed the expression of proteins involved in GABAergic neurotransmission in the striatum of the R6/1 transgenic mouse model. Western blot, quantitative PCR and immunohistochemical analyses were conducted on male R6/1 mice and age-matched wild type littermates. Analyses were performed on 2 and 6 month-old animals, respectively, before and after the onset of motor symptoms. Expression of GAD 67, GAD 65, NL2, or gephyrin proteins, involved in GABA synthesis or synapse formation did not display major changes. In contrast, expression of α1, α3 and α5 GABAAR subunits was increased while the expression of δ was decreased, suggesting a change in tonic- and phasic inhibitory transmission. Western blot analysis of the striatum from 8 month-old Hdh Q111, a knock-in mouse model of HD with mild deficits, confirmed the α1 subunit increased expression. From immunohistochemical analyses, we also found that α1 subunit expression is increased in medium-sized spiny projection neurons (MSN) and decreased in parvalbumin (PV)-expressing interneurons at 2 and 6 months in R6/1 mice. Moreover, α2 subunit labeling on the PV and MSN cell membranes was increased at 2 months and decreased at 6 months. Alteration of gene expression in the striatum and modification of GABAA receptor subtypes in both interneurons and projection neurons suggested that HD mutation has a profound effect on synaptic plasticity at an early stage, before the onset of motor symptoms. These results also indicate that cognitive and other behavioral deficits may be associated with changes in GABAergic neurotransmission that consequently could be a relevant target for early therapeutic treatment.

Early postnatal maturation in vestibulospinal pathways involved in neck and forelimb motor control.

To assess the organization and functional development of vestibulospinal inputs to cervical motoneurons (MNs), we have used electrophysiology (ventral root and electromyographic [EMG] recording), calcium imaging, trans-synaptic rabies virus (RV) and conventional retrograde tracing and immunohistochemistry in the neonatal mouse. By stimulating the VIIIth nerve electrically while recording synaptically mediated calcium responses in MNs, we characterized the inputs from the three vestibulospinal tracts, the separate ipsilateral and contralateral medial vestibulospinal tracts (iMVST/cMVST) and the lateral vestibulospinal tract (LVST), to MNs in the medial and lateral motor columns (MMC and LMC) of cervical segments. We found that ipsilateral inputs from the iMVST and LVST were differentially distributed to the MMC and LMC in the different segments, and that all contralateral inputs to MMC and LMC MNs in each segment derive from the cMVST. Using trans-synaptic RV retrograde tracing as well as pharmacological manipulation of VIIIth nerve-elicited synaptic responses, we found that a substantial proportion of inputs to both neck and forelimb extensor MNs was mediated monosynaptically, but that polysynaptic inputs were also significant. By recording EMG responses evoked by natural stimulation of the vestibular apparatus, we found that vestibular-mediated motor output to the neck and forelimb musculature became more robust during the first 10 postnatal days, concurrently with a decrease in the latency of MN discharge evoked by VIIIth nerve electrical stimulation. Together, these results provide insight into the complexity of vestibulospinal connectivity in the cervical spinal cord and a cogent demonstration of the functional maturation that vestibulospinal connections undergo postnatally. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1061-1077, 2016.

Age-Related Changes in Pre- and Postsynaptic Partners of the Cholinergic C-Boutons in Wild-Type and SOD1G93A Lumbar Motoneurons.

Large cholinergic synaptic terminals known as C-boutons densely innervate the soma and proximal dendrites of motoneurons that are prone to neurodegeneration in amyotrophic lateral sclerosis (ALS). Studies using the Cu/Zn-superoxide dismutase (SOD1) mouse model of ALS have generated conflicting data regarding C-bouton alterations exhibited during ALS pathogenesis. In the present work, a longitudinal study combining immunohistochemistry, biochemical approaches and extra- and intra-cellular electrophysiological recordings revealed that the whole spinal cholinergic system is modified in the SOD1 mouse model of ALS compared to wild type (WT) mice as early as the second postnatal week. In WT motoneurons, both C-bouton terminals and associated M2 postsynaptic receptors presented a complex age-related dynamic that appeared completely disrupted in SOD1 motoneurons. Indeed, parallel to C-bouton morphological alterations, analysis of confocal images revealed a clustering process of M2 receptors during WT motoneuron development and maturation that was absent in SOD1 motoneurons. Our data demonstrated for the first time that the lamina X cholinergic interneurons, the neuronal source of C-boutons, are over-abundant in high lumbar segments in SOD1 mice and are subject to neurodegeneration in the SOD1 animal model. Finally, we showed that early C-bouton system alterations have no physiological impact on the cholinergic neuromodulation of newborn motoneurons. Altogether, these data suggest a complete reconfiguration of the spinal cholinergic system in SOD1 spinal networks that could be part of the compensatory mechanisms established during spinal development.