Brainstem-spinal Cord Preparation Research Articles

Effect of chronic prenatal exposure to the food additive titanium dioxide E171 on respiratory activity in newborn mice.

Nanoparticles (NPs) possess unique properties that make their use valuable in all industries. Titanium dioxide (TiO2) NPs are extensively used as a white pigment in food (labeled under the European number E171) and personal care products, which creates a significant potential for chronic consumer exposure. Concerns about the potential toxic effects of TiO2 NPs have arisen, particularly in vulnerable populations, including pregnant women and infants. Recently, human materno-fetal transfer of E171 was demonstrated, and simultaneously, we reported that chronic prenatal exposure to reference P25 TiO2 NPs was found to alter the developing respiratory neural networks. In this study, using whole body plethysmography from postnatal day (P) 0 to P7, we assessed the respiratory function of newborn mice born to mothers fed with E171 during pregnancy. We also evaluated the potential alterations to respiratory centers by using brainstem-spinal cord electrophysiological recordings from P0 to P6. Our study reveals that E171-prenatally exposed animals displayed an abnormally elevated breathing rate from P3 onwards. From P5 to P6, the respiratory-related burst frequency generated by the isolated brainstem-spinal cord preparations was significantly higher in E171-exposed animals than in non-exposed animals. These findings demonstrate prenatal toxicity of E171 to the developing respiratory function and may contribute to policy-making regarding the use of TiO2 NPs.

Isoproterenol modulates expiratory activities in the brainstem spinal cord preparation in neonatal mice in vitro

Motor behaviors such as breathing required temporal coordination of different muscle groups to insured efficient ventilation and provide oxygen to the body. This action is the result of interactions between neural networks located within the brainstem. Inspiration and expiration depend at least in part on interactions between two separate oscillators: inspiration is driven by a neural network located in the preBötzinger complex (PreBötC) and active expiration is driven by a network in the parafacial respiratory group (pFRG). Neurons of the pFRG are silent at rest and become active when the respiratory drive increased. This study investigated the temporal coordination between the brainstem respiratory network and the lumbar spinal network that generates spontaneous activities that is different of the induced fictive locomotion. The remaining question is how these activities coordinate early during the development. Results of this study show that brainstem networks contribute to the temporal coordination of the lumbar spontaneous activity during inspiration since lumbar motor activity occurs exclusively during the expiratory time. This study also investigated the role of the β-noradrenergic modulation on the respiratory activities. β-noradrenergic receptors activation increased the frequency of the double bursts and increased expiratory activity at the lumbar level. These results suggest interactions between brainstem and spinal networks and reveal a descending drive that may contribute to the coordination of the respiratory and lumbar spontaneous activities.

The lesion site of organophosphorus-induced central apnea and the effects of antidotes

Organophosphorus poisoning kills individuals by causing central apnea; however, the underlying cause of death remains unclear. Following findings that the pre-Bötzinger complex impairment alone does not account for central apnea, we analyzed the effect of paraoxon on the brainstem-spinal cord preparation, spanning the lower medulla oblongata to phrenic nucleus. Respiratory bursts were recorded by connecting electrodes to the ventral 4th cervical nerve root of excised brainstem-spinal cord preparations obtained from 6-day-old Sprague–Dawley rats. We observed changes in respiratory bursts when paraoxon, neostigmine, atropine, and 2-pyridine aldoxime methiodide were administered via bath application. The percentage of burst extinction in the paraoxon-poisoning group was 50% compared with 0% and 18.2% in the atropine and 2-pyridine aldoxime methiodide treatment groups, respectively. Both treatments notably mitigated the paraoxon-induced reduction in respiratory bursts. In the neostigmine group, similar to paraoxon, bursts stopped in 66.7% of cases but were fully reversed by atropine. This indicates that the primary cause of central apnea is muscarinic receptor-mediated in response to acetylcholine excess. Paraoxon-induced central apnea is hypothesized to result from neural abnormalities within the inferior medulla oblongata to the phrenic nucleus, excluding pre-Bötzinger complex. These antidotes antagonize central apnea, suggesting that they may be beneficial therapeutic agents.

Disinhibition does not play a role in endomorphin-2-induced changes in inspiratory motoneuron output produced by in vitro neonatal rat preparations

Low level activation of mu-opioid receptors (MORs) in neonatal rat brainstem-spinal cord preparations increases inspiratory burst amplitude recorded on cervical spinal roots. We tested whether: (1) MOR activation with an endogenous ligand, such as endomorphin-2, increases inspiratory burst amplitude, (2) disinhibition of GABAergic or glycinergic inhibitory synaptic transmission is involved, and (3) inflammation alters endomorphin-2 effects. Using neonatal rat (P0-P3) brainstem-spinal cord preparations, bath-applied endomorphin-2 (10–200 nM) increased inspiratory burst amplitude and decreased burst frequency. Blockade of GABAA receptors (picrotoxin), glycine receptors (strychnine), or both (picrotoxin and strychnine) did not abolish endomorphin-2-induced effects. In preparations isolated from neonatal rats injected 3 h previously with lipopolysaccharide (LPS, 0.1 mg/kg), endomorphin-2 continued to decrease burst frequency but abolished the burst amplitude increase. Collectively, these data indicate that disinhibition of inhibitory synaptic transmission is unlikely to play a role in endomorphin-2-induced changes in inspiratory motor output, and that different mechanisms underlie the endomorphin-2-induced increases in inspiratory burst amplitude and decreases in burst frequency.

SGLT2 and SGLT1 inhibitors suppress the activities of the RVLM neurons in newborn Wistar rats.

Hypertension is well-known to often coexist with diabetes mellitus (DM) in humans. Treatment with sodium-glucose cotransporter 2 (SGLT2) inhibitors has been shown to decrease both the blood glucose and the blood pressure (BP) in such patients. Some reports show that SGLT2 inhibitors improve the BP by decreasing the activities of the sympathetic nervous system. Therefore, we hypothesized that SGLT2 inhibitors might alleviate hypertension via attenuating sympathetic nervous activity. Combined SGLT2/SGLT1 inhibitor therapy is also reported as being rather effective for decreasing the BP. In this study, we examined the effects of SGLT2 and SGLT1 inhibitors on the bulbospinal neurons of the rostral ventrolateral medulla (RVLM). To investigate whether bulbospinal RVLM neurons are sensitive to SGLT2 and SGLT1 inhibitors, we examined the changes in the neuronal membrane potentials (MPs) of these neurons using the whole-cell patch-clamp technique during superfusion of the cells with the SGLT2 and SGLT1 inhibitors. A brainstem-spinal cord preparation was used for the experiments. Our results showed that superfusion of the RVLM neurons with SGLT2 and SGLT1 inhibitor solutions induced hyperpolarization of the neurons. Histological examination revealed the presence of SGLT2s and SGLT1s in the RVLM neurons, and also colocalization of SGLT2s with SGLT1s. These results suggest the involvement of SGLT2s and SGLT1s in regulating the activities of the RVLM neurons, so that SGLT2 and SGLT1 inhibitors may inactivate the RVLM neurons hyperpolarized by empagliflozin. SGLT2 and SGLT1 inhibitors suppressed the activities of the bulbospinal RVLM neurons in the brainstem-spinal preparations, suggesting the possibilities of lowering BP by decreasing the sympathetic nerve activities. RVLM, rostral ventrolateral medulla. IML, intralateral cell column. aCSF, artificial cerebrospinal fluid.

Effects of aconitine on the respiratory activity of brainstem-spinal cord preparations isolated from newborn rats.

Aconitine is a sodium channel opener, but its effects on the respiratory center are not well understood. We investigated the dose-dependent effects of aconitine on central respiratory activity in brainstem-spinal cord preparations isolated from newborn rats. Bath application of 0.5-5μM aconitine caused an increase in respiratory rhythm and decrease in the inspiratory burst amplitude of the fourth cervical ventral root (C4). Separate application of aconitine revealed that medullary neurons were responsible for the respiratory rhythm increase, and neurons in both the medulla and spinal cord were involved in the decrease of C4 amplitude by aconitine. A local anesthetic, lidocaine (100μM), or a voltage-dependent sodium channel blocker, tetrodotoxin (0.1μM), partially antagonized the C4 amplitude decrease by aconitine. Tetrodotoxin treatment tentatively decreased the respiratory rhythm, but lidocaine tended to further increase the rhythm. Treatment with 100μM riluzole or 100μM flufenamic acid, which are known to inhibit respiratory pacemaker activity, did not reduce the respiratory rhythm enhanced by aconitine + lidocaine. The application of 1μM aconitine depolarized the preinspiratory, expiratory, and inspiratory motor neurons. The facilitated burst rhythm of inspiratory neurons after aconitine disappeared in a low Ca2+/high Mg2+ synaptic blockade solution. We showed the dose-dependent effects of aconitine on respiratory activity. The antagonists reversed the depressive effects of aconitine in different manners, possibly due to their actions on different sites of sodium channels. The burst-generating pacemaker properties of neurons may not be involved in the generation of the facilitated rhythm after aconitine treatment.

Cannabinoid receptors involved in descending inhibition on spinal seizure-like activity in the phrenic output.

Seizure-like burst activities are induced by blockade of GABAA and/or glycine receptors in various spinal ventral roots of brainstem-spinal cord preparation from neonatal rodents. We found that this is not applicable to the phrenic nerve and that a new inhibitory descending pathway may suppress seizure-like activity in the phrenic nerve. Experiments were performed in brainstem-spinal cord preparation from newborn rats (age: 0-1 day). Left phrenic nerve and right C4 activities were recorded simultaneously. When GABAA and glycine receptors were blocked by 10 μM bicuculline and 10 μM strychnine (Bic+Str), seizure-like burst activities appeared in the fourth cervical ventral root (C4) but not the phrenic nerve. After making a transverse section at C1, the inspiratory burst activity disappeared from both C4 and the phrenic nerve, whereas seizure-like activity appeared in both nerves. We hypothesized that inhibitory descending pathways other than those via GABAA and/or glycine receptors (from the medulla to the spinal cord) work to avoid disturbance of regular respiratory-related diaphragm contraction by seizure-like activity. We found that cannabinoid receptor antagonist, AM251 was effective for the induction of seizure-like activity by Bic+Str in the phrenic nerve in brainstem-spinal cord preparation. Cannabinoid receptors may be involved in this descending inhibitory system.

Deconstructing the modular organization and real-time dynamics of mammalian spinal locomotor networks

Locomotion empowers animals to move. Locomotor-initiating signals from the brain are funneled through descending neurons in the brainstem that act directly on spinal locomotor circuits. Little is known in mammals about which spinal circuits are targeted by the command and how this command is transformed into rhythmicity in the cord. Here we address these questions leveraging a mouse brainstem-spinal cord preparation from either sex that allows locating the locomotor command neurons with simultaneous Ca2+ imaging of spinal neurons. We show that a restricted brainstem area – encompassing the lateral paragigantocellular nucleus (LPGi) and caudal ventrolateral reticular nucleus (CVL) – contains glutamatergic neurons which directly initiate locomotion. Ca2+ imaging captures the direct LPGi/CVL locomotor initiating command in the spinal cord and visualizes spinal glutamatergic modules that execute the descending command and its transformation into rhythmic locomotor activity. Inhibitory spinal networks are recruited in a distinctly different pattern. Our study uncovers the principal logic of how spinal circuits implement the locomotor command using a distinct modular organization.

Glycine and GABAA receptors suppressively regulate the inspiratory-related calcium rise in the thoracic inspiratory cells of the neonatal rat

We previously demonstrated that in an isolated brainstem–spinal cord preparation from neonatal rats, a local bath application of strychnine (a broad antagonist of glycine and GABAA receptors) to the spinal cord enhances thoracic inspiratory motor activity. Herein, to investigate the involvement of the inspiratory spinal interneurons that provide excitatory input to the motoneuron, we conducted calcium imaging using this preparation. Oregon Green 488 BAPTA-1 AM, a fluorescent calcium indicator, was injected into the ventromedial surface of the thoracic cord. In all cells that showed inspiratory-related fluorescence changes > 2% of the baseline fluorescence intensity, the inspiratory-related fluorescence change decreased when the focal depth was deepened. The application of strychnine to the spinal cord increased the inspiratory-related intracellular calcium rise in these cells. These results suggest that the enhancement of inspiratory interneuron activity could be involved in this enhancement of inspiratory motor activity.

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

BackgroundOver 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.ResultsUsing 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.ConclusionsOur findings thus demonstrate that a maternal exposure to TiO2 NPs during pregnancy affects the normal development and operation of the respiratory centers in progeny.

Effects of ANP and BNP on the generation of respiratory rhythms in brainstem-spinal cord preparation isolated from newborn rats.

Natriuretic peptides (NPs) are a family of peptide hormones produced in cardiac muscle cells and consist mainly of three types: atrial NP (ANP), B-type (or brain) NP (BNP), and C-type NP. We herein report the effects of ANP and BNP on central respiratory activity in brainstem-spinal cord preparation isolated from newborn rats. Bath application of these peptides (100 nM) induced a weak transient depression of the respiratory rhythm followed by recovery. Respiratory-related neurons in the rostral ventrolateral medulla showed a tendency for transient hyperpolarization followed by recovery during the application of ANP or BNP. The application of a membrane-permeable cGMP, 8-Br-cGMP (10 or 20 μM), did not induce significant effects on respiratory rhythm, suggesting no involvement of guanylyl cyclase in effects of ANP or BNP. We also examined effects of BNP on respiratory depression induced by the sedative dexmedetomidine, which exerts an inhibitory influence on respiratory rhythm. When pretreated with 50 nM BNP, the inhibitory effect of 100 nM dexmedetomidine was significantly reduced. Our findings suggest that ANP and BNP act as mild excitatory agents with sustained effects on respiratory rhythm after an initial transient depression.

Mu-opioid receptor-dependent transformation of respiratory motor pattern in neonates in vitro.

Endogenous opioid peptides activating mu-opioid receptors (MORs) are part of an intricate neuromodulatory system that coordinates and optimizes respiratory motor output to maintain blood-gas homeostasis. MOR activation is typically associated with respiratory depression but also has excitatory effects on breathing and respiratory neurons. We hypothesized that low level MOR activation induces excitatory effects on the respiratory motor pattern. Thus, low concentrations of an MOR agonist drug (DAMGO, 10–200 nM) were bath-applied to neonatal rat brainstem-spinal cord preparations while recording inspiratory-related motor output on cervical spinal roots (C4-C5). Bath-applied DAMGO (50–200 nM) increased inspiratory motor burst amplitude by 40–60% during (and shortly following) drug application with decreased burst frequency and minute activity. Reciprocal changes in inspiratory burst amplitude and frequency were balanced such that 20 min after DAMGO (50–200 nM) application, minute activity was unaltered compared to pre-DAMGO levels. The DAMGO-induced inspiratory burst amplitude increase did not require crossed cervical spinal pathways, was expressed on thoracic ventral spinal roots (T4-T8) and remained unaltered by riluzole pretreatment (blocks persistent sodium currents associated with gasping). Split-bath experiments showed that the inspiratory burst amplitude increase was induced only when DAMGO was bath-applied to the brainstem and not the spinal cord. Thus, MOR activation in neonates induces a respiratory burst amplitude increase via brainstem-specific mechanisms. The burst amplitude increase counteracts the expected MOR-dependent frequency depression and may represent a new mechanism by which MOR activation influences respiratory motor output.

Activation of Brainstem mu‐Opioid Receptors (mORs) Increases Respiratory‐Related Motor Burst Amplitude in Neonatal Rat Brainstem‐Spinal Cord Preparations

Mu‐opioid receptors (mORs) located on brainstem respiratory neurons are part of an intricate peptidergic neuromodulatory system that regulates breathing. The endogenous role of mORs in respiratory motor control, however, is poorly understood. To explore this question, very low (nanomolar) concentrations of mOR agonist drugs were bath‐applied to neonatal rat (P1‐P3) brainstem‐spinal cord preparations while measuring respiratory‐related motor output from spinal C4‐C5 rootlets containing phrenic motor neuron axons. When DAMGO (synthetic mOR agonist) or endomorphin‐2 (endogenous mOR ligand) were bath‐applied for 15 min at 20‐100 nM, respiratory motor burst amplitude increased dramatically with a modest decrease in burst frequency. Burst amplitude facilitated by 45 ± 38% and 32 ± 24% (mean ± stdev) above baseline for 50 nM DAMGO (n=8) and 100 nM endomorphin‐2 (n=10), respectively (p<0.001). To test whether respiratory motor output on thoracic ventral roots was altered by mOR activation, brainstem‐spinal cord preparations were isolated to include spinal segments C1‐T8. Bath‐applied DAMGO (50 nM) induced burst amplitude facilitation on thoracic roots T4‐T6 and cervical roots C4‐C5 by 62 ± 19% and 38 ± 36%, respectively (p<0.012). To test whether mOR‐induced burst facilitation is due to brainstem or spinal mechanisms, a split‐bath chamber with a barrier at C1‐C2 was used to separately bathe the brainstem and spinal cord compartments. DAMGO (50 nM) application to the spinal cord (C2‐C8) had little effect on respiratory motor output measured from C4‐C5 rootlets, whereas DAMGO application to the brainstem increased C4‐C5 motor output by 34 ± 19% (p<0.004). These data suggest that mOR activation in the brainstem can transform the respiratory network to produce a motor pattern with decreased frequency balanced by increased amplitude, with relatively little change in overall neural output. Given that mOR‐induced facilitation was observed in both cervical and thoracic motor neuron pools, we hypothesize that burst amplitude facilitation is due to mOR activation on rhythm‐generating neurons or premotor neurons in the brainstem. This study reveals that endogenous mOR activation modulates the neural control of breathing in a manner that is potentially different from respiratory depression, which is more commonly associated with opioids.

Influence of cotinine on breathing related motor output

Cotinine, the primary metabolite of nicotine, is used as a marker of nicotine exposure due to its long plasma half‐life. Previous research shows that perinatal nicotine exposure alters the regions of the brain that control breathing and results in impaired protective breathing reflexes in exposed human and animal neonates. While it has long been known that nicotine has direct effects on brain function and development, it has only recently been established that cotinine may act on the brain as well. Preliminary data suggests that cotinine, the primary metabolite of nicotine, enhances the effects of nicotine on breathing, consistent with its presumed action as a modulator of nAChRs. Here, to build on this preliminary data, we test the hypothesis that cotinine enhances the effects of nicotine on the frequency and amplitude of respiratory motor bursting in the brainstem spinal cord preparation from 1‐5 day old control rat pups and rat pups that were exposed to nicotine in utero and after birth (developmental nicotine exposure, DNE). Respiratory motor nerve bursting was recorded from the 4th cervical ventral root in brainstem spinal‐cord preparations studied in two experimental groups. In the first group, preparations were perfused with traditional aCSF that did not contain cotinine (Control). In a second group, 400 nM cotinine was added to the aCSF during dissection and throughout the experiment (Cotinine). The frequency and amplitude of bursts were recorded using the following protocol: a 30‐minute baseline recording where amplitude and frequency of bursts were stable, 20‐minutes of 500 nM nicotine application, and a 30‐minute washout. To assess the effects of cotinine alone, additional control experiments were performed where preparations were perfused with traditional aCSF during the baseline period, followed by application of 400 nM cotinine aCSF for 60 minutes. Results from these experiments indicate that: (1)400 nM cotinine alone does not alter burst amplitude or frequency in either control (n=6, 2‐way ANOVA: Amplitude; P=0.4487, Frequency; P=0.1020) or DNE preparations (n=6, Amplitude; P=0.1338, Frequency; P=0.1034) (2) 500 nM nicotine decreased burst amplitude and increased burst frequency in both control (n=8, for Amplitude and Frequency; P<0.0001) and DNE preparations (n=8, for Amplitude and Frequency; P<0.0001); and (3)cotinine had no effect on the nicotine‐ mediated increase in frequency in either treatment group (n=8 for Control; P=0.5105, n=8 for DNE; P=0.0762), but enhanced the nicotine‐ mediated decrease in amplitude in DNE preparations (n=8, P=0.0227) but not in control preparations (n=8, P=0.2716). We conclude that cotinine may modulate the function of specific nAChR subtypes that mediate the inhibitory effects of nicotine on respiratory motor nerve burst amplitude, and that these receptors may be functionally upregulated by DNE.

Influence of developmental nicotine exposure on serotonergic control of breathing-related motor output.

Serotonin plays an important role in the development of brainstem circuits that control breathing. Here, we test the hypothesis that developmental nicotine exposure (DNE) alters the breathing-related motor response to serotonin (5HT). Pregnant rats were exposed to nicotine or saline, and brainstem-spinal cord preparations from 1- to 5-day-old pups were studied in a split-bath configuration, allowing drugs to be applied selectively to the medulla or spinal cord. The activity of the fourth cervical ventral nerve roots (C4VR), which contain axons of phrenic motoneurons, was recorded. We applied 5HT alone or together with antagonists of 5HT1A, 5HT2A, or 5HT7 receptor subtypes. In control preparations, 5HT applied to the medulla consistently reduced C4VR frequency and this reduction could not be blocked by any of the three antagonists. In DNE preparations, medullary 5HT caused a large and sustained frequency increase (10min), followed by a sustained decrease. Notably, the transient increase in frequency could be blocked by the independent addition of any of the antagonists. Experiments with subtype-specific agonists suggest that the 5HT7 subtype may contribute to the increased frequency response in the DNE preparations. Changes in C4VR burst amplitude in response to brainstem 5HT were uninfluenced by DNE. Addition of 5HT to the caudal chamber modestly increased phasic and greatly increased tonic C4VR activity, but there were no effects of DNE. The data show that DNE alters serotonergic signaling within brainstem circuits that control respiratory frequency but does not functionally alter serotonin signaling in the phrenic motoneuron pool.

Lactate ions induce synaptic plasticity to enhance output from the central respiratory network.

Lactate ion sensing has emerged as a process that regulates ventilation during metabolic challenges. Most work has focused on peripheral sensing of lactate for the control of breathing. However, lactate also rises in the central nervous system (CNS) during disturbances to blood gas homeostasis and exercise. Using an amphibian model, we recently showed that lactate ions, independently of pH and pyruvate metabolism, act directly in the brainstem to increase respiratory-related motor outflow. This response had a long washout time and corresponded with potentiated excitatory synaptic strength of respiratory motoneurons. Thus, we tested the hypothesis that lactate ions enhance respiratory output using cellular mechanisms associated with long-term synaptic plasticity within motoneurons. In this study, we confirm that 2mM sodium lactate, but not sodium pyruvate, increases respiratory motor output in brainstem-spinal cord preparations, persisting for 2h upon the removal of lactate. Lactate also led to prolonged increases in the amplitude of AMPA-glutamate receptor (AMPAR) currents in individual motoneurons from brainstem slices. Both motor facilitation and AMPAR potentiation by lactate required classic effectors of synaptic plasticity, L-type Ca2+ channels and NMDA receptors, as part of the transduction process but did not correspond with increased expression of immediate-early genes often associated with activity-dependent neuronal plasticity. Altogether these results show that lactate ions enhance respiratory motor output by inducing conserved mechanisms of synaptic plasticity and suggest a new mechanism that may contribute to coupling ventilation to metabolic demands in vertebrates. KEY POINTS: Lactate ions, independently of pH and metabolism, induce long-term increases in respiratory-related motor outflow in American bullfrogs. Lactate triggers a persistent increase in strength of AMPA-glutamatergic synapses onto respiratory motor neurons. Long-term plasticity of motor output and synaptic strength by lactate involves L-type Ca2+ channels and NMDA-receptors as part of the transduction process. Enhanced AMPA receptor function in response to lactate in the intact network is causal for motor plasticity. In sum, well-conserved synaptic plasticity mechanisms couple the brainstem lactate ion concentration to respiratory motor drive in vertebrates.

Conservation of locomotion-induced oculomotor activity through evolution in mammals

Efference copies are neural replicas of motor outputs used to anticipate the sensory consequences of a self-generated motor action or to coordinate neural networks involved in distinct motor behaviors.1 An established example of this motor-to-motor coupling is the efference copy of the propulsive motor command, which supplements classical visuo-vestibular reflexes to ensure gaze stabilization during amphibian larval locomotion.2 Such feedforward replica of spinal pattern-generating circuits produces a spino-extraocular motor coupled activity that evokes eye movements, spatiotemporally coordinated to tail undulation independently of any sensory signal.3,4 Exploiting the developmental stages of the frog,1 studies in metamorphing Xenopus demonstrated the persistence of this spino-extraocular motor command in adults and its developmental adaptation to tetrapodal locomotion.5,6 Here, we demonstrate for the first time the existence of a comparable locomotor-to-ocular motor coupling in the mouse. In neonates, exvivo nerve recordings of brainstem-spinal cord preparations reveal a spino-extraocular motor coupled activity similar to the one described in Xenopus. In adult mice, trans-synaptic rabies virus injections in lateral rectus eye muscle label cervical spinal cord neurons closely connected to abducens motor neurons. Finally, treadmill-elicited locomotion in decerebrated preparations7 evokes rhythmic eye movements in synchrony with the limb gait pattern. Overall, our data are evidence for the conservation of locomotor-induced eye movements in vertebrate lineages. Thus, in mammals as in amphibians, CPG-efference copy feedforward signals might interact with sensory feedback to ensure efficient gaze control during locomotion.

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.

Adenosine A2a receptors modulate TrkB receptor-dependent respiratory plasticity in neonatal rats

Neuroplasticity is a fundamental property of the respiratory control system, enabling critical adaptations in breathing to meet the challenges, but little is known whether neonates express neuroplasticity similar to adults. We tested the hypothesis that, similar to adults, tyrosine receptor kinase B (TrkB) or adenosine A2a receptor activation in neonates are independently sufficient to elicit respiratory motor facilitation, and that co-induction of TrkB and A2a receptor-dependent plasticity undermines respiratory motor facilitation. TrkB receptor activation with 7,8-dihydroxyflavone (DHF) in neonatal brainstem-spinal cord preparations induced a long-lasting increase in respiratory motor output in 55 % of preparations, whereas adenosine A2a receptor activation with CGS21680 only sporadically induced respiratory motor plasticity. CGS21680 and DHF co-application prevented DHF-dependent respiratory motor facilitation, whereas co-application of MSX-3 (adenosine A2a receptor antagonist) and DHF more rapidly induced respiratory motor plasticity. Collectively, these data suggest that mechanisms underlying respiratory neuroplasticity may be only partially operational in early neonatal life, and that adenosine A2a receptor activation undermines TrkB-induced respiratory plasticity.

Effects of acetylcholine on hypoglossal and C4 nerve activity in brainstem-spinal cord preparations from newborn rat

Effects of acetylcholine (ACh) on respiratory activity have been an intriguing theme especially in relation to central chemoreception and the control of hypoglossal nerve activity. We studied the effects of ACh on hypoglossal and phrenic (C4) nerve activities and inspiratory and pre-inspiratory neurons in the rostral ventrolateral medulla in brainstem-spinal cord preparations from newborn rats. ACh application increased respiratory rhythm, decreased inspiratory hypoglossal and C4 nerve burst amplitude, and enhanced pre-inspiratory hypoglossal activity. ACh induced membrane depolarization of pre-inspiratory neurons that might be involved in facilitation of respiratory rhythm by ACh. Effects of ACh on hypoglossal and C4 nerve activity were partially reversed by a nicotinic receptor blocker, mecamylamine. Further application of a muscarinic receptor antagonist, oxybutynin, resulted in slight increase of hypoglossal (but not C4) burst amplitude. Thus, ACh induced different effects on hypoglossal and C4 nerve activity in the brainstem-spinal cord preparation.