Medullary tachykinin precursor 1 neurons promote rhythmic breathing.

Rhythmic breathing is generated by neural circuits located in the brainstem. At its core is the preBötzinger Complex (preBötC), a region of the medulla, necessary for the generation of rhythmic breathing in mammals. The preBötC is comprised of various neuronal populations expressing neurokinin-1 receptors, the cognate G-protein-coupled receptor of the neuropeptide substance P (encoded by the tachykinin precursor 1 or Tac1). Neurokinin-1 receptors are highly expressed in the preBötC and destruction or deletion of neurokinin-1 receptor-expressing preBötC neurons severely impair rhythmic breathing. Although, the application of substance P to the preBötC stimulates breathing in rodents, substance P is also involved in nociception and locomotion in various brain regions, suggesting that Tac1 neurons found in the preBötC may have diverse functional roles. Here, we characterized the role of Tac1-expressing preBötC neurons in the generation of rhythmic breathing in vivo, as well as motor behaviors. Using a cre-lox recombination approach, we injected adeno-associated virus containing the excitatory channelrhodopsin-2 ChETA in the preBötC region of Tac1-cre mice. Employing a combination of histological, optogenetics, respiratory, and behavioral assays, we showed that stimulation of glutamatergic or Tac1 preBötC neurons promoted rhythmic breathing in both anesthetized and freely moving animals, but also triggered locomotion and overcame respiratory depression by opioid drugs. Overall, our study identified a population of excitatory preBötC with major roles in rhythmic breathing and behaviors.

Thyroid hormones during the perinatal period are necessary to respiratory network development of newborn rats.

Thyroid hormones (THs) are essential for foetal brain development. Because the gestating mother is the main source of THs to the foetus, maternal hypothyroidism and/or premature birth compromise neurological outcomes in the offspring. Respiratory instability and recurrent apneas due to immaturity of the respiratory control network are major causes of morbidity in infants. Inadequate TH supply may be sufficient to delay perinatal maturation of the respiratory control system; however, this hypothesis remains untested. To address this issue, maternal hypothyroidism was induced by adding methimazole (MMI; 0.02% w/v) to the drinking water of pregnant dams from conception to postpartum day 4 (P4). The effect of TH supplementation on respiratory function was tested by injecting levothyroxine (L-T4) in newborns at P1. Respiratory function was assessed by plethysmography (in vivo) and recording of phrenic output from medullary preparations (in vitro). By comparison with controls, TH deficiency increased the frequency of apneas and decreased basal ventilation in vivo and prevented the age-dependent increase in phrenic burst frequency normally observed in vitro. The effects of TH deficiency on GABAergic modulation of respiratory activity were measured by bath application of muscimol (GABAA agonist) or bicuculline (GABAA antagonist). The phrenic burst frequency responses to GABAergic agents were consistently greater in preparations from TH deficient pups. L-T4 supplementation reversed part of the respiratory anomalies related to MMI treatment in vitro. We conclude that TH deficiency during the perinatal period is sufficient to delay maturation of the respiratory control network development. Excessive GABAergic inhibition may contribute to this effect.

Relativistic-intensity near-single-cycle light waveforms at kHz repetition rate.

The development of ultra-intense and ultra-short light sources is currently a subject of intense research driven by the discovery of novel phenomena in the realm of relativistic optics, such as the production of ultrafast energetic particle and radiation beams for applications. It has been a long-standing challenge to unite two hitherto distinct classes of light sources: those achieving relativistic intensity and those with pulse durations approaching a single light cycle. While the former class traditionally involves large-scale amplification chains, the latter class places high demand on the spatiotemporal control of the electromagnetic laser field. Here, we present a light source producing waveform-controlled 1.5-cycle pulses with a 719 nm central wavelength that can be focused to relativistic intensity at a 1 kHz repetition rate based on nonlinear post-compression in a long hollow-core fiber. The unique capabilities of this source allow us to observe the first experimental indications of light waveform effects in laser wakefield acceleration of relativistic energy electrons.

Facilitation of microglial motility by thyroid hormones requires the presence of neurons in cell culture: A distinctive feature of the brainstem versus the cortex.

Microglia are critical for the refinement of neural networks that takes place during the perinatal period. Their phenotype and actions are guided by the signals produced by neighbouring cells and hormones present in their surrounding milieu. Cell populations and the signals they produce differ between regions. The fact that thyroid hormones (THs) promote the growth and morphological differentiation of microglia within the cortex contributes to the TH's powerful actions on the developing brain. The brainstem is especially active during early life owing to its role in generation of the rhythmic respiratory motor command. Despite evidences indicating that THs are necessary to proper development of the neural networks regulating this vital homeostatic function, their actions on microglia originating from the brainstem remain unknown. Using primary cultured microglia from newborn mice (C57BL/6J), we first report that regulation of microglial motility by THs is different between cortex and brainstem. Microglial motility (µm traveled over 3 h) was monitored with or without triiodothyronine (T3, 1µM). Exposure to T3 did not stimulate microglial motility from brainstem, but significantly stimulated (316 %) when they were co-cultured with neurons. Motility of cortex microglia was stimulated to the similar extent either with or without neurons. These data suggest that the microglial function in different regions of the brain is determined by the surrounding environment.

Development of central respiratory control in anurans: The role of neurochemicals in the emergence of air-breathing and the hypoxic response.

Physiological and environmental factors impacting respiratory homeostasis vary throughout the course of an animal's lifespan from embryo to adult and can shape respiratory development. The developmental emergence of complex neural networks for aerial breathing dates back to ancestral vertebrates, and represents the most important process for respiratory development in extant taxa ranging from fish to mammals. While substantial progress has been made towards elucidating the anatomical and physiological underpinnings of functional respiratory control networks for air-breathing, much less is known about the mechanisms establishing these networks during early neurodevelopment. This is especially true of the complex neurochemical ensembles key to the development of air-breathing. One approach to this issue has been to utilize comparative models such as anuran amphibians, which offer a unique perspective into early neurodevelopment. Here, we review the developmental emergence of respiratory behaviours in anuran amphibians with emphasis on contributions of neurochemicals to this process and highlight opportunities for future research.

Maternal thyroid hormone deficiency and cardiorespiratory disorder in rat pups.

During gestation, the mother is the main source of thyroid hormones for the foetus. Thus, hypothyroidism during pregnancy and/or preterm birth compromise thyroid hormone supply for the foetus. Maternal hypothyroidism increases risk of preterm birth and both conditions are associated with respiratory distress in infants. Since thyroid hormones are essential for normal brain development, it is plausible that maternal thyroid hormone deficiency plays a role in respiratory disorders related to neurological immaturity in the newborn; however, this hypothesis is yet to be tested. Here, we used methimazole treatment (MMI; 0.05% v/w) from the onset of pregnancy until two weeks postpartum to induce thyroid hormone deficiency in rat pups. At 14-15 days of age, we used plethysmography to measure breathing at rest and in response to hypoxia (12% O2, 20 min) in intact pups. We then used a urethane/chloralose anesthetised preparation to measure cardiorespiratory inhibition induced by laryngeal chemoreflex stimulation. In intact pups, basal breathing did not differ between groups but the breathing frequency response to hypoxia of MMI-treated pups was lower than controls. Following anesthesia, breathing frequency of MMI pups was 60% lower than controls; following laryngeal chemoreflex stimulation, the drop in O2 saturation that was 82% greater in MMI-treated pups than controls. Inactivation of GABAA receptors (bicuculline; 0.5 mg/kg) raised the frequency of anesthetised MMI pups but not control. We conclude that gestational thyroid hormone deficiency interferes with the respiratory and autonomic control systems of the offspring. Thyroid hormone supplementation could alleviate cardiorespiratory disorders in newborn, especially those born preterm.

Neuropathic pain inhibitor, RAP-103, is a potent inhibitor of microglial CCL1/CCR8.

Chemokine signaling is important in neuropathic pain, with microglial cells expressing chemokine (C-C motif) receptor CCR2, CCR5 and CCR8, all playing key roles. In the previous report (Padi et al., 2012), oral administration of a short peptide, RAP-103, for 7 days fully prevents mechanical allodynia and inhibits the development of thermal hyperalgesia after partial ligation of the sciatic nerve in rodents. As for the mechanism of the inhibiting effect of RAP-103, it was speculated to be due to dual blockade of CCR2 and CCR5. We report here that RAP-103 exhibits stronger antagonism for CCR8 (half maximal inhibitory concentration [IC50] 7.7 fM) compared to CCR5 (IC50 < 100 pM) in chemotaxis using primary cultured mouse microglia. In addition, RAP-103 at a concentration of 0.1 pM completely inhibits membrane ruffling and phagocytosis induced by chemokine (C-C motif) ligand 1 (CCL1), an agonist for CCR8. It has been shown that CCL1/CCR8 signaling is important in tactile allodynia induced by nerve ligation. Therefore, CCR8, among other chemokine receptors such as CCR2/CCR5, could be the most potent target for RAP-103. Inhibitory effects of RAP-103 on plural chemokine receptors may play important roles for broad clinical use in neuropathic pain treatment.

Respiratory consequences of targeted losses of Hoxa5 gene function in mice.

Fetal development of the respiratory tract and diaphragm requires strict coordination between genetically controlled signals and mechanical forces produced by the neural network that generates breathing. HOXA5, which is expressed in the mesenchyme of the trachea, lung and diaphragm, and in phrenic motor neurons, is a key transcription factor regulating lung development and function. Consequently, most Hoxa5-/- mutants die at birth from respiratory failure. However, the extensive effect of the null mutation makes it difficult to identify the origins of respiratory dysfunction in newborns. To address the physiological impact of Hoxa5 tissue-specific roles, we used conditional gene targeting with the Dermo1Cre and Olig2Cre mouse lines to produce specific Hoxa5 deletions in the mesenchyme and motor neurons, respectively. Hoxa5 expression in the mesenchyme is critical for trachea development, whereas its expression in phrenic motor neurons is essential for diaphragm formation. Breathing measurements in adult mice with whole-body plethysmography demonstrated that, at rest, only the motor neuron deletion affects respiration, resulting in higher breathing frequency and decreased tidal volume. But subsequent exposure to a moderate hypoxic challenge (FiO2 =0.12; 10 min) revealed that both mutant mice hyperventilate more than controls. Hoxa5flox/flox;Dermo1+/Cre mice showed augmented tidal volume while Hoxa5flox/flox;Olig2+/Cre mice had the largest increase in breathing frequency. No significant differences were observed between medulla-spinal cord preparations from E18.5 control and Hoxa5flox/flox;Olig2+/Cre mouse embryos that could support a role for Hoxa5 in fetal inspiratory motor command. According to our data, Hoxa5 expression in the mesenchyme and phrenic motor neurons controls distinct aspects of respiratory development.

On the origins of sex-based differences in respiratory disorders: Lessons and hypotheses from stress neuroendocrinology in developing rats.

The environment plays a critical role in shaping development and function of the brain. Stress, especially when experienced early in life, can interfere with these processes. In the context of respiratory control, perinatal stress can therefore alter the ability to achieve the "fine-tuning" necessary for proper detection of chemosensory stimuli and production of an adequate motor (respiratory) command. Depending on the timing, intensity, and duration, the detrimental consequences of perinatal exposure to adverse conditions on the respiratory network become manifest at various life stages and can persist into adulthood. During early life, respiratory diseases commonly associated with dysfunction of neural networks include apnea of prematurity (AOP) and cardio-respiratory failure leading to sudden infant death syndrome (SIDS). Sleep disordered breathing (SDB) can occur at various life stages, including adulthood. Regardless of age, a common element of these disorders is their greater prevalence in males. While this sexual dimorphism points to a potential role of sex hormones, our understanding of the neuroendocrine mechanisms remain poorly understood. In addition to their modulatory influence on breathing, gonadal hormones regulate sexual differentiation of the brain. Stress alters these effects, and over the years our laboratory has used various perinatal stress protocols to gain insight into the origins of sex-based differences in respiratory disorders. This review discusses our recent advances with a focus on the sex-specific impact of early life stress on O2-chemoreflex function both in newborn and adult rats. We conclude by discussing the basic principles emerging from this work, potential mechanisms, and clinical relevance.

Sex-specific response to hypoxia in a reduced brainstem preparation from Xenopus laevis.

Respiratory reflexes and tolerance to hypoxia show significant sexual dimorphism. However, the data supporting this notion originates exclusively from mammals. To determine whether this concept is limited to this group of vertebrates, we examined the sex-specific response to acute hypoxia in an adult reduced brainstem preparation from Xenopus laevis. Within the first 5min of exposure to hypoxic aCSF (98% N2/2% CO2), recordings of respiratory-related activity show a stronger increase in fictive breathing frequency in males than females. This initial response was followed by a decrease in respiratory-related activity; this depression occurred 6min sooner in males than females. These results represent new evidences of sexual dimorphism in respiratory control in amphibians and provide potential insight in understanding the homology with other groups of vertebrates, including mammals.

Aldosterone, corticosterone, and thyroid hormone and their influence on respiratory control development in Lithobates catesbeianus: An in vitro study.

The emergence of air breathing during Lithobates catesbeianus development requires significant changes to the brainstem circuits that generate and regulate breathing; however, the mechanisms responsible for initiating this transformation remain largely unknown. Because amphibian metamorphosis is regulated by hormones such as aldosterone, corticosterone, and thyroid hormone (T3), we tested the hypothesis that exposing the brainstem to these hormones augments the fictive air breathing frequency in pre-metamorphic tadpoles. Brainstems were isolated and were placed either in the recording chamber (acute; 1h+1h recovery) or in a bottle (chronic exposure; 24h) for treatment. Brainstems were exposed to artificial cerebrospinal fluid (aCSF; sham treatment) or one of the following hormones: aldosterone (100nM), corticosterone (100nM), or T3 (100nM). While acute exposure had limited effects on respiratory motor output, chronic incubation with any hormone significantly increased fictive air breathing; the burst frequencies observed following treatment were similar to those observed in adult bullfrogs. We conclude that through their long term effects, hormones regulating metamorphosis can initiate the maturation of the neural circuits that generate and regulate breathing in this species.

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording.

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

Spectral-phase interferometry for direct electric-field reconstruction applied to seeded extreme-ultraviolet free-electron lasers.

We present a setup for complete characterization of femtosecond pulses generated by seeded free-electron lasers (FELs) in the extreme-ultraviolet spectral region. Two delayed and spectrally shifted replicas are produced and used for spectral phase interferometry for direct electric field reconstruction (SPIDER). We show that it can be achieved by a simple arrangement of the seed laser. Temporal shape and phase obtained in FEL simulations are well retrieved by SPIDER reconstructions, allowing to foresee the implementation of this diagnostics tool on existing and future sources. This will be a significant step towards an experimental investigation and control of FEL spectral phase.

Carrier-envelope-phase stable, high-contrast, double chirped-pulse-amplification laser system.

We present the first carrier-envelope-phase stable chirped-pulse amplifier (CPA) featuring high temporal contrast for relativistic intensity laser-plasma interactions at 1 kHz repetition rate. The laser is based on a double-CPA architecture including cross-polarized wave (XPW) filtering technique and a high-energy grism-based compressor. The 8 mJ, 22 fs pulses feature 10⁻¹¹ temporal contrast at -20 ps and a carrier-envelope-phase drift of 240 mrad root mean square.

Control of breathing in in vitro brain stem preparation from goldfish (Carassius auratus; Linnaeus).

In vitro brain stem preparations from goldfish (Carassius auratus) were used to first determine whether this species possesses central chemoreceptors able to modulate respiratory activity. Preparations were superfused with an artificial cerebrospinal fluid (aCSF); fictive breathing was recorded extracellularly by placing a suction electrode on cranial nerve VII. Reducing the level of O2 in the gas mixture used to bubble the aCSF from a hyperoxic level (80% or 98.7% O2) to a relative hypoxic level (20% or 40% O2) increased the frequency of the fictive respiratory burst (P = 0.0002). Reducing the pH of the aCSF from 7.9 to 7.4 by increasing CO2 in the superfusate did not affect fictive breathing. Chloride-mediated neurotransmission (GABA/glycine) is a major modulator of respiratory activity; however, its effect on the neural circuits that regulate breathing in teleosts remains unknown. Bath application of GABA (0.5, 5.0 mM) decreased burst frequency but not amplitude; this effect was dose dependent (drug × concentration: P = 0.01). Superfusion of the preparations with aCSF containing 1.25 µM of bicuculline methochloride and 1.50 µM of strychnine hydrochloride (GABAA and glycine receptor antagonists, respectively) increased burst frequency (P = 0.002) and amplitude (P < 0.001). We conclude that respiratory activity produced by the goldfish brain stem is not responsive to the moderate CO2 levels used in this study; it may contain O2 chemoreceptors, but the relatively small response could also reflect nonspecific effects of hypoxia on the central nervous system. Cl(-)-mediated neurotransmission inhibits fictive breathing; this aspect of respiratory regulation is similar to other groups of vertebrates.

Energy-scalable temporal cleaning device for femtosecond laser pulses based on cross-polarized wave generation.

We report on a compact energy-scalable device for generating high-fidelity femtosecond laser pulses based on spatial filtering through a hollow-core fiber followed by a nonlinear crystal for cross-polarized wave (XPW) generation. This versatile device is suited for temporal pulse cleaning over a wide range of input energies (from 0.1 to >10 mJ) and is successfully qualified on different ultrafast laser systems. Full characterization of the XPW output is presented. In particular, we demonstrate the generation of 1.6 mJ energy pulses starting from 11 mJ input pulse energy. The temporal contrast of the pulses is enhanced by more than 4 orders of magnitude. In addition, pulse shortening from 40 fs down to 15 fs Fourier-transform limit yields an overall peak-power transmission of up to 50%. This device not only serves as an integrated pulse contrast filter inside an ultrafast laser amplifier but also as a simple back-end solution for temporal post-compression of amplified pulses.

Highly efficient nonlinear filter for femtosecond pulse contrast enhancement and pulse shortening.

We propose a highly efficient scheme for temporal filters devoted to femtosecond pulse contrast enhancement. The filter is based on cross-polarized wave generation with a spatially suger-Gaussian-shaped beam. In a single nonlinear crystal scheme the energy conversion to the cross-polarized pulse can reach 28%. We demonstrate that the process enables a significant spectral broadening. For an efficiency of 23% the pulse shortening is estimated to 2.2, leading to an intensity transmission of the nonlinear filter of 50%.

Betatron oscillations of electrons accelerated in laser wakefields characterized by spectral x-ray analysis.

Relativistic electrons accelerated by laser wakefields can produce x-ray beams from their motion in plasma termed betatron oscillations. Detailed spectral characterization is presented in which the amplitude of the betatron oscillations r is studied by numerical analysis of electron and x-ray spectra measured simultaneously. We find that r reaches as low as 1 mum in agreement with previous studies of radiation based on coherence and far-field spatial profile.

Imaging electron trajectories in a laser-wakefield cavity using betatron X-ray radiation.

We demonstrate that betatron x-ray radiation accurately provides direct imaging of electrons trajectories accelerated in laser wakefields. Experimental far field x-ray beam profiles reveal that electrons can follow similar transverse trajectories with typical excursions of 1.5 microm+/-0.5 microm in the plane of laser polarization and 0.7 microm+/-0.2 microm in the plane perpendicular.