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1. The transverse brainstem slice preparation containing the pre-Bötzinger complex (PBC) was used in mice to study developmental changes of the response of the in vitro respiratory network to hypoxia. This preparation generates at different postnatal stages (postnatal days (P) 0-22) spontaneous rhythmic activity in hypoglossal (XII) rootlets that occur in synchrony with periodic bursts of neurones in the PBC. 2. In slices from P0-4 mice, hypoxia did not significantly affect the amplitude of rhythmic synaptic drive potentials in four of five inspiratory neurones. Hypoxia reduced, but did not suppress, the amplitude of synaptic drive potentials in only one inspiratory neurone. Spike discharge and phasic 'inspiratory' hyperpolarizations of six expiratory neurones were suppressed during hypoxia revealing a phasic 'inspiratory' depolarization. 3. The coupling between rhythmic activity in PBC neurones and XII bursts occurred under control conditions in preparations from P0-4 mice in a 1:1 manner (n = 11) and from mice older than P5 in a 3:1 manner (n = 9). During hypoxia, PBC and XII activity were linked in a 1:1 manner in all slices. 4. In six of fourteen inspiratory PBC neurones, the amplitude of synaptic drive potentials of slices from mice older than P8 was increased during the period of augmentation, reduced during the period of depression and suppressed during a hypoxic response which we refer to as central apnoea. Augmentation led to a weak-to-moderate membrane depolarization which on average was 4.8 +/- 3.7 mV. This depolarization was followed by a hyperpolarization of 6.2 +/- 4.1 mV only in four inspiratory neurones. In the majority of neurones (n = 9), however, membrane depolarization remained stable and was not followed by hyperpolarization. In expiratory neurones (n = 12) from this age group hypoxia suppressed phasic hyperpolarizations that occurred in synchrony with XII bursts. As similarly seen in inspiratory neurones, membrane potentials were depolarized by 5.1 +/- 4.1 mV during the period of hypoxic augmentation. 5. The hypoxic response of respiratory neurones within the pre-Bötzinger complex resembles the response of neurones that were previously described under in vivo conditions. Thus we conclude that the 'transverse rhythmic slice' is a good model for studying the hypoxic response of the respiratory network under in vitro conditions.

Neuronal networks are endogenously modulated by aminergic and peptidergic substances. These modulatory processes are critical for maintaining normal activity and adapting networks to changes in metabolic, behavioral, and environmental conditions. However, disturbances in neuromodulation have also been associated with pathologies. Using whole animals (in vivo) and functional brainstem slices (in vitro) from mice, we demonstrate that exposure to acute intermittent hypoxia (AIH) leads to fundamental changes in the neuromodulatory response of the respiratory network located within the preBötzinger complex (preBötC), an area critical for breathing. Norepinephrine, which normally regularizes respiratory activity, renders respiratory activity irregular after AIH. Respiratory irregularities are caused bothin vitroandin vivoby AIH, which increases synaptic inhibition within the preBötC when norepinephrine is endogenously or exogenously increased. These irregularities are prevented by blocking synaptic inhibition before AIH. However, regular breathing cannot be reestablished if synaptic inhibition is blocked after AIH. We conclude that subtle changes in synaptic transmission can have dramatic consequences at the network level as endogenously released neuromodulators that are normally adaptive become the drivers of irregularity. Moreover, irregularities in the preBötC result in irregularities in the motor outputin vivoand in incomplete transmission of inspiratory activity to the hypoglossus motor nucleus. Our finding has basic science implications for understanding network functions in general, and it may be clinically relevant for understanding pathological disturbances associated with hypoxic episodes such as those associated with myocardial infarcts, obstructive sleep apneas, apneas of prematurity, Rett syndrome, and sudden infant death syndrome.

“Sighs, tears, grief, distress” expresses Johann Sebastian Bach in a musical example for the relationship between sighs and deep emotions. This review explores the neurobiological basis of the sigh and its relationship with psychology, physiology, and pathology. Sighs monitor changes in brain states, induce arousal, and reset breathing variability. These behavioral roles homeostatically regulate breathing stability under physiological and pathological conditions. Sighs evoked in hypoxia evoke arousal and thereby become critical for survival. Hypoarousal and failure to sigh have been associated with sudden infant death syndrome. Increased breathing irregularity may provoke excessive sighing and hyperarousal, a behavioral sequence that may play a role in panic disorders. Essential for generating sighs and breathing is the pre-Bötzinger complex. Modulatory and synaptic interactions within this local network and between networks located in the brainstem, cerebellum, cortex, hypothalamus, amygdala, and the periaqueductal gray may govern the relationships between physiology, psychology, and pathology. Unraveling these circuits will lead to a better understanding of how we balance emotions and how emotions become pathological.

Breathing is essential for survival. Clinical disorders that challenge breathing often have severe consequences, potentially leading to catastrophic ventilatory failure and death. In some cases, we believe that we know the “primary cause(s)” of a particular breathing disturbance, but that limited understanding is not always sufficient to explain the clinical phenotype. For example, as indicated by its name, airway obstruction seems to be the primary cause for breathing cessation (or apneas) in Obstructive Sleep Apnea. However, genetic predisposition, reflex mechanisms, neuromodulatory mechanisms associated with sleep states, hypoxic and oxidative stress, mechanisms of central respiratory rhythm generation and alterations at the level of motor nuclei play equally important roles in this disorder (Kheirandish-Gozal and Gozal, 2013; Plataki et al., 2013; Ramirez et al., 2013a). With Chronic Obstructive Pulmonary Disease (COPD), impaired breathing seems to result primarily from impaired pulmonary mechanics caused by lung/airway obstructions (Jacono, 2013). But, hypoxia and hypercapnic conditions alter intrinsic properties of muscles, chemoreceptor signaling and central respiratory control. Some breathing disorders result from specific gene mutations, such as Rett Syndrome, a neurological disorder caused by mutations in the Mecp2 gene (Ramirez et al., 2013b), but the mutation causes multiple alterations in synaptic transmission and neuromodulation and disturbed breathing, which in turn has secondary consequences to aspects of respiratory control. Many suspect that Sudden Infant Death Syndrome (SIDS) results from impaired function of the serotonergic nervous system, but the genetic predisposition and/or prior experiences (Paterson, 2013), make this a highly complex syndrome (Garcia et al., 2013). Thus, although in many instances we seem to know the primary cause of clinical disorders that impair breathing, we are beginning to realize that the clinical phenotype is the result of a complex interplay of multiple mechanisms that are still poorly understood– even in the best known examples. This Special Issue of Respiratory Physiology and Neurobiology on “Clinical Challenges to Ventilatory Control” was conceived and edited by Drs. Ramirez, Mitchell, Baker-Herman and Paydarfar. Our motivation was to review relevant clinical disorders and provide mechanistic insights into a broad range of breathing disorders with distinct etiology. Individual contributions to this special issue draw attention to common features in ventilatory control disorders, and the fact that the “primary causes” initiate complex, interactive processes in the peripheral and central nervous systems. This interplay ultimately impairs (or preserves) breathing and, in some instances, leads to forms of dysautonomia resulting from disturbed coupling between breathing and other physiological systems, such as the cardiovascular system. Many breathing disorders are associated with chronic intermittent hypoxia (CIH). For example, Di Fiore et al. (2013) discuss the “perfect storm” leading to apnea of prematurity. While premature birth may be the “primary cause” the article describes how immature development of the respiratory control system leads to respiratory instability that, together with underdeveloped lungs (and resultant lung injury), leads to recurrent apneas and CIH. In turn, CIH causes immediate and long-lasting morbidities (Di Fiore et al., 2013, Williamson et al., 2013). CIH and oxidative stress is also characteristic for central and obstructive sleep apneas, as well as Rett Syndrome. The immediate and long-term morbidities associated with these disorders are discussed in several articles of this special issue (Ramirez et al., 2013a,b; Kheirandish-Gozal and Gozal, 2013, Plataki et al., 2013). In each case, the clinical phenotype is not caused by factors considered to be their “primary cause” alone, but by a complex multimodal interplay. Understanding, this complex interplay will ultimately be of great importance as we develop treatments for distinct breathing disorders. Williamson et al. (2013) for example proposes that multimodal physiological measurements are critical for developing specific treatment of apnea of prematurity. These measurements include heart-rate variability, breathing variability, entropy measurements and sleep state. Considering ventilatory control as a non-linear dynamical system is one approach to address the complex nature of this interplay (Williamson et al. (2013), and it will help to unravel the vicious cycle associated with recurrent hypoxemia resulting from (central or obstructive) apneic events (Williamson et al., 2013). Although CIH can be an important cause of long-term detrimental changes that transcend immediate effects on the respiratory system, brief intermittent hypoxia elicits potentially beneficial effects such as up-regulation of growth factors that are neuro-protective, and respiratory motor plasticity. Respiratory plasticity may compensate for the “primary cause” of clinical disorders that impair breathing. Brief intermittent hypoxia (versus CIH) can also be harnessed to trigger long-lasting respiratory motor plasticity as a treatment for devastating clinical disorders that compromise breathing, such as cervical spinal injury (Johnson and Mitchell, 2013) or motor neuron disease (Nichols et al., 2013). Respiratory plasticity has considerable potential to compensate for, or induce functional recovery in a range of breathing disorders (Strey et al., 2013; Mantilla and Sieck, 2013; Johnson and Mitchell, 2013; Dimarco and Kowalski, 2013; Nichols et al., 2013). We hope that this Special Issue will inspire a serious dialog between basic scientists that study cellular and systems-level mechanisms controlling breathing, and clinicians that treat the clinical disorders that compromise breathing. Even though clinicians and physiologists share a deep interest in breathing, unfortunately there is often a disconnect between these disciplines. Whereas basic scientists may have a deep understanding of the impact of intermittent hypoxia, genetic contributions to neurological disorders, or mechanisms of respiratory rhythm generation and neuromodulation, they are often unaware of the significance of these mechanisms in the complex interplay underlying particular breathing disorders or their associated dysautonomia. Conversely, clinicians are most often fully aware of the complexity of a breathing disorder, but often fail to consider sometimes well-known mechanisms that drive this complexity. We are confident that a better integration between the basic sciences and clinicians is essential to truly understand, and ultimately treat and cure clinical disorders leading to severe breathing disturbances.

Sudden Infant Death Syndrome (SIDS) remains the leading cause of infant mortality in Western societies. A prior study identified an association between hearing suppression on the newborn hearing test and subsequent death from SIDS. This is the first finding of an abnormality in SIDS cases prior to death. A following study identified that inner ear dysfunction precipitates a marked suppression of the hypercapnic ventilatory response (HCVR). Failure of arousal has been proposed to be a key component in SIDS. The objective of the present study was to assess whether inner ear dysfunction not only weakens the hypercapnic response, but also plays a role in suppressing the arousal response to suffocating gas mixtures.Wild-type mice (n=28) received intra-tympanic gentamicin (IT-Gent) injections bilaterally or unilaterally to precipitate inner ear hair cell dysfunction. Three control groups (n=22) received intra-tympanic saline (IT-Saline) bilaterally or unilaterally (right or left), or intra-peritoneal gentamicin (IP-Gent). The body movement arousal responses to severe hypoxia-hypercarbia combined (5% CO2 in nitrogen) were tested under light anesthesia 8 days following the administration of gentamicin or saline.After injections, the bilateral and unilateral IT-Gent-treated animals behaved similarly to controls, however the HCVR as well as the arousal movements in response to severe hypoxia-hypercarbia were suppressed in IT-Gent-treated animals compared to control animals (P0.05). Thus the HCVR was significantly decreased in the bilateral (n=9) and unilateral IT-Gent-treated mice (n=19) compared to bilateral (n=7) and unilateral IT-Saline (n=9) control groups (p0.05). Arousal movements were suppressed in the bilateral IT-Gent group (n=9) compared to bilateral IT-Saline controls (n=7, P0.0001) and in the unilateral IT-Gent group (n=19) compared to unilateral IT-Saline controls (n=10, P0.0001).The findings support the theory that inner ear dysfunction could be relevant in the pathophysiology of SIDS. The inner ear appears to play a key role in arousal from suffocating gas mixtures that has not been previously identified.

The pre-Bötzinger complex (preBötC), an area that is critical for generating breathing (eupnea), gasps and sighs is continuously modulated by catecholamines. These amines and the generation of sighs have also been implicated in the regulation of arousal. Here we studied the catecholaminergic modulation of sighs not only in anesthetized freely breathing mice (in vivo), but also in medullary slice preparations that contain the preBötC and that generate fictive eupneic and sigh rhythms in vitro. We demonstrate that activating -noradrenergic receptors (B-NR) specifically increases the frequency of sighs, while eupnea remains unaffected both in vitro and in vivo. B-NR activation specifically increased the frequency of intrinsically bursting pacemaker neurons that rely on persistent sodium current (INap). By contrast, all parameters of bursting pacemakers that rely on the non-specific cation current (ICAN) remained unaffected. Moreover, riluzole, which blocks bursting in INap pacemakers abolished sighs altogether, while flufenamic acid which blocks the ICAN current did not alter the sigh-increasing effect caused by B-NR. Our results suggest that the selective B-NR action of sighs may result from the modulation of INap pacemaker activity and that disturbances in noradrenergic system may contribute to abnormal arousal response. The B-NR action on the preBötC may be an important mechanism in modulating behaviors that are specifically associated with sighs, such as the regulation of the early events leading to the arousal response.

Apnea, the cessation of breathing, is a common physiological and pathophysiological phenomenon with many basic scientific and clinical implications. Among the different forms of apnea, obstructive sleep apnea (OSA) is clinically the most prominent manifestation. OSA is characterized by repetitive airway occlusions that are typically associated with peripheral airway obstructions. However, it would be a gross oversimplification to conclude that OSA is caused by peripheral obstructions. OSA is the result of a dynamic interplay between chemo- and mechanosensory reflexes, neuromodulation, behavioral state and the differential activation of the central respiratory network and its motor outputs. This interplay has numerous neuronal and cardiovascular consequences that are initially adaptive but in the long-term become major contributors to the morbidity and mortality associated with OSA. However, not only OSA, but all forms of apnea have multiple, and partly overlapping mechanisms. In all cases the underlying mechanisms are neither “exclusively peripheral” nor “exclusively central” in origin. While the emphasis has long been on the role of peripheral reflex pathways in the case of OSA, and central mechanisms in the case of central apneas, we are learning that such a separation is inconsistent with the integration of these mechanisms in all cases of apneas. This review discusses the complex interplay of peripheral and central nervous components that characterizes the cessation of breathing.

It is well-established that environmental and biological risk factors contribute to Sudden Infant Death Syndrome (SIDS). There is also growing consensus that SIDS requires the intersection of multiple risk factors that result in the failure of an infant to overcome cardio-respiratory challenges. Thus, the critical next steps in understanding SIDS are to unravel the physiological determinants that actually cause the sudden death, to synthesize how these determinants are affected by the known risk factors, and to develop novel ideas for SIDS prevention. In this review, we will examine current and emerging perspectives related to cardio-respiratory dysfunctions in SIDS. Specifically, we will review: (1) the role of the preBötzinger complex (preBötC) as a multi-functional network that is critically involved in the failure to adequately respond to hypoxic and hypercapnic challenges; (2) the potential involvement of the pre-BötC in the gender and age distributions that are characteristic for SIDS; (3) the link between SIDS and prematurity; and (4) the potential relationship between SIDS, auditory function, and central chemosensitivity. Each section underscores the importance of marrying the epidemiological and pathological data to experimental data in order to understand the physiological determinants of this syndrome. We hope that a better understanding will lead to novel ways to reduce the risk to succumb to SIDS.

New, improved in vivo and in vitro approaches have led to a better understanding of the mechanisms that generate respiratory rhythm, which depends on a complex interaction between network and intrinsic membrane properties. The pre-Bötzinger complex in the ventrolateral medulla is particularly important for respiratory rhythm generation. This complex can be studied in isolation, and it contains all the known types of respiratory neurons that are now amenable to detailed cellular and molecular analyses.

1. Spontaneous rhythmic activity in hypoglossal (XII) rootlets is generated at all postnatal stages from postnatal day (P) 0 to P22 in the transverse brainstem slice of mice containing the pre-Bötzinger complex (PBC). The PBC is known to be a region essential for respiratory rhythm generation. It contains neurones generating periodic bursts that occur in synchrony with rhythmic XII activity. This synchrony indicates that the rhythmic PBC activity generated by the transverse slice is the central respiratory rhythm. 2. The strength of coupling between XII bursts and PBC bursts decreased during early postnatal development. In younger mice (P0-4) each burst in XII rootlets corresponded to one burst in the PBC. In older mice (P5-18) one burst in XII rootlets occurred only every third to fourth burst in PBC neurones. 3. Cycle length and burst duration of rhythmic XII activity did not change significantly during the first three postnatal weeks. However, the pattern of XII bursts changed from decrementing (P0-7) to bell shaped (P8-18) while the rate of rise of XII bursts decreased significantly. 4. The rate of rise of rhythmic depolarizations in neurones of the PBC discharging in phase with XII bursts ('inspiratory neurones') decreased with postnatal development. During interburst intervals, membrane potentials of neurones of older mice (P6-18) were characterized by waves of synaptic input that were not observed in neonatal animals (P0-5). 5. Blockade of glycine receptors by strychnine increased the frequency of rhythmic XII activity in neonatal and older mice (P0-22). Although in expiratory PBC neurones glycinergic transmission was blocked at 10 microM strychnine, in inspiratory PBC neurones and XII rootlets even higher concentrations of up to 50 microM strychnine failed to abolish rhythmic activity.

Cardiac and respiratory activities are intricately linked both functionally as well as anatomically through highly overlapping brainstem networks controlling these autonomic physiologies that are essential for survival. Cardiorespiratory coupling (CRC) has many potential benefits creating synergies that promote healthy physiology. However, when such coupling deteriorates autonomic dysautonomia may ensue. Unfortunately there is still an incomplete mechanistic understanding of both normal and pathophysiological interactions that respectively give rise to CRC and cardiorespiratory dysautonomia. Moreover, there is also a need for better quantitative methods to assess CRC. This review addresses the current understanding of CRC by discussing: (1) the neurobiological basis of respiratory sinus arrhythmia (RSA); (2) various disease states involving cardiorespiratory dysautonomia; and (3) methodologies measuring heart rate variability and RSA.

The dentate gyrus (DG) is a unique cortical region whose protracted development spans the embryonic and early postnatal periods. DG development involves large-scale reorganization of progenitor cell populations, ultimately leading to the establishment of the subgranular zone neurogenic niche. In the developing DG, the T-box transcription factorTbr2is expressed in both Cajal-Retzius cells derived from the cortical hem that guide migration of progenitors and neurons to the DG, and intermediate neuronal progenitors born in the dentate neuroepithelium that give rise to granule neurons. Here we show that in miceTbr2is required for proper migration of Cajal-Retzius cells to the DG; and, in the absence ofTbr2, formation of the hippocampal fissure is abnormal, leading to aberrant development of the transhilar radial glial scaffold and impaired migration of progenitors and neuroblasts to the developing DG. Furthermore, loss ofTbr2results in decreased expression ofCxcr4in migrating cells, leading to a premature burst of granule neurogenesis during early embryonic development accompanied by increased cell death in mutant animals. Formation of the transient subpial neurogenic zone was abnormal inTbr2conditional knock-outs, and the stem cell population in the DG was depleted before proper establishment of the subgranular zone. These studies indicate thatTbr2is explicitly required for morphogenesis of the DG and participates in multiple aspects of the intricate developmental process of this structure.

P/Q-type voltage-gated calcium channels (Cav2.1) play critical presynaptic and postsynaptic roles throughout the nervous system and have been implicated in a variety of neurological disorders. Here we report that mice with a genetic ablation of the Cav2.1 pore-forming α1Asubunit (α1A−/−) encoded by CACNA1a (Jun et al., 1999) suffer during postnatal development from increasing breathing disturbances that lead ultimately to death. Breathing abnormalities include decreased minute ventilation and a specific loss of sighs, which was associated with lung atelectasis. Similar respiratory alterations were preserved in the isolatedin vitrobrainstem slice preparation containing the pre-Bötzinger complex. The loss of Cav2.1 was associated with an alteration in the functional dependency on N-type calcium channels (Cav2.2). Blocking N-type calcium channels with conotoxin GVIA had only minor effects on respiratory activity in slices from control (CT) littermates, but abolished respiratory activity in all slices from α1A−/−mice. The amplitude of evoked EPSPs was smaller in inspiratory neurons from α1A−/−mice compared with CTs. Conotoxin GVIA abolished all EPSPs in inspiratory neurons from α1A−/−mice, while the EPSP amplitude was reduced by only 30% in CT mice. Moreover, neuromodulation was significantly altered as muscarine abolished respiratory network activity in α1A−/−mice but not in CT mice. We conclude that excitatory synaptic transmission dependent on N-type and P/Q-type calcium channels is required for stable breathing and sighing. In the absence of P/Q-type calcium channels, breathing, sighing, and neuromodulation are severely compromised, leading to early mortality.

Glioblastoma Multiforme (GBM) is the most prevalent and aggressive form of cancer in the adult central nervous system. Ion channels are increasingly being linked to cancer progression through the regulation of cell proliferation and migration. However, the role of ion channels in GBM tumor formation and progression is not well understood. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence, suggesting that ion channel expression may be perturbed in this population. Here, we used RNA-sequencing to assess the expression patterns of ion channels and transporters and identify uniquely enriched ion channels in GSCs. Twenty-two patient-derived GSC samples were expression profiled along with human neural stem cell (NSC) and astrocyte control cell populations. Differential expression analysis revealed a set of ion channels highly enriched in GSCs compared with controls. Real-time PCR analysis confirmed the increased expression of ion channels of interest across selected GSC lines compared to NSCs. The expression pattern of ion channel candidates was also more likely to be associated with distinct GBM molecular subtypes. Next, an integrative approach was taken to further identify ion channels unique to GSCs; the abovementioned findings were compared to results from transcriptome analyses of GBM bulk tumor cells (The Cancer Genome Atlas) and region-specific GBM cells (Ivy Glioblastoma Atlas Project). Ion channels that were identified in these analyses were associated with altered clinical outcomes. The functional implications of these expression changes were further assessed with targeted drug screening of GSCs and NSCs. Pharmacological antagonists of GSC-enriched ion channels suppressed stem cell viability. These antagonists similarly hindered BrdU incorporation of dividing GSCs, suggesting that these channels play a role in stem cell proliferative capacity. Finally, calcium imaging was used to test the real-time functional responses of GSCs to channel blockers, and these outcomes will be discussed. Collectively, these findings suggest the presence of ion channels that uniquely identify GSCs from other neural cell types and influence GSC proliferation, a hallmark of GBM tumor recurrence. Citation Format: Julia Pollak, Karan G. Rai, Patrick J. Paddison, Robert C. Rostomily, Jan-Marino Ramirez. Transcriptional profiling of glioblastoma stem-like cells reveals enrichment of ion channels with functional implications for malignancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2523.

Mechanistic descriptions of rhythmogenic neural networks have often relied on ball-and-stick diagrams, which define interactions between functional classes of cells assumed to be reasonably homogenous. Application of this formalism to networks underlying respiratory rhythm generation in mammals has produced increasingly intricate models that have generated significant insight, but the underlying assumption that individual cells within these network fall into distinct functional classes has not been rigorously tested. In the present study we used multiunit extracellular recording in the in vitro pre-Bötzinger complex to identify and characterize the rhythmic activity of 951 cells. Inspiratory phase-dependent activity was estimated for all cells, and the data set as a whole was analyzed with principal component analysis, nonlinear dimensionality reduction, and hierarchical clustering techniques. None of these techniques revealed categorically distinct functional cell classes, indicating instead that the behavior of these cells within the network falls along several continua of spiking behavior.

Rhythmically active networks are typically composed of neurons that can be classified as silent, tonic spiking, or rhythmic bursting based on their intrinsic activity patterns. Within these networks, neurons are thought to discharge in distinct phase relationships with their overall network output, and it has been hypothesized that bursting pacemaker neurons may lead and potentially trigger cycle onsets. We used multielectrode recording from 72 experiments to test these ideas in rhythmically active slices containing the pre-Bötzinger complex, a region critical for breathing. Following synaptic blockade, respiratory neurons exhibited a gradient of intrinsic spiking to rhythmic bursting activities and thus defied an easy classification into bursting pacemaker and nonbursting categories. Features of their firing activity within the functional network were analyzed for correlation with subsequent rhythmic bursting in synaptic isolation. Higher firing rates through all phases of fictive respiration statistically predicted bursting pacemaker behavior. However, a cycle-by-cycle analysis indicated that respiratory neurons were stochastically activated with each burst. Intrinsically bursting pacemakers led some population bursts and followed others. This variability was not reproduced in traditional fully interconnected computational models, while sparsely connected network models reproduced these results both qualitatively and quantitatively. We hypothesize that pacemaker neurons do not act as clock-like drivers of the respiratory rhythm but rather play a flexible and dynamic role in the initiation and stabilization of each burst. Thus, at the behavioral level, each breath can be thought of as de novo assembly of a stochastic collaboration of network topology and intrinsic properties.

Unraveling the mechanisms underlying oscillatory behavior is critical for understanding normal and pathological brain processes. Here we used electrophysiology in mouse neocortical slices and principles of nonlinear dynamics to demonstrate how an increase in the N-methyl-d-aspartic acid receptor (NMDAR) conductance can create a nonlinear whole-cell current-voltage (I-V) relationship which leads to changes in cellular stability. We discovered two behaviorally and morphologically distinct pyramidal cell populations. Under control conditions, both cell types responded to depolarizing current injection with regular spiking patterns. However, upon NMDAR activation, an intrinsic oscillatory (IO) cell type (n = 44) showed a nonlinear whole-cell I-V relationship, intrinsic voltage-dependent oscillations plus amplification of alternating input current, and these properties persisted after disabling action potential generation with tetrodotoxin (TTX). The other non-oscillatory (NO) neuronal population (n = 24) demonstrated none of these behaviors. Simultaneous intra- and extracellular recordings demonstrated the NMDAR's capacity to promote low-frequency seizure-like network oscillations via its effects on intrinsic neuronal properties. The two pyramidal cell types demonstrated different relationships with network oscillation--the IO cells were leaders that were activated early in the population activity cycle while the activation of the NO cell type was distributed across network bursts. The properties of IO neurons disappeared in a low-magnesium environment where the voltage dependence of the receptor is abolished; concurrently, the cellular contribution to network oscillation switched to synchronous firing. Thus, depending upon the efficacy of NMDAR in altering the linearity of the whole-cell I-V relationship, the two cell populations played different roles in sustaining network oscillation.

Observational studies in humans have found associations between overstimulation in infancy via excessive television viewing and subsequent deficits in cognition and attention. We developed and tested a mouse model of overstimulation whereby p10 mice were subjected to audio (70 db) and visual stimulation (flashing lights) for six hours per day for a total of 42 days. 10 days later cognition and behavior were tested using the following tests: Light Dark Latency, Elevated Plus Maze, Novel Object Recognition, and Barnes Maze. In all tests, overstimulated mice performed significantly worse compared to controls suggesting increased activity and risk taking, diminished short term memory, and decreased cognitive function. These findings suggest that excessive non-normative stimulation during critical periods of brain development can have demonstrable untoward effects on subsequent neurocognitive function.

Neural networks called central pattern generators (CPGs) generate repetitive motor behaviors such as locomotion and breathing. Glutamatergic neurons are required for the generation and inhibitory neurons for the patterning of the motor activity associated with repetitive motor behaviors. In the mouse, glutamatergic V2a neurons coordinate the activity of left and right leg CPGs in the spinal cord enabling mice to generate an alternating gait. Here, we investigate the role of V2a neurons in the neural control of breathing, an essential repetitive motor behavior. We find that, following the ablation of V2a neurons, newborn mice breathe at a lower frequency. Recordings of respiratory activity in brainstem–spinal cord and respiratory slice preparations demonstrate that mice lacking V2a neurons are deficient in central respiratory rhythm generation. The absence of V2a neurons in the respiratory slice preparation can be compensated for by bath application of neurochemicals known to accelerate the breathing rhythm. In this slice preparation, V2a neurons exhibit a tonic firing pattern. The existence of direct connections between V2a neurons in the medial reticular formation and neurons of the pre-Botzinger complex indicates that V2a neurons play a direct role in the function of the respiratory CPG in newborn mice. Thus, neurons of the embryonic V2a lineage appear to have been recruited to neural networks that control breathing and locomotion, two prominent CPG-driven, repetitive motor behaviors.

We report on wide-field optically detected magnetic resonance imaging of nitrogen-vacancy centers (NVs) in type IIa polycrystalline diamond. These studies reveal a heterogeneous crystalline environment that produces a varied density of NV centers, including preferential orientation within some individual crystal grains, but preserves long spin coherence times. Using the native NVs as nanoscale sensors, we introduce a three-dimensional strain imaging technique with high sensitivity (, Allen Institute for Brain Science, Howard Hughes Medical Institute

Familial dysautonomia (FD) is a profound sensory and autonomic nervous system disorder associated with an increased risk for sudden death. While bradycardia resulting from loss of sympathetic tone has been hypothesized to play a role in this mortality, extended in-home monitoring has failed to find evidence of low heart rates in children with FD. In order to better characterize the specific cardio-respiratory pathophysiology and autonomic dysregulation in patients with FD, 25 affected children and matched controls were studied with in-home technology, during day and night. Respiratory and heart rate timing and variability metrics were derived from inductance plethysmography and electrocardiogram signals. Selective shortening of inspiratory time produced an overall increase in respiratory frequency in children with FD, with higher daytime respiratory variability (vs. controls), suggesting alterations in central rhythm generating circuits that may contribute to the heightened risk for sudden death. Overall heart rate was increased and variability reduced in FD cases, with elevated heart rates during 20% of study time. Time and frequency domain measures of autonomic tone indicated lower parasympathetic drive in FD patients (vs. controls). These results suggest withdrawal of vagal, rather than sympathetic tone, as a cause for the sustained increase and dramatic lability in respiration and heart rates that characterize this disorder. Pediatr Pulmonol. 2012; 47:682–691. © 2011 Wiley Periodicals, Inc.

Biogenic amines are not just 'modulators', they are often essential for the execution of behaviors. Here, we explored the role of biogenic amines acting on the pre-Bötzinger complex (pre-BötC), an area located in the ventrolateral medulla which is critical for the generation of different forms of breathing. Isolated in transverse slices from mice, this region continues to spontaneously generate rhythmic activities that resemble normal (eupneic) inspiratory activity in normoxia and gasping in hypoxia. We refer to these as 'fictive eupneic' and 'fictive gasping' activity. When exposed to hypoxia, the pre-BötC transitions from a network state relying on calcium-activated nonspecific cation currents (I(CAN)) and persistent sodium currents (I(Nap)) to one that primarily depends on the I(Nap) current. Here we show that in inspiratory neurons I(Nap)-dependent bursting, blocked by riluzole, but not I(CAN) -dependent bursting, required endogenously released norepinephrine acting on alpha2-noradrenergic receptors (α2-NR). At the network level, fictive eupneic activity persisted while fictive gasping ceased following the blockade of α2-NR. Blockade of α2-NR eliminated fictive gasping even in slice preparations as well as in inspiratory island preparations. Blockade of fictive gasping by α2-NR antagonists was prevented by activation of 5-hydroxytryptamine type 2A receptors (5-HT2A). Our data suggest that gasping depends on the converging aminergic activation of 5-HT2AR and α2-NR acting on riluzole-sensitive mechanisms that have been shown to be crucial for gasping.

Breathing emerges through complex network interactions involving neurons distributed throughout the nervous system. The respiratory rhythm generating network is composed of micro networks functioning within larger networks to generate distinct rhythms and patterns that characterize breathing. The pre-Bötzinger complex, a rhythm generating network located within the ventrolateral medulla assumes a core function without which respiratory rhythm generation and breathing cease altogether. It contains subnetworks with distinct synaptic and intrinsic membrane properties that give rise to different types of respiratory rhythmic activities including eupneic, sigh, and gasping activities. While critical aspects of these rhythmic activities are preserved when isolated in in vitro preparations, the pre-Bötzinger complex functions in the behaving animal as part of a larger network that receives important inputs from areas such as the pons and parafacial nucleus. The respiratory network is also an integrator of modulatory and sensory inputs that imbue the network with the important ability to adapt to changes in the behavioral, metabolic, and developmental conditions of the organism. This review summarizes our current understanding of these interactions and relates the emerging concepts to insights gained in other rhythm generating networks.

Breathing emerges through complex network interactions involving neurons distributed throughout the nervous system. The respiratory rhythm generating network is composed of micro networks functioning within larger networks to generate distinct rhythms and patterns that characterize breathing. The pre-Botzinger complex, a rhythm generating network located within the ventrolateral medulla assumes a core function without which respiratory rhythm generation and breathing cease altogether. It contains subnetworks with distinct synaptic and intrinsic membrane properties that give rise to different types of respiratory rhythmic activities including eupneic, sigh, and gasping activities. While critical aspects of these rhythmic activities are preserved when isolated in in vitro preparations, the pre-Botzinger complex functions in the behaving animal as part of a larger network that receives important inputs from areas such as the pons and parafacial nucleus. The respiratory network is also an integrator of modulatory and sensory inputs that imbue the network with the important ability to adapt to changes in the behavioral, metabolic, and developmental conditions of the organism. This review summarizes our current understanding of these interactions and relates the emerging concepts to insights gained in other rhythm generating networks.

The modulatory actions of the biogenic amine octopamine on the femur tibia (FT) control loop in the stick insect Carausius morosus were examined. The response properties of the FT control loop were determined under open loop conditions. Mechanical stimulation of the femoral chordotonal organ (fCO) was the input and tibial movement and motoneuronal activity were measured as the output of the system. Following octopamine injection into the hemolymph of intact, inactive animals, two consecutive phases occurred at the behavioral level. Octopamine caused initially an activation of the animal. During this first phase (3.5–12 min duration) the response properties of the FT control loop were similar to those found in animals that were activated by tactile stimuli under normal conditions. Afterward, animals became inactive. During this second phase (15–20 min duration), the gain of the control loop was zero and no resistance reflex in the FT joint was generated in response to fCO stimulation. However, active movements of the tibia could still be elicited. As we could show in restrained animals, where dl-octopamine was applied topically onto the undesheated mesothoracic ganglion, the complete suppression of the resistance reflex on the motoneuronal level was dose dependent starting at concentrations of 5 ± 10−3M octopamine. We could show that octopamine specifically suppressed the pathways involved in the resistance reflex, while feedback loop responses to fCO stimuli typical for active animals could still be elicited. Our results indicate that an increase in the octopamine concentration mimicks activation of the animal: Properties being characteristic for the control of the FT joint in the inactive animal are inhibited by octopamine, while properties of the FT control loop typical for the active animal appear to be facilitated following octopamine injection. The results clearly demonstrate that different pathways in the neuronal network underlying the FT control loop are involved in the responses of the control loop to fCO stimuli in the inactive and active behavioral states of the stick insect. © 1993 John Wiley & Sons, Inc.

A complex cascade of neuronal, hormonal, muscular, and metabolic events leads to the initiation and maintenance of a behavioral act. Hormones are released to mobilize metabolic energy; muscle properties are altered to ensure the generation of the necessary force; neuronal circuits are activated to generate a specific locomotor pattern; and the neuronal circuit underlying respiration may also be altered to adapt the animal to increased oxygen consumption. All these events are part of a complex transition of the animal into a new behavioral state. A critical aspect of this transition is the need to alter these events at the appropriate time. This coordinating function may not only depend on neuronal interaction but may also be attributed to chemical substances that bias, at many levels, neuronal, hormonal, and muscular events towards the new functional state of the animal. For example, egg-laying by Aplysia spp. requires several peptides (38, 76) to elicit a complex of behaviors, including the cessation of locomotion, increase in heart and respiratory rate, egg laying, head waving, and mucous ecretion. Similarly, serotonin controls

To test the hypothesis that focal and parafocal neocortical tissue from pediatric patients with intractable epilepsy exhibits cellular and synap- tic differences, the authors characterized the propensity of these neurons to generate (a) voltage-dependent bursting and (b) synaptically driven parox- ysmal depolarization shifts. Neocortical slices were prepared from tissue resected from patients with intractable epilepsy. Multiunit network activity and simultaneous whole-cell patch recordings were made from neurons from three patient groups: (1) those with normal histology; (2) those with mild and severe cortical dysplasia; and (3) those with abnormal pathology but without cortical dysplasia. Seizure-like activity was characterized by population bursting with concomitant bursting in intracellularly recorded cortical neu- rons (n 59). The authors found significantly more N-methyl-D-aspartic acid-driven voltage-dependent bursting neurons in focal versus parafocal tissue in patients with severe cortical dysplasia (P 0.01). Occurrence of paroxysmal depolarization shifts and burst amplitude and burst duration were significantly related to tissue type: focal or parafocal (P 0.05). The authors show that functional differences between focal and parafocal tissue in patients with severe cortical dysplasia exist. There are functional differences between patient groups with different histology, and bursting properties can be significantly associated with the distinction between focal and parafocal tissue.

N-Methyl-D-aspartate (NMDA) receptors have been implicated in epileptogenesis, but how these receptors contribute to epilepsy remains unknown. In particular, their role is likely to be complicated because of their voltage-dependent behavior. Here, the authors investigate how activation of NMDA receptors can affect the intrinsic production of oscillation and the resonance properties of neocortical pyramidal neurons from children with intractable epilepsy. Intracellular whole-cell patch clamp recordings in cortical slices from these patients revealed that pyramidal neurons do not produce spontaneous oscillation under control conditions. However, they did exhibit resonance around 1.5 Hz. On NMDA receptor activation, with bath-applied NMDA (10 μM), the majority of neurons produced voltage-dependent intrinsic oscillation associated with a change in the stability of the neuronal system as reflected by the whole-cell I-V curve. Furthermore, the degree of resonance was amplified while the frequency of resonance was shifted to lower frequencies (∼1 Hz) in NMDA. These results suggest that NMDA receptors may both promote the production of low-frequency oscillation and sharpen the response of the cell to lower frequencies. Both these behaviors may be amplified in tissue from patients with epilepsy, resulting in an increased propensity to generate seizures.

Direct evidence that the mammalian neocortex is an important generator of intrinsic activity comes from isolated neocortical slices that spontaneously generate multiple rhythms including those in the beta, delta and gamma range. These oscillations are also seen in intact animals where they interact with other areas including the hippocampus, thalamus and basal ganglia. Here we show that thick isolated neocortical slices from hooded seals intrinsically generate persistent spontaneous activities, both repetitive non-rhythmic activity with activity states lasting for several minutes, and oscillating activity with rhythms that are much slower (

All neuronal networks are modulated by multiple neuropeptides and biogenic amines. Yet, few studies investigate how different modulators interact to regulate network activity. Here we explored the state-dependent functional interactions between three excitatory neuromodulators acting on neurokinin1 (NK1), alpha1 noradrenergic (alpha1 NE), and 5-HT2 serotonin receptors within the pre-Botzinger complex (pre-BotC), an area critical for the generation of breathing. In anesthetized, in vivo mice, the reliance on endogenous NK1 activation depended on spontaneous breathing frequency and the modulatory state of the animal. Endogenous NK1 activation had no significant respiratory effect when stimulating raphe magnus and/or locus ceruleus, but became critical when alpha1 NE and 5-HT2 receptors were pharmacologically blocked. The dependence of the centrally generated respiratory rhythm on NK1 activation was blunted in the presence of alpha1 NE and 5-HT2 agonists as demonstrated in slices containing the pre-BotC. We conclude that a modulator's action is determined by the concurrent modulation and interaction with other neuromodulators. Deficiencies in one neuromodulator are immediately compensated by the action of other neuromodulators. This interplay could play a role in the state dependency of certain breathing disorders.

The flight motor pattern of the adult locust (Locusta migratoria L.) is able to recover from the loss of the hindwing tegulae. This recovery is due to a functional substitution of the hindwing tegulae by the forewing tegulae (Büschges, Ramirez, and Pearson, 1992). To assess changes in the pathways from the forewing tegulae in the flight system, we investigated the pathways of the forewing tegula in intact locusts and in animals 2 weeks after hindwing tegula removal. The following physiological alterations in these pathways were found to be associated with the recovery: (1) In the intact locusts, the connections of forewing tegula afferents to flight interneurons are variable but this variability did not occur in recovered animals, and (2) larger numbers of forewing tegula afferents connect to interneurons that excite elevator motoneurons (interneurons 566 and 567) and to an interneuron that inhibits depressor motoneurons (interneuron 511). The size of unitary excitatory postsynaptic potentials (EPSPs) evoked by signal forewing tegula afferents was found not to be altered in recovered animals. The changes in connectivity of forewing tegula afferents are correlated with morphological alterations in the structure of the terminal processes of the afferents and with sprouting of some branches of interneurons receiving input from these afferents.

International audience; Disorders of respiratory control are a prominent feature of Rett syndrome (RTT), a severely debilitating condition caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2). RTT patients present with a complex respiratory phenotype that can include periods of hyperventilation, apnea, breath holds terminated by Valsalva maneuvers, forced and deep breathing and apneustic breathing, as well as abnormalities of heart rate control and cardiorespiratory integration. Recent studies of mouse models of RTT have begun to shed light on neurologic deficits that likely contribute to respiratory dysfunction including, in particular, defects in neurochemical signaling resulting from abnormal patterns of neurotransmitter and neuromodulator expression. The authors hypothesize that breathing dysregulation in RTT results from disturbances in mechanisms that modulate the respiratory rhythm, acting either alone or in combination with more subtle disturbances in rhythm and pattern generation. This article reviews the evidence underlying this hypothesis as well as recent efforts to translate our emerging understanding of neurochemical defects in mouse models of RTT into preclinical trials of potential treatments for respiratory dysfunction in this disease.

Deep diving mammals have developed strategies to cope with limited oxygen availability when submerged. These adaptations are associated with an increased neuronal hypoxia tolerance. Brain neurons of the hooded seal Cysto- phora cristata remain much longer active in hypoxic condi- tions than those of mice. To understand the cellular basis of neuronal hypoxia tolerance, we studied neuroglobin and cy- tochrome c in C. cristata brain. Neuroglobin, a respiratory protein typically found in vertebrate neurons, displays three unique amino acid substitutions in hooded seal. However, these substitutions unlikely contribute to a modulation of O2 affinity. Moreover, there is no significant difference in total neuroglobin protein levels in mouse, rat and seal brains. However, in terrestrial mammals neuroglobin resided exclu- sively in neurons, whereas in seals neuroglobin is mainly lo- cated in astrocytes. This unusual localization of neuroglobin is accompanied by a shift in the distribution of cytochrome c. In seals, this marker for oxidative metabolism is mainly localized in astrocytes, whereas in terrestrial mammals it is essentially found in neurons. Our results indicate that in seals aerobic ATP production depends significantly on astrocytes, while neurons rely less on aerobic energy metabolism. This adaptation may imbue seal neurons with an increased tolerance to hypoxia and potentially also to reactive oxygen species, and may explain in part the ability of deep diving mammals to sustain neuronal activity during prolonged dives. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

1. Modulatory effects of octopamine perfusion on identified central neurons in the flight system of the locust Locusta migratoria were examined by means of intracellular recordings from the isolated metathoracic ganglion. 2. Octopamine increased the excitatory response of elevator motoneurons to electrical stimulation of the hindwing tegula and increased the probability of triggering rhythmic activity in the flight system by current injection into single interneurons. 3. These effects of octopamine on the flight system are due in part to octopamine inducing intrinsic bursting properties in flight interneurons. Plateau potentials were evoked in these interneurons by synaptic input from tegula or by the injection of depolarizing current pulses. These potentials were prematurely terminated by hyperpolarizing currents, and their generation was voltage sensitive in that they were suppressed with hyperpolarizing offset currents. 4. Longer depolarizing current pulses evoked endogenous bursting in a number of flight interneurons. This rhythmic bursting was reset by the injection of pulses of hyperpolarizing currents. The frequency of bursting was dependent on the injected current strength. 5. The injection of hyperpolarizing current into flight interneurons during octopamine-induced rhythmic activity lead to sudden decreases in the amplitude of the depolarizations thus indicating that plateau potentials contribute to the generation of the rhythmic depolarizations. 6. The shape of the depolarizations, the duration of the bursts (50–75 ms), and the frequency range of endogenous bursting (4-16 Hz) as seen in individual interneurons during octopamine perfusion were similar to the corresponding characteristics in the same neurons during wind-induced flight activity in deafferented locusts. This correspondence suggests that intrinsic bursting properties may play an important role in generating the normal motor pattern for flight.