Silvia Pagliardini

Active Expiration Induced by Excitation of Ventral Medulla in Adult Anesthetized Rats

Data from perinatal and juvenile rodents support our hypothesis that the preBötzinger complex generates inspiratory rhythm and the retrotrapezoid nucleus–parafacial respiratory group (RTN/pFRG) generates active expiration (AE). Although the role of the RTN/pFRG in adulthood is disputed, we hypothesized that its rhythmogenicity persists but is typically silenced by synaptic inhibition. We show in adult anesthetized rats that local pharmacological disinhibition or optogenetic excitation of the RTN/pFRG can generate AE and transforms previously silent RTN/pFRG neurons into rhythmically active cells whose firing is correlated with late-phase active expiration. Brief excitatory stimuli also reset the respiratory rhythm, indicating strong coupling of AE to inspiration. The AE network location in adult rats overlaps with the perinatal pFRG and appears lateral to the chemosensitive region of adult RTN. We suggest that (1) the RTN/pFRG contains a conditional oscillator that generates AE, and (2) at rest and in anesthesia, synaptic inhibition of RTN/pFRG suppresses AE.

Ontogeny of the pre-Bötzinger complex in perinatal rats.

The aim of this study was to provide a systematic examination of the ontogenesis of the mammalian respiratory rhythm generating center, the pre-Bötzinger complex (pre-BötC). A combination of immunohistochemical markers and electrophysiological recordings was used to determine the time of inception of the pre-BötC and the developmental changes during the perinatal period in rats spanning from embryonic day 15 (E15) to postnatal day 7. The first clear indication of neurons immunopositive for neurokinin-1 receptors (NK1Rs) and somatostatin expression, two proposed markers for pre-BötC neurons, was at ∼E17. Birth dating of neurons in the ventrolateral medulla using 5-bromo-2′-deoxyuridine demonstrated that NK1R-positive neurons populating the area of the pre-BötC during late E16-E18 are born at E12.5-E13.5, ∼2 d later than adjacent NK1R-positive neurons in the ventrolateral medulla. Extracellular recordings of neuronal populations within the pre-BötC of perinatal medullary slice preparations demonstrated that the onset of rhythmical respiratory discharge commences at ∼E17. Application of substance P, a ligand for NK1R receptors, to the media bathing E17 medullary slice and brainstem-spinal cord preparations resulted in a marked increase in respiratory frequency. These data provide insights into the ontogeny of the pre-BötC, giving fundamental information on the genesis, settlement, and inception of rhythmic activity within the group of neurons proposed to be responsible for the respiratory rhythm generation. Furthermore, this provides the foundation for further analyses of cell lineage, the transcriptional control of respiratory neuronal development, and electrophysiological and pharmacological properties of the pre-BötC during the prenatal period.

Projections of PreBötzinger Complex Neurons in Adult Rats

The preBötzinger Complex (preBötC) contains neural microcircuitry essential for normal respiratory rhythm generation in rodents. A subpopulation of preBötC neurons expresses somatostatin, a neuropeptide with a modulatory action on breathing. Acute silencing of a subpopulation of preBötC neurons transfected by a virus driving protein expression under the somatostatin promoter results in persistent apnea in awake adult rats. Given the profound effect of silencing these neurons, their projections are of interest. We used an adeno‐associated virus to overexpress enhanced green fluorescent protein driven by the somatostatin promoter in preBötC neurons to label their axons and terminal fields. These neurons send brainstem projections to: 1) contralateral preBötC; 2) ipsi‐ and contralateral Bötzinger Complex; 3) ventral respiratory column caudal to preBötC; 4) parafacial respiratory group / retrotrapezoid nucleus; 5) parahypoglossal nucleus/nucleus of the solitary tract; 6) parabrachial/Kölliker‐Fuse nuclei; and 7) periaqueductal gray. We did not find major projections to either cerebellum or spinal cord. We conclude that there are widespread projections from preBötC somatostatin‐expressing neurons specifically targeted to brainstem regions implicated in control of breathing, and provide a network basis for the profound effects and the essential role of the preBötC in breathing. J. Comp. Neurol. 518:1862–1878, 2010.

Developmental abnormalities of neuronal structure and function in prenatal mice lacking the prader-willi syndrome gene necdin.

Necdin (Ndn) is one of a cluster of genes deleted in the neurodevelopmental disorder Prader-Willi syndrome (PWS). Ndntm2Stw mutant mice die shortly after birth because of abnormal respiratory rhythmogenesis generated by a key medullary nucleus, the pre-Bötzinger complex (preBötC). Here, we address two fundamental issues relevant to its pathogenesis. First, we performed a detailed anatomical study of the developing medulla to determine whether there were defects within the preBötC or synaptic inputs that regulate respiratory rhythmogenesis. Second, in vitro studies determined if the unstable respiratory rhythm in Ndntm2Stw mice could be normalized by neuromodulators. Anatomical defects in Ndntm2Stw mice included defasciculation and irregular projections of axonal tracts, aberrant neuronal migration, and a major defect in the cytoarchitecture of the cuneate/gracile nuclei, including dystrophic axons. Exogenous application of neuromodulators alleviated the long periods of slow respiratory rhythms and apnea, but some instability of rhythmogenesis persisted. We conclude that deficiencies in the neuromodulatory drive necessary for preBötC function contribute to respiratory dysfunction of Ndntm2Stw mice. These abnormalities are part of a more widespread deficit in neuronal migration and the extension, arborization, and fasciculation of axons during early stages of central nervous system development that may account for respiratory, sensory, motor, and behavioral problems associated with PWS.

Fluorescent Tagging of Rhythmically Active Respiratory Neurons within the Pre-Bötzinger Complex of Rat Medullary Slice Preparations

Elucidation of the neuronal mechanisms underlying respiratory rhythmogenesis is a major focal point in respiratory physiology. An area of the ventrolateral medulla, the pre-Bötzinger complex (preBötC), is a critical site. Attention is now focused on understanding the cellular and network properties within the preBötC that underlie this critical function. The inability to clearly identify key “rhythm-generating” neurons within the heterogeneous population of preBötC neurons has been a significant limitation. Here we report an advancement allowing precise targeting of neurons expressing neurokinin-1 receptors (NK1Rs), which are hypothesized to be essential for respiratory rhythmogenesis. The internalization of tetramethylrhodamine conjugated substance P in rhythmically active medullary slice preparations provided clear visualization of NK1R-expressing neurons for subsequent whole-cell patch-clamp recordings. Among labeled neurons, 82% were inspiratory modulated, and 25% had pacemaker properties. We propose that this approach can be used to greatly expedite progress toward understanding the neuronal processes underlying the control of breathing.

Tripartite Purinergic Modulation of Central Respiratory Networks during Perinatal Development: The Influence of ATP, Ectonucleotidases, and ATP Metabolites

ATP released during hypoxia from the ventrolateral medulla activates purinergic receptors (P2Rs) to attenuate the secondary hypoxic depression of breathing by a mechanism that likely involves a P2Y1R-mediated excitation of preBötzinger complex (preBötC) inspiratory rhythm-generating networks. In this study, we used rhythmically active in vitro preparations from embryonic and postnatal rats and ATP microinjection into the rostral ventral respiratory group (rVRG)/preBötC to reveal that these networks are sensitive to ATP when rhythm emerges at embryonic day 17 (E17). The peak frequency elicited by ATP at E19 and postnatally was the same (∼45 bursts/min), but relative sensitivity was threefold greater at E19, reflecting a lower baseline frequency (5.6 ± 0.9 vs 19.0 ± 1.3 bursts/min). Combining microinjection techniques with ATP biosensors revealed that ATP concentration in the rVRG/preBötC falls rapidly as a result of active processes and closely correlates with inspiratory frequency. A phosphate assay established that preBötC-containing tissue punches degrade ATP at rates that increase perinatally. Thus, the agonist profile [ATP/ADP/adenosine (ADO)] produced after ATP release in the rVRG/preBötC will change perinatally. Electrophysiology further established that the ATP metabolite ADP is excitatory and that, in fetal but not postnatal animals, ADO at A1 receptors exerts a tonic depressive action on rhythm, whereas A1 antagonists extend the excitatory action of ATP on inspiratory rhythm. These data demonstrate that ATP is a potent excitatory modulator of the rVRG/preBötC inspiratory network from the time it becomes active and that ATP actions are determined by a dynamic interaction between the actions of ATP at P2 receptors, ectonucleotidases that degrade ATP, and ATP metabolites on P2Y and P1 receptors.

State-Dependent Modulation of Breathing in Urethane-Anesthetized Rats

Respiratory activity is most fragile during sleep, in particular during paradoxical [or rapid eye movement (REM)] sleep and sleep state transitions. Rats are commonly used to study respiratory neuromodulation, but rodent sleep is characterized by a highly fragmented sleep pattern, thus making it very challenging to examine different sleep states and potential pharmacological manipulations within them. Sleep-like brain-state alternations occur in rats under urethane anesthesia and may be an effective and efficient model for sleep itself. The present study assessed state-dependent changes in breathing and respiratory muscle modulation under urethane anesthesia to determine their similarity to those occurring during natural sleep. Rats were anesthetized with urethane and respiratory airflow, as well as electromyographic activity in respiratory muscles were recorded in combination with local field potentials in neocortex and hippocampus to determine how breathing pattern and muscle activity are modulated with brain state. Measurements were made in normoxic, hypoxic, and hypercapnic conditions. Results were compared with recordings made from rats during natural sleep. Brain-state alternations under urethane anesthesia were closely correlated with changes in breathing rate and variability and with modulation of respiratory muscle tone. These changes closely mimicked those observed in natural sleep. Of great interest was that, during both REM and REM-like states, genioglossus muscle activity was strongly depressed and abdominal muscle activity showed potent expiratory modulation. We demonstrate that, in urethane-anesthetized rats, respiratory airflow and muscle activity are closely correlated with brain-state transitions and parallel those shown in natural sleep, providing a useful model to systematically study sleep-related changes in respiratory control.

Central Respiratory Rhythmogenesis Is Abnormal in Lbx1- Deficient Mice

Lbx1 is a transcription factor that determines neuronal cell fate and identity in the developing medulla and spinal cord. Newborn Lbx1 mutant mice die of respiratory distress during the early postnatal period. Using in vitro brainstem–spinal cord preparations we tested the hypothesis that Lbx1 is necessary for the inception, development and modulation of central respiratory rhythmogenesis. The inception of respiratory rhythmogenesis at embryonic day 15 (E15) was not perturbed in Lbx1 mutant mice. However, the typical age-dependent increase in respiratory frequency observed in wild-type from E15 to P0 was not observed in Lbx1 mutant mice. The slow respiratory rhythms in E18.5 Lbx1 mutant preparations were increased to wild-type frequencies by application of substance P, thyrotropin releasing hormone, serotonin, noradrenaline, or the ampakine drug 1-(1,4-benzodioxan-6-yl-carbonyl) piperidine. Those data suggest that respiratory rhythm generation within the pre-Bötzinger complex (preBötC) is presumably functional in Lbx1 mutant mice with additional neurochemical drive. This was supported by anatomical data showing that the gross structure of the preBötC was normal, although there were major defects in neuronal populations that provide important modulatory drive to the preBötC including the retrotrapezoid nucleus, catecholaminergic brainstem nuclei, nucleus of the solitary tract, and populations of inhibitory neurons in the ventrolateral and dorsomedial medullary nuclei. Finally, we determined that those defects were caused by abnormalities of neuronal specification early in development or subsequent neuronal migration.

The peptidergic control circuit for sighing

Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B (Nmb) or gastrin-releasing peptide (Grp). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs.

Spontaneous sleep-like brain state alternations and breathing characteristics in urethane anesthetized mice.

Brain state alternations resembling those of sleep spontaneously occur in rats under urethane anesthesia and they are closely linked with sleep-like respiratory changes. Although rats are a common model for both sleep and respiratory physiology, we sought to determine if similar brain state and respiratory changes occur in mice under urethane. We made local field potential recordings from the hippocampus and measured respiratory activity by means of EMG recordings in intercostal, genioglossus, and abdominal muscles. Similar to results in adult rats, urethane anesthetized mice displayed quasi-periodic spontaneous forebrain state alternations between deactivated patterns resembling slow wave sleep (SWS) and activated patterns resembling rapid eye movement (REM) sleep. These alternations were associated with an increase in breathing rate, respiratory variability, a depression of inspiratory related activity in genioglossus muscle and an increase in expiratory-related abdominal muscle activity when comparing deactivated (SWS-like) to activated (REM-like) states. These results demonstrate that urethane anesthesia consistently induces sleep-like brain state alternations and correlated changes in respiratory activity across different rodent species. They open up the powerful possibility of utilizing transgenic mouse technology for the advancement and translation of knowledge regarding sleep cycle alternations and their impact on respiration.