Anatomical distribution of µ-opioid receptors, neurokinin-1 receptors, and vesicular glutamate transporter 2 in the mouse brainstem respiratory network.

µ-Opioid receptors (MORs) are responsible for mediating both the analgesic and respiratory effects of opioid drugs. By binding to MORs in brainstem regions involved in controlling breathing, opioids produce respiratory depressive effects characterized by slow and shallow breathing, with potential cardiorespiratory arrest and death during overdose. To better understand the mechanisms underlying opioid-induced respiratory depression, thorough knowledge of the regions and cellular subpopulations that may be vulnerable to modulation by opioid drugs is needed. Using in situ hybridization, we determined the distribution and coexpression of Oprm1 (gene encoding MORs) mRNA with glutamatergic (Vglut2) and neurokinin-1 receptor (Tacr1) mRNA in medullary and pontine regions involved in breathing control and modulation. We found that >50% of cells expressed Oprm1 mRNA in the preBötzinger complex (preBötC), nucleus tractus solitarius (NTS), nucleus ambiguus (NA), postinspiratory complex (PiCo), locus coeruleus (LC), Kölliker-Fuse nucleus (KF), and the lateral and medial parabrachial nuclei (LBPN and MPBN, respectively). Among Tacr1 mRNA-expressing cells, >50% coexpressed Oprm1 mRNA in the preBötC, NTS, NA, Bötzinger complex (BötC), PiCo, LC, raphe magnus nucleus, KF, LPBN, and MPBN, whereas among Vglut2 mRNA-expressing cells, >50% coexpressed Oprm1 mRNA in the preBötC, NTS, NA, BötC, PiCo, LC, KF, LPBN, and MPBN. Taken together, our study provides a comprehensive map of the distribution and coexpression of Oprm1, Tacr1, and Vglut2 mRNA in brainstem regions that control and modulate breathing and identifies Tacr1 and Vglut2 mRNA-expressing cells as subpopulations with potential vulnerability to modulation by opioid drugs.NEW & NOTEWORTHY Opioid drugs can cause serious respiratory side-effects by binding to µ-opioid receptors (MORs) in brainstem regions that control breathing. To better understand the regions and their cellular subpopulations that may be vulnerable to modulation by opioids, we provide a comprehensive map of Oprm1 (gene encoding MORs) mRNA expression throughout brainstem regions that control and modulate breathing. Notably, we identify glutamatergic and neurokinin-1 receptor-expressing cells as potentially vulnerable to modulation by opioid drugs and worthy of further investigation using targeted approaches.

Galaninergic and hypercapnia-activated neuronal projections to the ventral respiratory column.

In mammals, the ventral respiratory column (VRC) plays a pivotal role in integrating neurochemically diverse inputs from brainstem and forebrain regions to generate respiratory motor patterns. VRC microinjection of the neuropeptide galanin has been reported to dampen carbon dioxide (CO2)-mediated chemoreflex responses. Additionally, we previously demonstrated that galaninergic neurons in the retrotrapezoid nucleus (RTN) are implicated in the adaptive response to hypercapnic stimuli, suggesting a link between RTN neuroplasticity and increased neuronal drive to the VRC. VRC neurons express galanin receptor 1, suggesting potential regulatory action by galanin, however, the precise galaninergic chemoreceptor-VRC circuitry remains to be determined. This study aimed to identify sources of galaninergic input to the VRC that contribute to central respiratory chemoreception. We employed a combination of retrograde neuronal tracing, in situ hybridisation and immunohistochemistry to investigate VRC-projecting neurons that synthesise galanin mRNA. In an additional series of experiments, we used acute hypercapnia exposure (10% CO2, 1 h) and c-Fos immunohistochemistry to ascertain which galaninergic nuclei projecting to the VRC are activated. Our findings reveal that a total of 30 brain nuclei and 51 subnuclei project to the VRC, with 12 of these containing galaninergic neurons, including the RTN. Among these galaninergic populations, only a subset of the RTN neurons (approximately 55%) exhibited activation in response to acute hypercapnia. Our findings highlight that the RTN is the likely source of galaninergic transmission to the VRC in response to hypercapnic stimuli.

Phase- and state-dependent modulation of breathing pattern by preBötzinger complex somatostatin expressing neurons.

As neuronal subtypes are increasingly categorized, delineating their functional role is paramount. The preBötzinger complex (preBötC) subpopulation expressing the neuropeptide somatostatin (SST) is classified as mostly excitatory, inspiratory-modulated and not rhythmogenic. We further characterized their phenotypic identity: 87% were glutamatergic and the balance were glycinergic and/or GABAergic. We then used optogenetics to investigate their modulatory role in both anaesthetized and freely moving mice. In anaesthetized mice, short photostimulation (100 ms) of preBötC SST+ neurons modulated breathing-related variables in a combinatory phase- and state-dependent manner; changes in inspiratory duration, inspiratory peak amplitude (Amp), and phase were different at higher (≥2.5 Hz) vs. lower (<2.5 Hz) breathing frequency (f). Moreover, we observed a biphasic effect of photostimulation during expiration that is probabilistic, that is photostimulation given at the same phase in consecutive cycles can evoke opposite responses (lengthening vs. shortening of the phase). These unexpected probabilistic state- and phase-dependent responses to photostimulation exposed properties of the preBötC that were not predicted and cannot be readily accounted for in current models of preBötC pattern generation. In freely moving mice, prolonged photostimulation decreased f in normoxia, hypoxia or hypercapnia, and increased Amp and produced a phase advance, which was similar to the results in anaesthetized mice when f ≥ 2.5 Hz. We conclude that preBötC SST+ neurons are a key mediator of the extraordinary and essential lability of breathing pattern. KEY POINTS: PreBötzinger complex (preBötC) SST+ neurons, which modulate respiratory pattern but are not rhythmogenic, were transfected with channelrhodopsin to investigate phase- and state-dependent modulation of breathing pattern in anaesthetized and freely behaving mice in normoxia, hypoxia and hypercapnia. In anaesthetized mice, photostimulation during inspiration increased inspiratory duration and amplitude regardless of baseline f, yet the effects were more robust at higher f. In anaesthetized mice with low f (<2.5 Hz), photostimulation during expiration evoked either phase advance or phase delay, whereas in anaesthetized mice with high f (≥2.5 Hz) and in freely behaving mice in normoxia, hypoxia or hypercapnia, photostimulation always evoked phase advance. Phase- and state-dependency is a function of overall breathing network excitability. The f-dependent probabilistic modulation of breathing pattern by preBötC SST+ neurons was unexpected, requiring reconsideration of current models of preBötC function, which neither predict nor can readily account for such responses.

Systemic 8-OH-DPAT challenge causes hyperventilation largely via activating pre-botzinger complex 5-HT1A receptors.

Systemic 8-OH-DPAT (a 5-HT1A receptor agonist) challenge evokes hyperventilation independent of peripheral 5-HT1A receptors. Though the pre-Botzinger Complex (PBC) is critical in generating respiratory rhythm and activation of local 5-HT1A receptors induces tachypnea via disinhibition of local GABAA neurons, its role in the respiratory response to systemic 8-OH-DPAT challenge is still unclear. In anesthetized rats, 8-OH-DPAT (100 µg/kg, iv) was injected twice to confirm the reproducibility of the evoked responses. The same challenges were performed after bilateral microinjections of (S)-WAY-100135 (a 5-HT1A receptor antagonist) or gabazine (a GABAA receptor antagonist) into the PBC. Our results showed that: 1) 8-OH-DPAT caused reproducible hyperventilation associated with hypotension and bradycardia; 2) microinjections of (S)-WAY-100135 into the PBC attenuated the hyperventilation by ˜60 % without effect on the evoked hypotension and bradycardia; and 3) the same hyperventilatory attenuation was also observed after microinjections of gabazine into the PBC. Our data suggest that PBC 5-HT1A receptors play a key role in the respiratory response to systemic 8-OH-DPAT challenge likely via disinhibiting local GABAergic neurons.

Respiratory disturbances and high risk of sudden death in the neonatal connexin-36 knockout mouse.

Neural circuits at the brainstem involved in the central generation of the motor patterns of respiration and cardiorespiratory chemoreflexes organize as cell assemblies connected by chemical and electrical synapses. However, the role played by the electrical connectivity mainly mediated by connexin36 (Cx36), which expression reaches peak value during the postnatal period, is still unknown. To address this issue, we analyzed here the respiratory phenotype of a mouse strain devoid constitutively of Cx36 at P14. Male Cx36-knockout mice at rest showed respiratory instability of variable degree, including a periodic Cheyne-Stokes breathing. Moreover, mice lacking Cx36 exhibited exacerbated chemoreflexes to normoxic and hypoxic hypercapnia characterized by a stronger inspiratory/expiratory coupling due to an increased sensitivity to CO2 . Deletion of Cx36 also impaired the generation of the recurrent episodes of transient bradycardia (ETBs) evoked during hypercapnic chemoreflexes; these EBTs constituted a powerful mechanism of cardiorespiratory coupling capable of improving alveolar gaseous exchange under hypoxic hypercapnia conditions. Approximately half of the homo- and heterozygous Cx36KO, but none WT, mice succumbed by respiratory arrest when submitted to hypoxia-hypercapnia, the principal exogenous stressor causing sudden infant death syndrome (SIDS). The early suppression of EBTs, which worsened arterial O2  saturation, and the generation of a paroxysmal generalized clonic-tonic activity, which provoked the transition from eupneic to gasping respiration, were the critical events causing sudden death in the Cx36KO mice. These results indicate that Cx36 expression plays a pivotal role in respiratory control, cardiorespiratory coordination, and protection against SIDS at the postnatal period.

Astrocytic contribution to glutamate-related central respiratory chemoreception in vertebrates.

Central respiratory chemoreceptors play a key role in the respiratory homeostasis by sensing CO2 and H+ in brain and activating the respiratory neural network. This ability of specific brain regions to respond to acidosis and hypercapnia is based on neuronal and glial mechanisms. Several decades ago, glutamatergic transmission was proposed to be involved as a main mechanism in central chemoreception. However, a complete identification of mechanism has been elusive. At the rostral medulla, chemosensitive neurons of the retrotrapezoid nucleus (RTN) are glutamatergic and they are stimulated by ATP released by RTN astrocytes in response to hypercapnia. In addition, recent findings show that caudal medullary astrocytes in brainstem can also contribute as CO2 and H+ sensors that release D-serine and glutamate, both gliotransmitters able to activate the respiratory neural network. In this review, we describe the mammalian astrocytic glutamatergic contribution to the central respiratory chemoreception trying to trace in vertebrates the emergence of several components involved in this process.

Ablation of Zfhx4 results in early postnatal lethality by disrupting the respiratory center in mice.

Breathing is an integrated motor behavior that is driven and controlled by a network of brainstem neurons. Zfhx4 is a zinc finger transcription factor and our results showed that it was specifically expressed in several regions of the mouse brainstem. Mice lacking Zfhx4 died shortly after birth from an apparent inability to initiate respiration. We also found that the electrical rhythm of brainstem‒spinal cord preparations was significantly depressed in Zfhx4-null mice compared to wild-type mice. Immunofluorescence staining revealed that Zfhx4 was coexpressed with Phox2b and Math1 in the brainstem and that Zfhx4 ablation greatly decreased the expression of these proteins, especially in the retrotrapezoid nucleus. Combined ChIP‒seq and mRNA expression microarray analysis identified Phox2b as the direct downstream target gene of Zfhx4, and this finding was validated by ChIP‒qPCR. Previous studies have reported that both Phox2b and Math1 play key roles in the development of the respiratory center, and Phox2b and Math1 knockout mice are neonatal lethal due to severe central apnea. On top of this, our study revealed that Zfhx4 is a critical regulator of Phox2b expression and essential for perinatal breathing.

Endogenous hydrogen sulfide maintains eupnea in an in situ arterially perfused preparation of rats.

Hydrogen sulfide (H2S) is constitutively generated in the human body and works as a gasotransmitter in synaptic transmission. In this study, we aimed to evaluate the roles of endogenous H2S in generating eupnea at the respiratory center. We employed an in situ arterially perfused preparation of decerebrated rats and recorded the central respiratory outputs. When the H2S-producing enzyme cystathionine ß-synthase (CBS) was inhibited, respiration switched from the 3-phase eupneic pattern, which consists of inspiration, postinspiration, and expiration, to gasping-like respiration, which consists of inspiration only. On the other hand, when H2S synthesis was inhibited via cystathionine γ-lyase (CSE) or when H2S synthesis was activated via CBS, eupnea remained unchanged. These results suggest that H2S produced by CBS has crucial roles in maintaining the neuronal network to generate eupnea. The mechanism of respiratory pattern generation might be switched from a network-based system to a pacemaker cell-based system in low H2S conditions.

Computing the Effects of SARS-CoV-2 on Respiration Regulatory Mechanisms in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been established as a cause of severe alveolar damage and pneumonia in patients with advanced Coronavirus disease (COVID-19). The consolidation of lung parenchyma precipitates the alterations in blood gases in COVID-19 patients that are known to complicate and cause hypoxemic respiratory failure. With SARS-CoV-2 damaging multiple organs in COVID-19, including the central nervous system that regulates the breathing process, it is a daunting task to compute the extent to which the failure of the central regulation of the breathing process contributes to the mortality of COVID-19 affected patients. Emerging data on COVID-19 cases from hospitals and autopsies in the last few months have helped in the understanding of the pathogenesis of respiratory failures in COVID-19. Recent reports have provided overwhelming evidence of the occurrence of acute respiratory failures in COVID-19 due to neurotropism of the brainstem by SARS-CoV-2. In this review, a cascade of events that may follow the alterations in blood gases and possible neurological damage to the respiratory regulation centers in the central nervous system (CNS) in COVID-19 are related to the basic mechanism of respiratory regulation in order to understand the acute respiratory failure reported in this disease. Though a complex metabolic and respiratory dysregulation also occurs with infections caused by SARS-CoV-1 and MERS that are known to contribute toward deaths of the patients in the past, we highlight here the role of systemic dysregulation and the CNS respiratory regulation mechanisms in the causation of mortalities seen in COVID-19. The invasion of the CNS by SARS-CoV-2, as shown recently in areas like the brainstem that control the normal breathing process with nuclei like the pre-Bötzinger complex (pre-BÖTC), may explain why some of the patients with COVID-19, who have been reported to have recovered from pneumonia, could not be weaned from invasive mechanical ventilation and the occurrences of acute respiratory arrests seen in COVID-19. This debate is important for many reasons, one of which is the fact that permanent damage to the medullary respiratory centers by SARS-CoV-2 would not benefit from mechanical ventilators, as is possibly occurring during the management of COVID-19 patients.

Activation of µ-opioid receptors differentially affects the preBötzinger Complex and neighbouring regions of the respiratory network in the adult rabbit.

The role of the different components of the respiratory network in the mediation of opioid-induced respiratory depression is still unclear. We investigated the contribution of the preBötzinger Complex (preBötC) and the neighbouring Bötzinger Complex (BötC) and inspiratory portion of the ventral respiratory group (iVRG) in anesthetized, vagotomized, paralyzed and artificially ventilated adult rabbits making use of bilateral microinjections (30-50 nl) of the µ-opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO). Dose-dependent effects were observed. In the preBötC and the BötC 0.1 mM DAMGO microinjections caused mainly reductions in peak phrenic amplitude associated with tonic phrenic activity and irregular (ataxic) patterns of breathing that were more pronounced in the preBötC. Apneic effects developed at 0.5 mM. In the iVRG DAMGO provoked decreases in amplitude and frequency of phrenic bursts at 0.1 mM and apnea at 0.5 mM. Local 5 mM naloxone reversed the apneic effects. The results imply that different components of the respiratory network may contribute to opioid-induced respiratory disorders.

Maternal cigarette smoke exposure disturbs glutamate/GABA balance in pFRG of neonatal rats.

We previously found that maternal cigarette smoke (CS) exposure resulted in impairment of central chemoreception and oxidative stress and mitochondrial dysfunction of parafacial respiratory group (pFRG, a critical site for mammalian central chemoreception) in neonatal rats. The present work was carried out to identify if maternal CS exposure could disturb the glutamate (GLU)-ergic and γ-aminobutyric acid (GABA)-ergic balance in pFRG of neonatal rats. We found that maternal CS exposure induced a decrease in GLU content and consequently in GLU/GABA ratio in pFRG of neonatal rats. Maternal CS exposure also decreased glutamine content and glutaminase and glutamine synthetase activity in offspring pFRG. In addition, expression of vesicular glutamate transporter 2 was depressed, and those of glutamate transporter 1 and GABA transporter 3 were elevated by maternal CS exposure. These results indicate that maternal CS exposure leads to a disturbance of GLU/GABA balance in pFRG of the neonatal rats, which might contribute to the suppression of central chemoreception in maternal CS-exposed offspring.

Development of the brainstem respiratory circuit.

The respiratory circuit is comprised of over a dozen functionally and anatomically segregated brainstem nuclei that work together to control respiratory rhythms. These respiratory rhythms emerge prenatally but only acquire vital importance at birth, which is the first time the respiratory circuit faces the sole responsibility for O2 /CO2 homeostasis. Hence, the respiratory circuit has little room for trial-and-error-dependent fine tuning and relies on a detailed genetic blueprint for development. This blueprint is provided by transcription factors that have specific spatiotemporal expression patterns along the rostral-caudal or dorsal-ventral axis of the developing brainstem, in proliferating precursor cells and postmitotic neurons. Studying these transcription factors in mice has provided key insights into the functional segregation of respiratory control and the vital importance of specific respiratory nuclei. Many studies converge on just two respiratory nuclei that each have rhythmogenic properties during the prenatal period: the preBötzinger complex (preBötC) and retrotrapezoid nucleus/parafacial nucleus (RTN/pF). Here, we discuss the transcriptional regulation that guides the development of these nuclei. We also summarize evidence showing that normal preBötC development is necessary for neonatal survival, and that neither the preBötC nor the RTN/pF alone is sufficient to sustain normal postnatal respiratory rhythms. Last, we highlight several studies that use intersectional genetics to assess the necessity of transcription factors only in subregions of their expression domain. These studies independently demonstrate that lack of RTN/pF neurons weakens the respiratory circuit, yet these neurons are not necessary for neonatal survival because developmentally related populations can compensate for abnormal RTN/pF function at birth. This article is categorized under: Nervous System Development > Vertebrates: Regional Development.

Optogenetic analysis of respiratory neuronal networks in the ventral medulla of neonatal rats producing channelrhodopsin in Phox2b-positive cells.

Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem-spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.5 s) of the rostral pFRG reset the respiratory rhythm. At the cellular level, photostimulation depolarised Phox2b-positive pre-inspiratory, inspiratory and respiratory-modulated tonic neurons and Phox2b-negative pre-inspiratory neurons. In contrast, changes in membrane potential of Phox2b-negative inspiratory and expiratory neurons varied depending on characteristics of ongoing synaptic connections in local respiratory networks in the rostral medulla. In the presence of tetrodotoxin, photostimulation depolarised Phox2b-positive cells, but caused no significant changes in membrane potential of Phox2b-negative cells. We concluded that depolarisation of Phox2b-positive neurons was due to cell-autonomous photo-activation and summation of excitatory postsynaptic potentials, whereas membrane potential changes of Phox2b-negative neurons depended on the network configuration. Our findings shed further light on local networks among respiratory-related neurons in the rostral ventrolateral medulla and emphasise the important role of pre-inspiratory neurons in respiratory rhythm generation in the neonatal rat en bloc preparation.

Characterisation of medullary astrocytic populations in respiratory nuclei and alterations in sudden unexpected death in epilepsy.

Central failure of respiration during a seizure is one possible mechanism for sudden unexpected death in epilepsy (SUDEP). Neuroimaging studies indicate volume loss in the medulla in SUDEP and a post mortem study has shown reduction in neuromodulatory neuropeptidergic and monoaminergic neurones in medullary respiratory nuclear groups. Specialised glial cells identified in the medulla are considered essential for normal respiratory regulation including astrocytes with pacemaker properties in the pre-Botzinger complex and populations of subpial and perivascular astrocytes, sensitive to increased pCO2, that excite respiratory neurones. Our aim was to explore niches of medullary astrocytes in SUDEP cases compared to controls. In 48 brainstems from three groups, SUDEP (20), epilepsy controls (10) and non-epilepsy controls (18), sections through the medulla were labelled for GFAP, vimentin and functional markers, astrocytic gap junction protein connexin43 (Cx43) and adenosine A1 receptor (A1R). Regions including the ventro-lateral medulla (VLM; for the pre-Bötzinger complex), Median Raphe (MR) and lateral medullary subpial layer (MSPL) were quantified using image analysis for glial cell populations and compared between groups. Findings included morphologically and regionally distinct vimentin/Cx34-positive glial cells in the VLM and MR in close proximity to neurones. We noted a reduction of vimentin-positive glia in the VLM and MSPL and Cx43 glia in the MR in SUDEP cases compared to control groups (p < 0.05-0.005). In addition, we identified vimentin, Cx43 and A1R positive glial cells in the MSPL region which likely correspond to chemosensory glia identified experimentally. In conclusion, altered medullary glial cell populations could contribute to impaired respiratory regulatory capacity and vulnerability to SUDEP and warrant further investigation.

Structural and functional defects of the respiratory neural system in the medulla and spinal cord of Pax6 mutant rats.

Pax6 is an important transcription factor expressed in several discrete domains of the developing central nervous system. It has been reported that Pax6 is involved in the specification of subtypes of hindbrain motor neurons. Pax6 homozygous mutant (rSey2/rSey2) rats die soon after birth, probably due to impaired respiratory movement. To determine whether the respiratory center in the medulla functions normally, we analyzed the histological and neurophysiological properties of the medulla and spinal cord in fetal rats with this mutation. First, the medulla of rSey2/rSey2 at embryonic (E) 21.5-E22.5 tended to be smaller than those from heterozygous mutant (rSey2/+) and wild-type (+/+) littermates. Through immunohistochemical analysis, we confirmed normal distribution of Phox2b-expressing cells in the parafacial respiratory group (pFRG) of rSey2/rSey2 rats. Expression of neurokinin-1 receptor (NK-1R) was weak and dispersed in rSey2/rSey2 rats. In addition, rSey2/rSey2 rats have a defect of the hypoglossal nerve root. Electrophysiological analysis using brainstem-spinal cord preparations (E21.5-E22.5) revealed that rSey2/rSey2 rats showed larger fluctuation of the amplitude of inspiratory activity monitored from the fourth cervical root although there was no significant difference in the respiratory rate among rSey2/rSey2, rSey2/+, and +/+ littermates. The response of respiratory rhythm to high CO2 was similar among all genotypes. Optical recordings of neuronal activity revealed that the activity of the pFRG tended to be weaker and inspiratory activity appeared in more scattered areas in the caudal ventral medulla in the rSey2/rSey2 rats. These results suggest that the basal activity of the respiratory system was preserved with mild impairment of the inspiratory activity in the rSey2/rSey2 rats and that the Pax6 gene is involved in the functional development of the neuronal system producing effective inspiratory motor outputs for survival.

Glutamate receptor plasticity in brainstem respiratory nuclei following chronic hypercapnia in goats.

Patients that retain CO2 in respiratory diseases such as chronic obstructive pulmonary disease (COPD) have worse prognoses and higher mortality rates than those with equal impairment of lung function without hypercapnia. We recently characterized the time-dependent physiologic effects of chronic hypercapnia in goats, which suggested potential neuroplastic shifts in ventilatory control mechanisms. However, little is known about how chronic hypercapnia affects brainstem respiratory nuclei (BRN) that control multiple physiologic functions including breathing. Since many CNS neuroplastic mechanisms include changes in glutamate (AMPA (GluR) and NMDA (GluN)) receptor expression and/or phosphorylation state to modulate synaptic strength and network excitability, herein we tested the hypothesis that changes occur in glutamatergic signaling within BRN during chronically elevated inspired CO2 (InCO2 )-hypercapnia. Healthy goats were euthanized after either 24 h or 30 days of chronic exposure to 6% InCO2 or room air, and brainstems were rapidly extracted for western blot analyses to assess GluR and GluN receptor expression within BRN. Following 24-hr exposure to 6% InCO2 , GluR or GluN receptor expression were changed from control (P < 0.05) in the solitary complex (NTS & DMV),ventrolateral medulla (VLM), medullary raphe (MR), ventral respiratory column (VRC), hypoglossal motor nucleus (HMN), and retrotrapezoid nucleus (RTN). These neuroplastic changes were not found following 30 days of chronic hypercapnia. However, at 30 days of chronic hypercapnia, there was overall increased (P < 0.05) expression of glutamate receptors in the VRC and RTN. We conclude that time- and site-specific glutamate receptor neuroplasticity may contribute to the concomitant physiologic changes that occur during chronic hypercapnia.

Serotonergic projections to the ventral respiratory column from raphe nuclei in rats.

The ventral respiratory column (VRC) generates rhythmical respiration and is divided into four compartments: the Bötzinger complex (BC), pre-Bötzinger complex (PBC), rostral ventral respiratory group (rVRG), and caudal ventral respiratory group (cVRG). Serotonergic nerve fibers are densely distributed in the rostral to caudal VRC and serotonin would be one of the important modulators for the respiratory control in the VRC. In the present study, to elucidate detailed distribution of serotonergic neurons in raphe nuclei projecting to the various rostrocaudal levels of VRC, we performed combination of retrograde tracing technique by cholera toxin B subunit (CTB) with immunohistochemistry for tryptophan hydroxylase 2 (TPH2). The double-immunoreactive neurons with CTB and TPH2 were distributed in the both rostral and caudal raphe nuclei, i.e. dorsal raphe nucleus, raphe magnus nucleus, gigantocellular reticular nucleus alpha and ventral parts, lateral paragigantocellular nucleus, parapyramidal area, raphe obscurus nucleus, and raphe pallidus nucleus. The distributions of double-immunoreactive neurons were similar among injection groups of BC, PBC, anterior rVRG, and posterior rVRG/cVRG. In conclusion, serotonergic neurons in both rostral and caudal raphe nuclei projected throughout the VRC and these serotonergic projections may contribute to respiratory responses to various environmental and vital changes.

Analysis of the neuronal network of the medullary respiratory center in transgenic rats expressing archaerhodopsin-3 in Phox2b-expressing cells.

Preinspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG) comprise one of the respiratory rhythm generators in the medulla of the neonatal rat. A subgroup of pFRG/Pre-I neurons expresses the transcription factor Phox2b. To further analyze detailed neuronal mechanisms of respiratory rhythm generation in the neonatal rat, we developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch). Brainstem-spinal cord preparations were isolated from 0-2-day-old Tg newborn rats and were superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25-26 °C. Inspiratory fourth cervical ventral root (C4) activity was monitored, and membrane potentials of neurons in the pFRG including Pre-I and inspiratory neurons were recorded. Phox2b-positive cells in the Tg rats were essentially positive for enhanced green fluorescent protein (EGFP) signals (reporter for Arch) in the pFRG. Continuous photo-stimulation of the rostral ventral medulla for up to 90 s by covering the pFRG with green laser light (532 nm) induced a decrease of respiratory rate measured at C4 accompanied by membrane hyperpolarization of Phox2b-positive pFRG/Pre-I neurons. In contrast, Phox2b-negative inspiratory neurons were not hyperpolarized during the photo-stimulation. Our findings showed that Phox2b-expressing pFRG/Pre-I neurons are involved in the maintenance of the basic respiratory rhythm in neonatal rat.

Impaired chemoreflex correlates with decreased c-Fos in respiratory brainstem centers of the streptozotocin-induced Alzheimer's disease rat model.

Besides impairment in cognition and memory, patients with Alzheimer's disease (AD) often exhibit marked dysfunction in respiratory control. Sleep-disordered breathing (SDB) is commonly found in cases of AD, resulting in periods of hypoxia during sleep. Early structural changes in brainstem areas controlling respiratory function may account for SDB in the course of AD. However, to date the underlying mechanisms for these complications are not known. The streptozotocin (STZ)-induced rat model of AD exhibits abnormal responses to hypoxia and increased astrogliosis in a key region for respiratory control. In this study we further defined the pathophysiological respiratory response of STZ-AD rats to 10% O2. In addition, we analyzed hypoxia-induced neuronal activation in respiratory and cardiovascular nuclei of the dorsal and ventral brainstem. Two hours of hypoxia induced a transient increase in tidal volume that was followed by a prolonged increase in respiratory rate. Only respiratory rate was significantly blunted in the STZ-AD model, which continued over the entire duration of the hypoxic episode. Analysis of c-Fos expression as a marker for neuronal activation showed abundant labeling throughout the nTS, nuclei of the ventral respiratory column, and A1/C1 cells of cardiovascular centers in the ventral brainstem. STZ-AD rats showed a significant decrease of c-Fos labeling in the caudal/medial nTS, rostral ventral respiratory group, and Bötzinger complex. c-Fos in other respiratory centers and A1/C1 cells was unaltered when compared to control. The results of this study document a region-specific impact of STZ-induced AD in respiratory brainstem nuclei. This decrease in c-Fos expression correlates with the observed blunting of respiration to hypoxia in the STZ-AD rat model.