Somatostatin selectively ablates post-inspiratory activity after injection into the Bötzinger complex

Somatostatin (SST) neurons in the ventral respiratory column (VRC) are essential for the generation of normal breathing. Little is known about the neuromodulatory role of SST on ventral respiratory neurons other than that local administration induces apnoea. Here, we describe the cardiorespiratory effects of microinjecting SST into the preBötzinger and Bötzinger complexes which together elaborate a normal inspiratory augmenting and expiratory respiratory pattern, and on spinally projecting respiratory subnuclei (rostral ventral respiratory group; rVRG). Microinjections (20–50 nl) of SST (0.15, 0.45, 1.5 mM) were made into respiratory subnuclei of urethane-anaesthetized, paralysed, vagotomized and artificially ventilated Sprague–Dawley rats (n=46). Unilateral microinjection of SST into the Bötzinger complex converted the augmenting activity of phrenic nerve discharge into a square-wave apneustic pattern associated with a lengthening of inspiratory period and shortening of expiratory time. Following bilateral microinjection the apneusis became pronounced and was associated with a dramatic variability in inspiratory duration. Microinjection of SST into the Bötzinger complex also abolished the post-inspiratory (post-I) motor activity normally observed in vagal and sympathetic nerves. In the preBötzinger complex SST caused bradypnoea and with increasing dose, apnoea. In the rVRG SST reduced phrenic nerve amplitude, eventually causing apnoea. In conclusion, SST powerfully inhibits respiratory neurons throughout the VRC. Of particular interest is the finding that chemical inhibition of the Bötzinger complex with SST ablates the post-I activity that is normally seen in respiratory activity and leads to apneusis. This loss of post-I activity is a unique feature of inhibition with SST and is not seen following inhibition with other agents such as galanin, GABA and endomorphin. The effect seen on post-I activity is similar to the effect of inhibiting the Kölliker–Fuse nucleus in the pons. The mechanism by which SST exerts this effect on Bötzinger neurons remains to be determined.     

Effects of sevoflurane on respiratory rhythm oscillators in the medulla oblongata

Using in vitro newborn rat brainstem–spinal cord preparations with and without the parafacial respiratory group (pFRG), we examined the effects of the volatile anaesthetic sevoflurane on the respiratory rhythm oscillators of the pFRG and the preBötzinger complex (preBötC). Our study indicated that sevoflurane depressed pre-inspiratory neurons (Pre-Is) in the pFRG via γ-aminobutyric acid-A (GABA_A)ergic and glycinergic inhibition and that it depressed preBötC inspiratory neurons via GABA_Aergic but not via glycinergic inhibition. We also found that sevoflurane had stimulant effects on the respiratory rhythm oscillators. Our results shed light on respiratory rhythm generation. In all preparations (n = 16) in which Pre-Is activity was recorded, inspiratory-related cervical motor output remained after application of 0.47 mM sevoflurane, despite the disappearance of the burst activity of Pre-Is. This finding shows that Pre-Is are not essential for respiratory rhythm generation and suggests that sevoflurane, when applied at a proper concentration, might offer a pharmacological means to eliminate pFRG function while preserving preBötC activity.     

Silencing by raised extracellular Ca of pre-Bötzinger complex neurons in newborn rat brainstem slices without change of membrane potential or input resistance

Breathing is controlled by inspiratory pre-Bötzinger complex (preBötC) networks that remain active in transversal brainstem slices from perinatal rodents. In 600 μm thick preBötC slices, inspiratory-related bursting in physiological (3 mM) [K⁺] is depressed by <1 mM elevation of superfusate [Ca²⁺]. Here, we studied underlying cellular mechanisms in whole-cell-recorded neurons of 400 μm thin newborn rat slices with the <200 μm thin preBötC in the middle (“m-preBötC[400]” slices). Extracellular activity in the ventrolateral slice area in 3 mM K⁺ and a most common physiological Ca²⁺ range (1–1.2 mM) stopped spontaneously within 2 h (“in vitro apnea”). Contrary, rhythm was stable for >3 h at 6–8 bursts/min in 7 mM K⁺ and 1.2 mM Ca²⁺ solution. In non-pacemaker preBötC inspiratory cells and neighboring inspiratory or tonically active neurons, block or frequency depression by >90% of rhythm in the latter solution by 2–3 mM Ca²⁺ changed neither resting potential nor input resistance. High Ca²⁺ silenced inspiratory neurons and depressed tonic discharge of non-respiratory neurons. However, in both cell types current injection evoked normal action potentials with unchanged threshold potential. The findings show that m-preBötC[400] slices represent a good compromise between long term viability of rhythmogenic preBötC neurons and minimal modulation of these cells by adjacent tissue, but need to be studied in elevated K⁺. The lack of postsynaptic K⁺ channel-mediated hyperpolarization suggests that saturation of surface charges, presynaptic block of transmission and/or inhibition of postsynaptic burst-promoting conductances such as Ca²⁺ activated non-selective cation channels are involved in inspiratory depression by high Ca²⁺.     

K and Ca dependence of inspiratory-related rhythm in novel “calibrated” mouse brainstem slices

Recently developed transversal newborn rat brainstem slices with “calibrated” rostrocaudal margins unraveled novel features of rhythmogenic inspiratory active pre-Bötzinger complex (preBötC) neural networks (Ballanyi and Ruangkittisakul, 2009). For example, slice rhythm in physiological (3 mM) superfusate K⁺ is depressed by modestly raised Ca²⁺ and restored by raised K⁺. Correspondingly, we generated here calibrated preBötC slices from commonly used newborn C57BL/6 mice in which rostrocaudal extents of respiratory marker structures, e.g., the inferior olive, turned out to be smaller than in newborn rats. Slices of 400–600 μm thickness with likely centered preBötC kernel (“m-preBötC slices”) generated rhythm in 3 mM K⁺ and 1 mM Ca²⁺ for several hours although its rate decreased to <5 bursts/min after >1 h. Rhythm was stable at 8–12 bursts/min in 6–7 mM K⁺, depressed by 2 mM Ca²⁺, and restored by 9 mM K⁺. Our findings provide the basis for future structure–function analyses of the mouse preBötC, whose activity depends critically on a “Ca⁺/K⁺ antagonism” as in rats.     

Galanin microinjection into the PreBötzinger or the Bötzinger Complex terminates central inspiratory activity and reduces responses to hypoxia and hypercapnia in rat

Respiratory rhythm is generated and shaped by the synaptic interaction of neurons in the Bötzinger Complex (BötC) and PreBötzinger Complex (PreBötC) located in the ventral respiratory column of the medulla. Metabotropic receptors are important modulators of fast neurotransmission in the generation and shaping of respiratory rhythm. Microinjection of the neuropeptide galanin (1 mM, 50 nL, 50 pmol) into functionally identified BötC or PreBötC in urethane anesthetized, mechanically ventilated and vagotomized rats caused severe dysrhythmia or persistent apnea. In the BötC and PreBötC, galanin reduced the ventilatory response to hypercapnia (5% CO₂) by 21% (P < 0.001) and 38% (P < 0.01) respectively. In the BötC and PreBötC, galanin reduced the ventilatory response to hypoxia (10% O₂) by 15% (P < 0.05) and 23% (P < 0.01) respectively. These results indicate that microinjection of galanin into the BötC or PreBötC depresses a neural substrate required for the generation of respiratory motor output and reflex responses to hypercapnea and hypoxia.     

Cross-correlation of augmenting expiratory neurons of the Bötzinger complex in the cat

Ipsilateral and contralateral pairs of augmenting expiratory neurons were recorded simultaneously from the Bötzinger complex using glass-coated tungsten microelectrodes in pentobarbitone-anaesthetized cats. The neurons were identified both by firing pattern and by antidromic activation from the contralateral site of the dorsal respiratory group. Cross-correlation histograms of the extracellularly recorded action potentials were calculated in order to detect short time-scale synchronizations of firing indicative of synaptic connections between the neurons. The cross-correlation histograms for 40 ipsilateral pairs of neurons less than 1 mm apart showed eight (20%) narrow troughs (mean half-amplitude width ±SD, 1.1±0.37 ms) at short latencies (mean latency±SD, 1.0±0.35 ms) suggestive of monosynaptic inhibition. These included two cross-correlation histograms which showed troughs on both sides of time zero, indicating a mutual inhibition. For another four pairs of neurons (10%), a central broad peak suggestive of common activation due to either excitation or release from inhibition was evident. Contralateral pairs of expiratory neurons of the Bötzinger complex were examined in a similar manner. The cross-correlation histograms for 43 pairs of neurons showed five (12%) narrow troughs (mean half-amplitude width±SD, 1.2±0.67 ms) at short latencies (mean latency±SD, 2.7±1.47 ms) suggestive of monosynaptic inhibition. These included one cross-correlation histogram which showed troughs (one not statistically significant) on both sides of time zero, indicating a mutual inhibition. For another two pairs of neurons (4.6%) a central, broad peak suggestive of common activation due to either excitation or release from inhibition was evident. We conclude that inhibitory interconnections exist between augmenting expiratory neurons of the Bötzinger complex ipsilaterally and contralaterally. These connections may synchronize the expiratory burst of activity within this population and assist in the patterning of the burst.     

Large-conductance calcium-activated potassium channels in the neurons of pre-Bötzinger complex and their participation in the regulation of central respiratory activity in neonatal rats

The present study was conducted to test our hypothesis that the large-conductance calcium-activated potassium channels (BK_Ca channels) exist in the neurons of the pre-Bötzinger complex (PBC), a brainstem region that may generate respiratory rhythm in mammals, and play roles in central regulation of respiratory activity in neonatal rats. Immunohistochemical technique revealed that BK_Ca channels expressed in the neurons of PBC region. Whole cell voltage clamp recordings from the neurons in the PBC showed that BK_Ca channels could be activated by membrane depolarization and blocked by 1 mM tetraethylammonium (TEA) or 10 μM paxilline in the preparation of thin (about 300 μm) medullary slices of neonatal rats. The rhythmic respiratory-like discharge of hypoglossal rootlets could be changed by perfusing the thick (700–900 μm) medullary slices with 1 mM TEA or 10 μM paxilline. Both TEA and paxilline could prolong the inspiratory duration, shorten the expiratory duration and increase the respiratory frequency. The results suggest that BK_Ca channels exist in the PBC neurons and may be involved in the central control of rhythmic respiration in the neonatal rats.     

The effect of hyperpolarizing inputs on the dynamics of a bursting pacemaker neuron model in the pre-Bötzinger complex

The pre-Bötzinger complex (pre-BötC), a subregion of the ventrolateral medulla involved in respiratory rhythm generation, contains intrinsically bursting pacemaker neurons. A previous study proposed Hodgkin–Huxley type minimal models for pacemaker neurons and predicted the effect of a hyperpolarizing input on the dynamics of a model under certain conditions. In this model, bursting is explained by the dynamics of a persistent sodium current. In the present study, the effect of a hyperpolarizing input on the dynamics of a model was investigated under variable conditions. It was observed that immediately after an input of sufficient intensity and duration, an increase in the maximal value of the gating variable h of a persistent sodium current was brought about by a decrease in the timing of the hyperpolarizing input. This corresponds to an observation that immediately after the input, a monotonic increase in the number of spikes in the neuron model was brought about by a decrease in the timing of the hyperpolarizing input. In addition, the dependency of burst duration immediately after the input on the timing of the hyperpolarizing input varied depending on the condition of input. The present study is the first to elucidate that the influence of hyperpolarizing inputs on the number of spikes within a burst in a pacemaker neuron model in the pre-BötC is dependent on the timing of the hyperpolarizing input and to clarify the possible mechanism of this influence, thereby facilitating a detailed understanding of the dynamics of a pacemaker neuron model in the pre-BötC.     

Structure–function analysis of rhythmogenic inspiratory pre-Bötzinger complex networks in “calibrated” newborn rat brainstem slices

Inspiratory pre-Bötzinger complex (preBötC) networks remain active in perinatal rodent brainstem slices. Our analysis of (crescendo-like) inspiratory-related population and cellular bursting in novel histologically identified transversal preBötC slices in physiological (3 mM) superfusate [K⁺] revealed: (i) the preBötC extent sufficient for rhythm in thin slices is at most 175 μm. (ii) In 700 μm thick slices with unilaterally exposed preBötC, a <100 μm kernel generates a eupnea-like inspiratory pattern under predominant influence of caudally adjacent structures or thyrotropin-releasing hormone-like transmitters, but a mixed eupnea-sigh-like pattern when influence of rostral structures or substance-P-like transmitters dominates. (iii) Primarily presynaptic processes may underlie inhibition of rhythm by opioids or raising superfusate [Ca²⁺] from lower to upper physiological limits (1–1.5 mM). (iv) High K⁺ reverses depression of rhythm by raised Ca²⁺, opioids and anoxia. In summary, distinct activity patterns of spatiochemically organized isolated inspiratory networks depend on both an extracellular “Ca²⁺–K⁺ antagonism” and slice dimensions. This explains some discrepant findings between studies and suggests use of “calibrated” slices and more uniform experimental conditions.     

The pre-Bötzinger complex participates in generating the respiratory effects of thyroliberin

Experiments on anesthetized rats were performed to study the effects of microinjection of thyroliberin (10 fM–100 μM) into the area of the pre-Bötzinger complex on respiratory and circulatory parameters. Thyroliberin dose-dependently increased respiration frequency, with shortening of inspiration and expiration. Tidal volume and the amplitude of the integrated EMG recorded from the inspiratory muscles decreased after administration of concentrated solutions. Using this dosage method, thyroliberin had weak effects on systemic hemodynamics. The data suggest that structures located in the area of the pre-Bötzinger complex take part in generating the respiratory effects of thyroliberin.     

Respiratory responses to ionotropic glutamate receptor antagonists in the ventral respiratory group of the rabbit

Selective excitatory amino acid receptor antagonists acting on either N-methyl-D-aspartic acid (NMDA) or non-NMDA receptors were microinjected (30-50 nl) bilaterally into different subregions of the ventral respiratory group (VRG) of alpha-chloralose-urethane anaesthetized, vagotomized, paralysed and artificially ventilated rabbits. Blockade of NMDA receptors by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 or 10 mM) within the inspiratory portion of the VRG (iVRG) dose-dependently decreased the peak amplitude and rate of rise of phrenic nerve activity, without significant changes in respiratory timing. Decreases in respiratory frequency and peak phrenic amplitude up to apnoea were evoked by 20 mM D-AP5; phrenic nerve activity was restored transiently by hypoxic or hypercapnic stimulation during D-AP5-induced apnoea. Microinjections of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1, 10 or 20 mM) into the iVRG provoked less intense depressant respiratory effects. No significant respiratory responses were evoked by microinjections of these antagonists into more caudal VRG subregions. The results suggest that ionotropic glutamate receptors within the iVRG are involved mainly in the control of the intensity of inspiratory activity, with a major role played by NMDA receptors. Glutamate receptor antagonism in the iVRG does not seem to impair the basic mechanisms underlying respiratory rhythm generation.     

Parvalbumin in respiratory neurons of the ventrolateral medulla of the adult rat

A column of parvalbumin immunoreactive neurons is closely associated with the location of respiratory neurons in the ventrolateral medulla of the rat. The majority (66%) of bulbospinal neurons in the medullary ventral respiratory column (VRC) that were retrogradely labeled by tracer injections in the phrenic nucleus were also positive for parvalbumin. In contrast, only 18.8% of VRC neurons retrogradely labeled after a tracer injection in the VRC, also expressed parvalbumin. The average cross-sectional area of VRC neurons retrogradely labeled after VRC injections was 193.8 m² ± 6.6 SE. These were significantly smaller than VRC parvalbumin neurons (271.9 m² ± 12.3 SE). Parvalbumin neurons were found in the Bötzinger Complex, the rostral ventral respiratory group (VRG), and the caudal VRG, areas which all contribute to the bulbospinal projection. In contrast, parvalbumin neurons were sparse or absent in the preBötzinger Complex and in the vicinity of the retrotrapezoid nucleus, areas that have few bulbospinal projections. Parvalbumin was rarely colocalized within Neurokinin-1 receptor positive (NK1R) VRC neurons, which are found in the preBötzinger complex and in the anteroventral part of the rostral VRG. Parvalbumin neurons in the Bötzinger Complex and rostral VRG help define the rostrocaudal extent of these regions. The absence of parvalbumin neurons from the intervening preBötzinger complex also helps establish the boundaries of this region. Regional boundaries described in this manner are in good agreement with earlier physiological and anatomical studies. Taken together, the distributions of parvalbumin, NK1R and bulbospinal neurons suggest that the rostral VRG may be subdivided into distinct, anterodorsal, anteroventral, and posterior subdivisions.     

Axonal projections from Bötzinger expiratory neurons to contralateral ventral and dorsal respiratory groups in the cat

Summary We studied projection patterns of the augmenting expiratory neurons of the Bötzinger complex (BÖT) in the contralateral brainstem. Three experimental approaches were used: 1) electrophysiological analysis using antidromic microstimulation, and morphological analyses using 2) intraaxonal injection of HRP, and 3) application of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L). Taken together, the three methods revealed morphological details of the axonal arborizations of the expiratory neurons in the BÖT and the ventral respiratory group (VRG). The majority of augmenting expiratory neurons of the BÖT had axonal collaterals in the contralateral brainstem. The stem axons to the contralateral side crossed the midline almost at the level of the cell somata. They descended dorsomedial to the ventral spinocerebellar tract and gave off collateral branches directed dorsomedially. Terminal boutons were distributed abundantly in the caudal part of the BÖT and in the more caudally situated VRG. Axon collaterals sometimes ran to the dorsal respiratory group (DRG) and distributed terminal boutons there. Together with the fact of extensive ipsilateral arborizations shown previously, the present results indicate that the augmenting expiratory neurons of the BÖT have wide bilateral influence on the BÖT, VRG, DRG, and spinal cord.Abbreviations VII facial nucleus - XII hypoglossal nucleus - AMB nucleus ambiguus - AP area postrema - CX external cuneate nucleus - D descending vestibular nucleus - DX dorsal-motor nucleus of the vagus - M medial vestibular nucleus - NTS nucleus of the solitary tract - R nucleus of Roller - S solitary tract - RFN retrofacial nucleus This paper is dedicated to Professor Hajime Mannen on the occasion of his 65th birthdaySupported by grants-in-aid for Scientific Research nos. 60304044, 62570068, 62770043, and 63570027 from the Japan Ministry of Education, Science and Culture     

Neurons in the preBötzinger complex and VRG are located in proximity to arterioles in newborn mice

The constant cyclic respiratory activity in the brainstem requires an un-interrupted blood flow providing glucose and O₂ to neurons generating respiratory rhythm. Here we used a combination of classical vascular visualization techniques, and calcium imaging, to compare the microvascular structure and localization of active respiratory neurons in the brainstem of newborn mice at the level of the preBötzinger complex (PBC) and ventral respiratory group. The brainstem is supplied with perforating arteries, which enter primarily in the midline and in a circumscribed region mid-laterally in the medulla. Presumed arterioles then pass dorso-medially with a high density immediately lateral to the midline, and mid-laterally at 60% of the midline-to-lateral edge distance. Calcium imaging, using Fluo-8, AM, showed that active PBC/VRG neurons are located in the same region where a high density of arterioles is found. We conclude that the striking co-localization of medullary arterioles and the PBC/VRG could imply that respiratory neurons may derive part of their glucose and oxygen consumption directly from arterioles, and that humoral factors affecting ventilation may reach respiratory neurons by precapillary transport mechanisms.     

Expressions of 5-HT/5-HT2A receptors and phospho-protein kinase C theta in the pre-Bötzinger complex in normal and chronic intermittent hypoxic rats

The pre-Bötzinger complex (pre-BötC), a functionally defined subregion in the ventrolateral medulla oblongata, is a presumed kernel of normal respiratory rhythmogenesis. However, less is known about the pre-BötC's contribution to respiratory neuroplasticity. The most frequently studied model for respiratory neuroplasticity is episodic hypoxia-induced phrenic long-term facilitation, which is 5-HT_2A receptors (5-HT_2AR)-dependent. We hypothesized that preconditioning with chronic intermittent hypoxic (CIH) would activate the 5-HT/5-HT_2AR system and the downstream protein kinase C (PKC) pathway in the pre-BötC. Animals were exposed to alternating 5 min of hypobaric hypoxia and 5 min of normoxia for 10 h/day for 7 days. Hypobaric hypoxia was achieved by continuous air evacuation to reach a pressure of 210–220 mm Hg, corresponding to an altitude of 9000–10000 m. In contrast to the CIH model, a group of animals were pretreated with chronic sustaining hypoxia (CSH), a protocol of continuous hypobaric hypoxia at 360 mm Hg, corresponding to an altitude of about 6000 m, for 10 h/day for 7 days. Immunoreactivity of 5-HT and 5-HT_2AR was examined in the pre-BötC, identified by the presence of neurokinin-1 receptor (NK1R). We found that 15.5% of 5-HT-immunoreactive (ir) terminals were in contact with NK1R-ir neurons. Asymmetric synapses could be identified between them. 38.7% of NK1R-ir dendrites were also immunoreactive for 5-HT_2AR, which was distributed along the inner surface of the plasma membrane in control animals. CIH challenge increased the expressions of 5-HT and 5-HT_2AR in the pre-BötC, an increase in the expressed 5-HT_2AR that was not detected in this region in CSH animals. Specifically, 5-HT_2AR was distributed not only along the inner surface, but also along the outer surface, or directly on the plasma membrane, a pattern not detectable in control animals. 5-HT_2AR was also detectable in the invaginations of the plasma membrane, where receptor endocytosis or exocytosis might occur, indicating CIH-induced higher trafficking of 5-HT_2AR. Concurrently, there was an up-regulation of phospho-PKC theta (P-PKCθ) in the pre-BötC, suggesting a 5-HT/5-HT_2AR-activated PKC mechanism that may contribute to hypoxia-induced respiratory neuroplasticity in the pre-BötC. The close association of P-PKCθ with the postsynaptic density implicates a postsynaptic mechanism mediating respiratory neuroplasticity in the pre-BötC.     

Fluorescence imaging of active respiratory networks

Breathing in mammals is controlled by neural networks in the brainstem such as the pre-Bötzinger complex (preBötC) and the parafacial respiratory group (pFRG). Exploring these rhythmogenic networks and their interactions is greatly facilitated by live fluorescence imaging that enables analysis of (i) spatiotemporal patterns of respiratory (population) activities, (ii) (sub)cellular signaling in identified respiratory neurons, and (iii) membrane properties of respiratory neurons that are fluorescence-tagged for characteristic markers. Transversal medullary slices containing the preBötC and “en bloc” brainstem-spinal cord preparations with a functional preBötC/pFRG dual respiratory center which interacts, e.g., with pontine structures, are used for respiratory imaging in perinatal rodents. Imaging of less reduced (mature) respiratory networks is feasible in arterially-perfused “working-heart-brainstem” preparations from rodents. In these in situ models, imaging with voltage and Ca²⁺ sensitive dyes is established for assessment of respiratory (population) activities. Here, we summarize findings from diverse live imaging approaches in these models and point out potential pitfalls and future perspectives of respiratory-related optical recording.     

Pre-Bötzinger complex functions as a central hypoxia chemosensor for respiration in vivo

Recently, we identified a region located in the pre-B?tzinger complex (pre-B?tC; the proposed locus of respiratory rhythm generation) in which activation of ionotropic excitatory amino acid receptors using DL-homocysteic acid (DLH) elicits a variety of excitatory responses in the phrenic neurogram, ranging from tonic firing to a rapid series of high-amplitude, rapid rate of rise, short-duration inspiratory bursts that are indistinguishable from gasps produced by severe systemic hypoxia. Therefore we hypothesized that this unique region is chemosensitive to hypoxia. To test this hypothesis, we examined the response to unilateral microinjection of sodium cyanide (NaCN) into the pre-B?tC in chloralose- or chloralose/urethan-anesthetized vagotomized, paralyzed, mechanically ventilated cats. In all experiments, sites in the pre-B?tC were functionally identified using DLH (10 mM, 21 nl) as we have previously described. All sites were histologically confirmed to be in the pre-B?tC after completion of the experiment. Unilateral microinjection of NaCN (1 mM, 21 nl) into the pre-B?tC produced excitation of phrenic nerve discharge in 49 of the 81 sites examined. This augmentation of inspiratory output exhibited one of the following changes in cycle timing and/or pattern: 1) a series of high-amplitude, short-duration bursts in the phrenic neurogram (a discharge similar to a gasp), 2) a tonic excitation of phrenic neurogram output, 3) augmented bursts in the phrenic neurogram (i.e., eupneic breath ending with a gasplike burst), or 4) an increase in frequency of phrenic bursts accompanied by small increases or decreases in the amplitude of integrated phrenic nerve discharge. Our findings identify a locus in the brain stem in which focal hypoxia augments respiratory output. We propose that the respiratory rhythm generator in the pre-B?tC has intrinsic hypoxic chemosensitivity that may play a role in hypoxia-induced gasping.     

Mu opioid receptors in rat ventral medulla: effects of endomorphin-1 on phrenic nerve activity

Anatomical and in vitro studies suggest that mu opioid receptors (MOR) on pre-B?tzinger complex neurons are responsible for opioid induced respiratory depression (Grey et al., Science 286 (1999) 1566). However, mu opioid agonists injected in vivo, in other regions of the ventral respiratory group (VRG), produce respiratory depression, suggesting that opioids are widely distributed in the VRG. We therefore re-examined the distribution of the MOR in the ventral medulla and found MOR-immunoreactive neurons and terminals in all subdivisions of the VRG. Furthermore, we determined, in rats, the effects of a MOR agonist (endomorphin-1, 10 mM, 60 nl, unilateral), microinjected into different subdivisions of the VRG, on phrenic nerve activity. Endomorphin-1 produced changes in phrenic nerve frequency and amplitude, throughout the VRG. Unexpectedly, endomorphin-1 microinjected into the B?tzinger and pre-B?tzinger complexes consistently increased phrenic nerve frequency. These results support the widespread distribution of MOR in the VRG and also indicate that endomorphin-1, a postulated endogenous ligand, may differentially regulate respiration.     

Chemical activation of caudal medullary expiratory neurones alters the pattern of breathing in the cat.

1. The purpose of this work was to ascertain whether the activation of caudal expiratory neurones located in the caudal part of the ventral respiratory group (VRG) may affect the pattern of breathing via medullary axon collaterals. 2. We used microinjections of DL-homocysteic acid (DLH) to activate this population of neurones in pentobarbitone-anaesthetized, vagotomized, paralysed and artificially ventilated cats. Both phrenic and abdominal nerve activities were monitored; extracellular recordings from medullary and upper cervical cord respiratory neurones were performed. 3. DLH (160 mM) microinjected (10-30 nl for a total of 1.6-4.8 nmol) into the caudal VRG, into sites where expiratory activity was encountered, provoked an intense and sustained activation of the expiratory motor output associated with a corresponding period of silence in phrenic nerve activity. During the progressive decline of the activation of abdominal motoneurones, rhythmic inspiratory activity resumed, displaying a decrease in frequency and a marked reduction or the complete suppression of postinspiratory activity as its most consistent features. 4. Medullary and upper cervical cord inspiratory neurones exhibited inhibitory responses consistent with those observed in phrenic nerve activity, while expiratory neurones in the caudal VRG on the side contralateral to the injection showed excitation patterns similar to those of abdominal motoneurones. On the other hand, in correspondence to expiratory motor output activation, expiratory neurones of the Bötzinger complex displayed tonic discharges whose intensity was markedly lower than the peak level of control breaths. 5. Bilateral lignocaine blockades of neural transmission at C2-C3 affecting the expiratory and, to a varying extent, the inspiratory bulbospinal pathways as well as spinal cord transections at C2-C3 or C1-C2, did not suppress the inhibitory effect on inspiratory neurones of either the ipsi- or contralateral VRG in response to DLH microinjections into the caudal VRG. 6. The results show that neurones within the column of caudal VRG expiratory neurones promote inhibitory effects on phrenic nerve activity and resetting of the respiratory rhythm. We suggest that these effects are mediated by medullary bulbospinal expiratory neurones, which may, therefore, have a function in the control of breathing through medullary axon collaterals.     

NMDA receptors in preBötzinger complex neurons can drive respiratory rhythm independent of AMPA receptors

The role of AMPA receptors (AMPARs) in generation and propagation of respiratory rhythm is well documented both in vivo and in vitro , whereas the functional significance of NMDA receptors (NMDARs) in preBötzinger complex (preBötC) neurons has not been explored. Here we examined the interactions between AMPARs and NMDARs during spontaneous respiratory rhythm generation in slices from neonatal rats in vitro . We tested the hypothesis that activation of NMDARs can drive respiratory rhythm in the absence of other excitatory drives. Blockade of NMDARs with dizocilpine hydrogen maleate (MK-801, 20 μ m ) had a negligible effect on respiratory rhythm and pattern under standard conditions in vitro , whereas blockade of AMPARs with NBQX (0.5 μ m ) completely abolished respiratory activity. Removal of extracellular Mg²⁺ to relieve the voltage-dependent block of NMDARs maintained respiratory rhythm without a significant effect on period, even in the presence of high NBQX concentrations (≤ 100 μ m ). Removal of Mg²⁺ increased inspiratory-modulated inward current peak ( I _I) and charge ( Q _I) in preBötC neurons voltage-clamped at −60 mV by 245% and 309%, respectively, with respect to basal values. We conclude that the normal AMPAR-mediated postsynaptic current underlying respiratory drive can be replaced by NMDAR-mediated postsynaptic current when the voltage-dependent Mg²⁺ block is removed. Under this condition, respiratory-related frequency is unaffected by changes in I _I, suggesting that the two can be independently regulated.