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.     

Modeling the ponto-medullary respiratory network

The generation and shaping of the respiratory motor pattern are performed in the lower brainstem and involve neuronal interactions within the medulla and between the medulla and pons. A computational model of the ponto-medullary respiratory network has been developed by incorporating existing experimental data on the medullary neural circuits and possible interactions between the medulla and pons. The model reproduces a number of experimental findings concerning alterations of the respiratory pattern following various perturbations/stimulations applied to the pons and pulmonary afferents. The results of modeling support the concept that eupneic respiratory rhythm generation requires contribution of the pons whereas a gasping-like rhythm (and the rhythm observed in vitro) may be generated within the medulla and involve pacemaker-driven mechanisms localized within the medullary pre-Bötzinger Complex. The model and experimental data described support the concept that during eupnea the respiration-related pontine structures control the medullary network mechanisms for respiratory phase transitions, suppress the intrinsic pacemaker-driven oscillations in the pre-BötC and provide inspiration-inhibitory and expiration-facilitatory reflexes which are independent of the pulmonary Hering–Breuer reflex but operate through the same medullary phase switching circuits.     

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.     

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.     

Respiratory responses to somatostatin microinjections into the Bötzinger complex and the pre-Bötzinger complex of the rabbit

The respiratory responses to bilateral microinjections (30–50 nl) of 5 mM somatostatin (SOM) or 10 mM cyclosomatostatin (c-SOM, a SOM antagonist) into the Bötzinger complex (BötC), the pre-Bötzinger complex (preBötC) and the rostral inspiratory portion of the ventral respiratory group (iVRG) were investigated in urethane–chloralose anesthetized, vagotomized, paralysed and artificially ventilated rabbits. SOM microinjections into the BötC decreased respiratory frequency and the rate of rise of phrenic nerve activity without obvious changes in its peak amplitude. SOM microinjected into the preBötC caused increases in respiratory frequency and decreases in peak phrenic activity associated with increases in its rate of rise. No changes in respiration were induced by SOM microinjections into the iVRG. Microinjections of c-SOM into the preBötC caused decreases in respiratory frequency as well as in peak amplitude and rate of rise of phrenic nerve activity. The results show that endogenously released SOM within the preBötC contributes to shape the pattern of baseline respiratory activity and that SOM receptors within the BötC and the preBötC have a role in the modulation of respiration in the rabbit.     

Multiple pontomedullary mechanisms of respiratory rhythmogenesis

Mammalian central pattern generators producing rhythmic movements exhibit robust but flexible behavior. However, brainstem network architectures that enable these features are not well understood. Using precise sequential transections through the pons to medulla, it was observed that there was compartmentalization of distinct rhythmogenic mechanisms in the ponto-medullary respiratory network, which has rostro-caudal organization. The eupneic 3-phase respiratory pattern was transformed to a 2-phase and then to a 1-phase pattern as the network was physically reduced. The pons, the retrotrapezoid nucleus and glycine mediated inhibition are all essential for expression of the 3-phase rhythm. The 2-phase rhythm depends on inhibitory interactions (reciprocal) between Bötzinger and pre-Bötzinger complexes, whereas the 1-phase-pattern is generated within the pre-Bötzinger complex and is reliant on the persistent sodium current. In conditions of forced expiration, the RTN region was found to be essential for the expression of abdominal late expiratory activity. However, it is unknown whether the RTN generates or simply relays this activity. Entrained with the central respiratory network is the sympathetic nervous system, which exhibits patterns of discharge coupled with the respiratory cycle (in terms of both gain and phase of coupling) and dysfunctions in this coupling appear to underpin pathological conditions. In conclusion, the respiratory network has rhythmogenic capabilities at multiple levels of network organization, allowing expression of motor patterns specific for various physiological and pathophysiological respiratory behaviors.     

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.     

Depletion of substance P and glutamate by capsaicin blocks respiratory rhythm in neonatal rat in vitro

The specific role of the neuromodulator substance P (SP) and its target, the neurokinin 1 receptor (NK1R), in the generation and regulation of respiratory activity is not known. The preBötzinger complex (preBötC), an essential site for respiratory rhythm generation, contains glutamatergic NK1R-expressing neurones that are strongly modulated by exogenously applied SP or acute pharmacological blockade of NK1Rs. We investigated the effects of capsaicin, which depletes neuropeptides (including SP) and glutamate from presynaptic terminals, on respiratory motor output in medullary slice preparations of neonatal rat that generate respiratory-related activity. Bath application of capsaicin slowed respiratory motor output in a dose- and time-dependent manner. Respiratory rhythm could be restored by bath application of SP or glutamate transporter blockers. Capsaicin also evoked dose-dependent glutamate release and depleted SP in fibres within the preBötC. Our results suggest that depletion of SP (or other peptides) and/or glutamate by capsaicin causes a cessation of respiratory rhythm in neonatal rat slices.     

Opioid-induced depression in the lamprey respiratory network

The role of opioid receptors in modulating respiratory activity was investigated in in vitro brainstem preparations of adult lampreys by bath application of agonists and antagonists. The vagal motor output was used to monitor respiratory activity. Neuronal recordings were also performed to characterize the rostrolateral trigeminal region that has been suggested to be critical for respiratory rhythmogenesis. Microinjections of the micro-opioid receptor agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) were also made into this region and at different locations within the brainstem. Bath application of DAMGO (0.5-2 microM) caused marked decreases in respiratory frequency up to complete apnea. Bath application of the delta-opioid receptor agonist [d-Pen(2,5)]-enkephalin (DPDPE) at 10-40 microM induced less pronounced depressant respiratory effects, while no changes in respiratory activity were induced by the kappa-opioid receptor agonist trans-(1S,2S)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide (U50488) at 10-40 microM. Bath application of the opioid receptor antagonists naloxone and naltrindole did not affect baseline respiratory activity, but prevented agonist-induced effects. DAMGO microinjections (1 mM; 0.5-1 nl) at sites rostrolateral to the trigeminal motor nucleus, where respiration-related neuronal activity was recorded, abolished the respiratory rhythm. The results show that opioids may have an important role in the lamprey respiratory network and that micro-opioid receptor activation is the most effective in causing respiratory depression. They also indicate that endogenous opioids are not required for the generation of baseline respiratory activity. Apneic responses induced by DAMGO microinjections support the hypothesis that a specific opioid-sensitive region rostrolateral to the trigeminal motor nucleus, that we have termed the paratrigeminal respiratory group (pTRG), likely has a pivotal role in respiratory rhythmogenesis. Since the lamprey diverged from the main vertebrate line around 450 million years ago, our results also imply that the inhibitory role of opioids on respiration is present at an early stage of vertebrate evolution.     

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.     

Hypoglossal motoneurons in newborn mice receive respiratory drive from both sides of the medulla

Respiratory motor output in bilateral cranial nerves is synchronized, but the underlying synchronizing mechanisms are not clear. We used an in vitro slice preparation from newborn mice to investigate the effect of systematic transsections on respiratory activity in bilateral XII nerves. Complete transsection at the midline resulted in desynchronized rhythm with reduced XII burst amplitude and duration. Transsections in the ventral or dorsal 1/3 of the midline did not desynchronize rhythm. However, transsections in the ventral 2/3 of the midline desynchronized rhythm with characteristic amplitude correlations, where large-amplitude XII-bursts on one side was synchronized with small-amplitude XII-burst on the contralateral side. These characteristic amplitude correlations suggest that hypoglossal motoneurons receive respiratory drive from bilateral sources. Retrograde labeling confirmed that commissural fibers from the pre-Bötzinger complex cross in the mid-1/3 of the midline, and that dendrites of hypoglossal motoneurons project into the contralateral XII nucleus. In conclusion, commissural fibers crossing in the mid-1/3 of the midline are required for synchronization of respiratory activity in bilateral XII nerves. Hypoglossal motoneurons receive respiratory drive from both sides of the medulla, possibly mediated by contralaterally projecting dendrites.     

Role of persistent sodium current in mouse preBötzinger Complex neurons and respiratory rhythm generation

Breathing movements in mammals depend on respiratory neurons in the preBötzinger Complex (preBötC), which comprise a rhythmic network and generate robust bursts that form the basis for inspiration. Persistent Na⁺ current ( I _NaP) is widespread in the preBötC and is hypothesized to play a critical role in rhythm generation because of its subthreshold activation and slow inactivation properties that putatively promote long-lasting burst depolarizations. In neonatal mouse slice preparations that retain the preBötC and generate a respiratory-related rhythm, we tested the role of I _NaP with multiple Na⁺ channel antagonists: tetrodotoxin (TTX; 20 n m ), riluzole (RIL; 10 μ m ), and the intracellular Na⁺ channel antagonist QX-314 (2 m m ). Here we show that I _NaP promotes intraburst spiking in preBötC neurons but surprisingly does not contribute to the depolarization that underlies inspiratory bursts, i.e. the inspiratory drive potential. Local microinjection in the preBötC of 10 μ m RIL or 20 n m TTX does not perturb respiratory frequency, even in the presence of bath-applied 100 μ m flufenamic acid (FFA), which attenuates a Ca²⁺-activated non-specific cation current ( I _CAN) that may also have burst-generating functionality. These data contradict the hypothesis that I _NaP in preBötC neurons is obligatory for rhythmogenesis. However, in the presence of FFA, local microinjection of 10 μ m RIL in the raphe obscurus causes rhythm cessation, which suggests that I _NaP regulates the excitability of neurons outside the preBötC, including serotonergic raphe neurons that project to, and help maintain, rhythmic preBötC function.     

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.     

Postnatal changes in the expressions of serotonin 1A, 1B,and 2A receptors in ten brain stem nuclei of the rat: implication for a sensitive period

A critical period in respiratory network development occurs in the rat around postnatal days (P) 12–13, when abrupt neurochemical, metabolic, and physiological changes were evident. As serotonin and its receptors are involved in respiratory modulation, and serotonergic abnormality is implicated in sudden infant death syndrome, we hypothesized that 5-HT receptors are significantly downregulated during the critical period. This was documented recently for 5-HT_2AR in several respiratory nuclei. The present study represents a comprehensive analysis of postnatal development of 5-HT_1AR and 5-HT_1BR in 10 brain stem nuclei and 5-HT_2AR in six nuclei not previously examined. Optical densitometric analysis of immunohistochemically-reacted neurons from P2 to P21 indicated four developmental patterns of expression: (1) Pattern I: a high level of expression at P2-P11, an abrupt and significant reduction at P12, followed by a plateau until P21 (5-HT_1AR and 5-HT_1BR in raphé magnus [RM], raphé obscurus [ROb], raphé pallidus [RP], pre-Bötzinger complex [PBC], nucleus ambiguus [Amb], and hypoglossal nucleus [XII; 5-HT_1AR only]). (2) Pattern II: a high level at P2-P9, a gradual decline from P9 to P12, followed by a plateau until P21 (5-HT_1AR and 5-HT_1BR in the retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG)). (3) Pattern III: a high level at P2-P11, followed by a gradual decline until P21 (5-HT_1AR in the ventrolateral subnucleus of solitary tract nucleus [NTS_VL] and the non-respiratory cuneate nucleus [CN]). (4) Pattern IV: a relatively constant level maintained from P2 to P21 (5-HT_1AR in the commissural subnucleus of solitary tract nucleus (NTS_COM); 5-HT_1BR in XII, NTS_VL, NTS_COM, and CN; and 5-HT_2AR in RM, ROb, RP, RTN/pFRG, NTS_VL, and NTS_COM). Thus, a significant reduction in the expression of 5-HT_1AR, 5-HT_1BR, and 5-HT_2AR in multiple respiratory-related nuclei at P12 is consistent with reduced serotonergic transmission during the critical period, thereby rendering the animals less able to respond adequately to ventilatory distress.     

Prenatal nicotine exposure alters the response of the mouse in vitro respiratory rhythm to hypoxia

Prenatal nicotine exposure is associated with deficiencies in the ability to respond to life threatening stressors such as hypoxia. Although many of these deficiencies appear to originate from defects in the brainstem respiratory network, the specific effects of prenatal nicotine exposure on the brainstem respiratory network are not well understood. We have tested the effects of prenatal nicotine exposure on the respiratory rhythm using an in vitro mouse brainstem slice preparation containing the pre-Bötzinger Complex, a region of the ventral respiratory group that is the postulated site of inspiratory rhythm generation. We found that nicotine exposure during pre- and early postnatal development led to a lower frequency of baseline fictive respiratory discharges from rhythmic slices and a reduction in the ability of the slice to maintain a respiratory rhythm during exposure to severe hypoxia compared to controls. These impairments of the central respiratory rhythm could potentially affect the ability to survive a period of exposure to severe hypoxia in vivo.     

Effects of riluzole and flufenamic acid on eupnea and gasping of neonatal mice in vivo

The pre-Bötzinger complex (PBC), part of the ventral respiratory group that is responsible for inspiratory rhythm generation, contains at least two types of pacemaker neurons. In vitro studies have shown that bursting properties of one type of pacemaker relies on a riluzole-sensitive persistent sodium current, whereas bursting of a second type is sensitive to flufenamic acid (FFA), a calcium-dependent nonspecific cationic current blocker. In vitro, under control conditions, the PBC generates fictive eupneic activity that depends on both riluzole-sensitive and FFA-sensitive pacemaker neurons. During hypoxia the PBC generates fictive gasping activity and only riluzole-sensitive pacemaker neurons appear to be necessary for this rhythm. We carried out pharmacological experiments to test the role of respiratory pacemaker neurons in vivo by performing plethysmographic recordings on neonate mice. As reported in vitro, eupnea activity in vivo is abolished only if both FFA and riluzole are coadministered intracisternally, but not when either of them is administered independently. On the other hand riluzole, but not FFA, drastically reduced gasping generation and compromised the ability of mice to autoresucitate. Neither substance P nor forskolin was able to reestablish respiratory activity after riluzole and FFA coapplication. Our results confirm in vitro reports and suggest that eupnea generation in neonates requires a complex neuronal network that includes riluzole- and FFA-sensitive elements and that gasping activity depends mostly on a riluzole-sensitive mechanism.     

Spatial and functional architecture of the mammalian brain stem respiratory network: a hierarchy of three oscillatory mechanisms

Mammalian central pattern generators (CPGs) producing rhythmic movements exhibit extremely robust and flexible behavior. Network architectures that enable these features are not well understood. Here we studied organization of the brain stem respiratory CPG. By sequential rostral to caudal transections through the pontine-medullary respiratory network within an in situ perfused rat brain stem-spinal cord preparation, we showed that network dynamics reorganized and new rhythmogenic mechanisms emerged. The normal three-phase respiratory rhythm transformed to a two-phase and then to a one-phase rhythm as the network was reduced. Expression of the three-phase rhythm required the presence of the pons, generation of the two-phase rhythm depended on the integrity of B?tzinger and pre-B?tzinger complexes and interactions between them, and the one-phase rhythm was generated within the pre-B?tzinger complex. Transformation from the three-phase to a two-phase pattern also occurred in intact preparations when chloride-mediated synaptic inhibition was reduced. In contrast to the three-phase and two-phase rhythms, the one-phase rhythm was abolished by blockade of persistent sodium current (I(NaP)). A model of the respiratory network was developed to reproduce and explain these observations. The model incorporated interacting populations of respiratory neurons within spatially organized brain stem compartments. Our simulations reproduced the respiratory patterns recorded from intact and sequentially reduced preparations. Our results suggest that the three-phase and two-phase rhythms involve inhibitory network interactions, whereas the one-phase rhythm depends on I(NaP). We conclude that the respiratory network has rhythmogenic capabilities at multiple levels of network organization, allowing expression of motor patterns specific for various physiological and pathophysiological respiratory behaviors.     

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⁺.     

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.