Parameters of discharges of respiratory neurons of the ventrolateral medullary regions in early postnatal rats

In experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of newborn (1st day of life) and 4- to 5-day-old rats, we studied the parameters of extracellularly recorded spike activity of respiratory neurons of the ventrolateral medullary regions (VLMR). In SIMSP of 4- to 5-day-old rats, the frequency of discharges of pre-inspiratory, inspiratory, and expiratory neurons is shown to be significantly higher, while the dispersion of its values is considerably lower, as compared with the corresponding values for newborn animals. In the majority of pre-inspiratory and inspiratory neurons of SIMSP of newborn rats, irregular low-frequency discharges are usually generated within the interinspiration phase. The relative intensity of suppression of discharges of pre-inspiratory and expiratory neurons within an inspiration phase is much lower in SIMSP of newborn rats, as compared with that in 4- to 5-day-old preparations. The activity of most pre-inspiratory neurons manifests a trend toward transformation from a two-phase pattern in newborn rats (with two frequency peaks, pre- and post-inspiratory) to a monophasic pattern (with one pre-inspiratory frequency peak) typical of 4- to 5-day-old animals. The effects of electrical stimulation of the site of localization of pre-inspiratory neurons showed that in SIMSP of both age groups of rats an inspiratory response could be evoked in then. phrenicus only in the case when stimulation was applied within the second half of an interinspiratory phase. Therefore, it can be supposed that the respiratory network in newborn animals is to a considerable extent immature in the morphofunctional aspect. It seems probable that in early postnatal rats pre-inspiratory neurons are involved in the medullary mechanisms foron-off switching of the inspiratory and expiratory phases.Neirofiziologiya/Neurophysiology, Vol. 28, No. 4/5, pp. 207–217, July–October, 1996.     

Participation of the opioid- and serotonergic systems of the ventrolateral medullary regions in the control of respiratory activity in early postnatal rats

The effects of blockers of opioid and serotonin receptors (naloxone and methisergide, respectively) on the respiratory activity recorded from then. phrenicus of semi-isolated medullo-spinal preparations (SIMSP) were studied in newborn (1-day-old) and 4- to 5-day-old rats. The preparations were superfusedin situ, and inspiratory discharges (ID) were recorded before and after transverse sections of the ventrolateral medullary regions (VLMR) at different levels. Naloxone evoked an increase in the ID frequency in then. phrenicus both at the initial configuration of preparations and after successive transections of the VLMR between theM andS chemosensitive zones and between theS andL zones. The relative intensity of this effect was significantly higher in newborn rats than in 4- to 5-day-old ones. In contrast, methisergide made the ID frequency lower at all configurations of the SIMSP, and this effect was more intensive in 4- to 5-day-old animals. In the course of progressive separations of more rostral VLMR zones, the effects of naloxone and methisergide applications on the ID frequency became weaker. This allows us to suppose that in newborn rats the rostral VLMR portions provide more intensive opioidergic inhibitory control influences on the activity of respiratory networks than those in 4- to 5-day-old animals. At the same time, tonic activating influences from serotonergic VLMR neurons on the mechanisms generating respiratory rhythm are more intensive in 4- to 5-day-old animals than those in newborn ones. Thus, it can be supposed that the levels of maturation of the opioid- and serotonergic neurotransmitter systems, which provide tonic control of respiratory activity generated in the VLMR, are different in newborn and 4- to 5-day-old animals. The problems of how the opioid- and serotonergic mechanisms controlling respiratory rhythm generation are formed in the course of early stages of ontogenetic development are discussed.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 62–73, January–February, 1996.     

Effects of hypoxia on the respiratory activity generated by semi-isolated medullo-spinal preparations of early postnatal rats

The effects of short- and long-lasting (2-min-long and up to 30-min-long) hypoxia episodes on the inspiratory activity (IA) recorded from then. phrenicus were tested in experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of newborn (the lst day of life) and 4- to 5-day-old rats. Hypoxia was provided by superfusion of the preparations with low-O₂ solution. Short-lasting hypoxia evoked no significant modulation of the IA in preparations of newborn animals, while two-phase responses (an initial, up to 30 sec, increase in the frequency of inspiratory discharges, followed by their longer, up to 4 min, suppression) were observed in 4- to 5-day-old preparations. Long-lasting hypoxia suppressed activity in then. phrenicus of 1-day-old preparations, and this effect was replaced in five cases by the development of sporadic low-amplitude and short-lasting inspiratory discharges. These shortened discharges were qualified as gasps. The responses of 4- to 5-day-old SIMSP to long-lasting hypoxia episodes were more complex. An initial increase in the IA frequency lasted up to 30 sec, and in 4–6 min it was followed by complete suppression of the activity. In some of the SIMSP, permanent tonic activity appeared in then. phrenicus within the period of total absence of inspiratory discharges, which could be followed by generation of short low-amplitude gasping discharges. Against the background of gasping pattern, eupnoe-like discharges appeared in four preparations. Under control conditions, transerve section of the ventrolateral medullary regions (VLMR) at a middle level of then. hypoglossus root abolished respiratory activity in all studied SIMSP. Yet, in some of the SIMSP of both newborn and 4- to 5-day-old animals long-lasting hypoxia testing evoked weak tonic activity in then. phrenicus followed by the appearance of gasping discharges. After the transection of the VLMR at the caudal edge of then. hypoglossus root, long-lasting hypoxia evoked only weak tonic responses in some SIMSP of both age groups, and there were no phasic discharges in this case. The results of our experiments, first, show that the respiratory activity in newborn animals is more resistant to hypoxia than that in 4-to 5-day-old rats, and, second, they allow us to suppose that the gasp-generating medullary structures are localized in more caudal medullary regions. We discuss the questions of how the eupnoe-generating and gasp-generating medullary structures are formed in rats during their initial four to five postnatal days, and what specific features are typical of hypoxia-related respiratory responses in these animals.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 121–131, March–June, 1996.     

Roles of NMDA receptors and nitric oxide in responses of the medullary respiratory generator of early postnatal rats to hypoxia and acidosis

The dynamics of changes in the frequency of the respiratory activity recorded from the n. phrenicus under conditions of 3-min-long applications of 5 μM N-methyl-D-aspartate (NMDA), an anoxic gas mixture-saturated saline, or an acidified (pH 7.0) solution were studied in the experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of 3- to 4-day-old rats. Test applications were performed on the intact SIMSP or on those preliminarily influenced by the following substances: a non-competitive NMDA receptor blocker, ketamine (10 μM); an inhibitor of NO synthase, methyl ester of N^G-nitro-L-arginine (MENA, l0 μM); hemoglobin, which binds NO (Hb, 0.3 μM); an NO donor, sodium nitroprusside (SNP, 10 μM); or/and a competitive blocker of non-NMDA receptors, CNQX (1.0 μM). Application of NMDA increased the frequency of the respiratory discharges, and the effect was blocked by MENA, Hb, and SNP. Addition of Hb to the SNP-containing solution neutralized the effect of the latter. In hypoxia, ketamine blocked an increase in the respiratory frequency within the initial 90-sec segment of the test and decreased the rhythm suppression within the second test half. MENA increased the respiration discharge frequency throughout the test. CNQX exerted no Influence on the frequency in the initial period and decreased its suppression within the second test half. Preliminary ketamine and MENA applications made smaller the increment of the discharge frequency at application of the solution with pH 7.0; the MENA effect was stronger. In addition, using a histochemical technique, we studied spatial distribution of the neurons containing an NO synthase marker, NADPH-diaphorase (NADPH-d), in frontal sections of the medulla of 4-day-old rats. NADPH-d-positive cells were observed within the limits of the dorsal and ventral respiratory neuronal groups (DRG and VRG, respectively). Their density was the highest in the rostral VRG part (in the region of the lateral paragigantocellular nucleus). Our results show that in early postnatal rats NMDA receptors and endogenous NO are actively involved in the control of respiratory rhythm generated by SIMSP under hypoxic and acidotic conditions. The results of morphohistochemical study can be considered a neuroanatomical support for the active NO role in the control of medullary respiratory rhythm in the early postnatal period.     

Respiratory activity generated by semi-isolated medullo-spinal preparation and recorded from the phrenic nerve of newborn rats

Inspiratory activity generated by superfusedin situ semi-isolated medullo-spinal preparations of newborn (one-day-old) and four- to five-day-old rats was recorded from then. phrenicus before and after transverse sectioning of the ventrolateral part of the medulla (VLPM) at different levels. Under similar experimental conditions, the frequency of inspiratory discharges (ID) and their integral intensity, reflecting the volume and temporal parameters of inspiration, are much lower in one-day-old rats, as compared with those in four- to five-day-old animals. Specific roles of different VLPM levels in respiration control in young rats are demonstrated. Transection of the VLPM below the most rostral VLPM portion, corresponding to theM chemosensitive zone, caused a significant increase in the ID frequency and a decrease in the ID integral intensity. Transection performed below the intermediate VLPM region, corresponding to theS chemosensitive zone, resulted in a significant decrease in both ID frequency and ID integral intensity, up to total ID blockade in 5 of 12 1-day-old preparations. This finding can be interpreted as an indication of morphofunctional immaturity of the respiratory network in the caudal VLPM regions in newborn animals. Comparative analysis of ID pattern showed that this activity in one-day-old rats is more or less gasping-like, while that in four- to five-day-old animals is eupnoe-like. The results allow us to conclude that the level of maturity of morphofunctional organization of medullary respiratory networks considerably differs in newborn and older animals. The mechanisms responsible for formation and control of respiratory activity in early postnatal period of rats are discussed.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 387–395, September–December, 1995.     

Impact of NMDA Receptors and Nitric Oxide on pH Sensitivity of Medullary Mechanisms Controlling Respiratory Rhythm

We studied the dynamics of modifications of the respiratory activity generated by semi-isolated medullo-spinal preparations (SIMSP) of 3- to 4-day-old rats related to a drop in the pH of superfusing solution from 7.4 to 7.0. Reactions were recorded in the norm and under conditions of preliminary applications of a noncompetitive blocker of NMDA receptors, ketamine; an inhibitor of nitric oxide synthase (NOS), N^G-nitro-L-arginine methyl ester (L-NAME); a substrate for NO synthesis, L-arginine; or an exogenous NO donor, sodium nitroprusside (SN). Under control conditions, test applications of the solution with pH 7.0 resulted in a significant increase in the frequency of inspiratory discharges (ID) recorded from the phrenic nerve and drops in their amplitude and integral intensity. Such SIMSP extracellular acidification-induced responses were inhibited in a dose-dependent manner by ketamine and L-NAME (the effect of the latter was more intensive). The effects of agents increasing the NO level in the tissues were not uniform: L-arginine potentiated an increase in the ID frequency related to application of the acidified solution, while SN inhibited such a reaction. Our findings allow us to suppose that the stimulating influences of the pH-sensitive chemoreceptor structures of the ventrolateral medulla (VLM) on the activity of the medullary respiratory generator of early postnatal rats are realized with the involvement of NMDA receptors of excitatory amino acids and the process of enzyme-mediated NO production. It seems probable that endogenous synthesis of NO in VLM structures mediates and potentiates the effect of activation of the NMDA receptors on the medullary generator of the respiratory rhythm.     

Effect of nitric oxide on the respiratory activity generated under hypoxic conditions by medullo-spinal preparations from early postnatal rats

Experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of 3- to 4-day-old rats were carried out to study the effects of a blocker of nitric oxide synthase (NO synthase), methyl ester of N^G-nitro-L-arginine (MENA), and an exogenic NO donor, nitroglycerin, on the respiratory activity. Inspiratory discharges (ID) were recorded from the phrenic nerve under superfusion of SIMSP with a standard saline and a solution saturated with anoxic isocapnic gas mixture. Under normal conditions, 3-min-long applications of 1.0 μM MENA evoked no significant changes in the parameters of inspiratory activity; yet 10.0 μM of this blocker evoked a significant drop in the amplitude and an increase in the ID frequency. Three-min-long applications of 1.0 μM nitroglycerin significantly decreased the ID frequency and somewhat increased their amplitude and integral intensity. Higher doses of nitroglycerin (10.0 μM) significantly increased the amplitude and integral intensity of ID and in a lesser extent lowered their frequency. Under conditions of 3-min-long hypoxia, 10-min-long preliminary superfusion of SIMSP with the 1.0 μM MENA-containing saline resulted in no significant changes of respiratory activity, as compared with the hypoxia effect in the norm. Applied before the hypoxic test, 10 μM MENA resulted in significant decreases in the amplitude and integral intensity of ID; concurrently their frequency became higher, as compared with the respective parameters measured at hypoxic testing of the intact preparations. Ten-min-long superfusion with 1.0 μM nitroglycerin-containing solution at subsequent hypoxic testing significantly increased the amplitude and integral intensity of ID and decreased their frequency; these shifts developed during the first half of exposure to the hypoxic solution. Increased (to 10 μM) nitroglycerin concentration resulted in less intensive shifts in the ID frequency within the first half of a hypoxic episode. In a part of the tests, the second half of exposure of SIMSP to the hypoxic solution was characterized by the appearance of low-amplitude short ID against the background of suppressed eupnea-like respiratory activity; we qualified such discharges as gasping discharges. The experimental data confirm the involvement, of NO in the central regulation of the frequency and amplitude parameters of inspiratory activity generated by SIMSP of early postnatal rats both under normoxic and hypoxic conditions. The role NO plays under hypoxic conditions in modifications of parameters of respiratory activity and in modulation of the functional, levels of the bulbar respiratory generator is discussed.     

Blocking of glutamate ionotropic receptors: Influence on the respiratory activity generated by medullo-spinal preparations of rats in hypoxia

We studied the influences of a non-competitive blocker of glutamate NMDA-receptors ketamune and of a competitive blocker of AMPA-kainate non-NMDA receptors, CNQX, on the respiratory activity generelated by superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of 3- to 4-day-old rats. We compared the ampes recorded under conditions of superfusion, a standard solution and the solution saturated with an anoxic isocapine gas mixture were compared; pO₂ in these solutions were 440±22 and 41±8 mm Hg, respectively. The experments were carried out with the ventrolateral medullary region (VLMR) left intact or after separation of its rostral part, which propertchonally corresponded to the chemosensitiveM zone. A 3-min-long hypoxic test initially evoked an increase in the frequency of inspiratory discharges (IR) in the phrenic nerve followed by a frequency drop within the final half of the test. After the rostral VLMR had been separated, the hypoxic test did not elicit a significant decrease in the IR frequency. After preliminary application of 1.0 or 10.0 μM ketamine or CNQX on intact preparations, the IR frequency under hypoxic conditions dropped within the first half of the test and increased in the second half, while the amplitude and integral intensity of these discharges were depressed more intensively than in hypoxia with no applications. Using ketamme and CNQX in the same concentrations resulted in significant drops in the amplitude, frequency, and integral intensity of IR recorde din the hypoxic test. Our experiments showed that in the early postnatal period glutamate ionotropic receptors of rostral VLMR neurons are involved in the control of IR frequency under hypoxic conditions. The possible role of glutamatergic control of the respiratory rhythm and mechanisms of the influences resulting from blocking of NMDA and non-NMDA receptors on the parameters of respiratory activity are discussed.     

Involvement of NMDA receptors in the control of respiratory rhythm generated by medullo-spinal preparations of early postnatal rats

Effects of a non-competetive blocker of glutamate NMDA receptors, ketamine, on respiratory activity recorded from the phrenic nerve were studied in experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of 3− to 4-day-old rats. The experiments were carried out under conditions where the ventrolateral medullary region (VLM) was left intact, or its rostral portion (projectionally corresponding to the chemosensitiveM zone) was separated by transection. Three-min-long application of 1.0 μM ketamine evoked a slight increase in the duration of inspiratory discharges (ID) and a statistically significant increase in their frequency. After the rostral VLM had been separated, similar ketamine application resulted in significant increases in the duration, amplitude, and integral intensity of ID and some drop in their frequency. An increase to 10 μM ketamine concentration in the superfusing solution determined a significant rise of the ID duration, which indicates the possibility of inhibition of the mechanisms switching inspiration to expiration. Concurrently, the ID frequency significantly dropped, while their amplitude and integral intensity increased. After separation of the rostral VLM, the latter ketamine concentration ceased to increase the ID duration, and their frequency and amplitude significantly dropped. Application of ketamine in the concentration of 100 μM resulted in rather profound decreases of all measured ID parameters, and separation of the rostral VLM exerted no influence on the direction of the above modifications. Thus, we obtained evidence of the involvement of NMDA receptors of the VLM in the control of temporal and frequency-amplitude parameters of respiratory activity of early postnatal rats. Possible localization of NMDA receptors and mechanisms of their involvement in inspiration-expiration switching and tonic inhibitory control of respiratory rhythms are discussed.     

Involvement of nitric oxide in realization of the NMDA influence on the respiratory rhythm generated by medullo-spinal preparations of early postnatal rats

The effects of blocking of NO synthase on the dynamics of NMDA-induced changes in the respiratory activity were studied in experiments on semi-isolatedin situ medullo-spinal preparations of 3- to 4-day-old rats. The experiments were carried out with the ventrolateral regions of the medulla (VLMR) left intact or when the rostral portion of this region (corresponding to the chemosensitive zoneM) had been separated by a transversial transection. Three-min-long application of 5.0 μM NMDA increased the frequency of inspiratory discharges (ID); the shifts were more intensive after separation of the VLM rostral portion. Superfusion of preparations with a solution containing 10.0 μM of an NO synthase inhibitor, methyl ester of N^G-nitro-L-arginine (MENA), increased the frequency and decreased the amplitude and integral intensity of ID generated by the preparations with the intact VLM, whereas after separation of the rostral VLM portion this inhibitor decreased the ID frequency. Application of 5 μM NMDA against the background of 10-min-long superfusion with the 10 μM MENA-containing solution resulted in no significant increase in the ID frequency. After the rostral VLM portion had been removed, NMDA application after superfusion with the MENA-containing solution led to frequency shifts which did not significantly differ from those in the absence of the blockade of NO synthase. Against the MENA influence, NMDA-induced depression of the ID amplitude became significantly more intensive. The experiments showed that during the early postnatal period endogenous NO is involved in realization of the NMDA influence on the parameters of respiratory activity of rats. Possible mechanisms of the influences exerted by activation of NMDA receptors and by the associated NO synthesis on regulation of the respiratory rhythmogenesis and their specificity within the early postnatal period are discussed.     

d-serine regulation of the timing and architecture of the inspiratory burst in neonatal mice

d-serine, released from mouse medullary astrocytes in response to increased CO₂ levels, boosts the respiratory frequency to adapt breathing to physiological demands. We analyzed in mouse neonates, the influence of d-serine upon inspiratory/expiratory durations and the architecture of the inspiratory burst, assessed by pwelch's power spectrum density (PSD) and continuous wavelet transform (CWT) analyses. Suction electrode recordings were performed in slices from the ventral respiratory column (VRC), site of generation of the respiratory rhythm, and in brainstem-spinal cord (en bloc) preparations, from the C5 ventral roots, containing phrenic fibers that in vivo innervate and drive the diaphragm, the main inspiratory muscle.In en bloc and slice preparations, d-serine (100 μM) reduced the expiratory, but not the inspiratory duration, and increased the frequency and the regularity of the respiratory rhythm. In en bloc preparations, d-serine (100 μM) also increased slightly the amplitude of the integrated inspiratory burst and the area under the curve of the integrated inspiratory burst, suggesting a change in the recruitment or the firing pattern of neurons within the burst. Time-frequency analyses revealed that d-serine changed the burst architecture of phrenic roots, widening their frequency spectrum and shifting the position of the core of firing frequencies towards the onset of the inspiratory burst. At the VRC, no clear d-serine induced changes in the frequency-time domain could be established. Our results show that d-serine not only regulates the timing of the respiratory cycle, but also the recruitment strategy of phrenic motoneurons within the inspiratory burst.     

Spectral characteristics of electrical activity of the respiratory center in brain processes in fetus and newborn rats in vitro

Power spectral analysis of inspiratory discharges of C3-C5 ventral roots in brainstem-spinal cord preparation from foetal (18 and 20 gestation days) and newborn (0-1 and 2-3 postnatal days) rats was performed. The respiratory centre perinatal development manifests itself by decreasing of respiratory rhythm variability and increasing of inspiratory burst duration. In foetal inspiratory bursts, low-frequency oscillations (1-10 Hz) dominate. In early postnatal stage, the relative power of low-frequency oscillations begin to decrease, and medium frequency oscillations (10-50 Hz) start to dominate over the inspiratory discharge. The data obtained suggests, that perinatal maturation of respiratory centre is characterised by stabilisation of the respiratory rhythm generation and developmental alteration of inspiratory activity's spectral and temporary parameters.     

NADPH-Diaphorase-containing neurons in the medullary structures involved in generation of the respiratory activity in neonatal rats

Using a histochemical technique, we examined distribution of the neurons containing a marker of nitric oxide synthase (NOS), NADPH-diaphorase (NADPH-d), on frontal slices of the medulla and upper cervical spinal segments of 4-day-old rats. It was demonstrated that NADPH-d-positive cells are present within the dorsal and ventral medullary respiratory groups. The highest density of the labeled middle-size multipolar neurons (27.9±2.6 cells per 0.1 mm² of the slice) was observed in the rostral part of the ventral respiratory group, within the reticular lateral paragigantocellular nucleus. Similar NADPH-d-positive neurons were also observed in other reticular formation structures: rostroventrolateral reticular, gigantocellular, and ventral medullary nuclei, and in the ventral part of the paramedial nucleus. There were no labeled neurons in the lateral reticular nucleus. Single small and medium-size labeled neurons were found at all rostro-caudal levels of thenucl. ambiguous (nuclei retrofacialis, ambiguous, andretroam-biguous). Groups of NADPH-d-positive neurons were also revealed within the dorsal respiratory group, along the whole length of thenucl. tractus solitarii (mostly in its ventrolateral parts). Single labeled neurons were also observed in thenucl. n. hypoglossi, and their groups were observed in the dorsal motor part of thenucl. n. vagus. Involvement of the structures containing NADPH-d-positive neurons in the processes related to generation of the respiratory activity is discussed. Our neuroanatomical experiments prove that in early postnatal mammals NO is actively involved in generation and regulation of the medullary respiratory rhythm. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 128–136, March–April, 2000.     

Association of nicotinic acetylcholine receptors with central respiratory control in isolated brainstem-spinal cord preparation of neonatal rats

Nicotine exposure is a risk factor in several breathing disorders Nicotinic acetylcholine receptors (nAChRs) exist in the ventrolateral medulla, an important site for respiratory control. We examined the effects of nicotinic acetylcholine neurotransmission on central respiratory control by addition of a nAChR agonist or one of various antagonists into superfusion medium in the isolated brainstem-spinal cord from neonatal rats. Ventral C4 neuronal activity was monitored as central respiratory output, and activities of respiratory neurons in the ventrolateral medulla were recorded in whole-cell configuration. RJR-2403 (0.1-10 mM), alpha4beta2 nAChR agonist induced dose-dependent increases in respiratory frequency. Non-selective nAChR antagonist mecamylamine (0.1-100 mM), alpha4beta2 antagonist dihydro-beta-erythroidine (0.1-100 mM), alpha7 antagonist methyllycaconitine (0.1-100 mM), and a-bungarotoxin (0.01-10 mM) all induced dose-dependent reductions in C4 respiratory rate. We next examined effects of 20 mM dihydro-beta-erythroidine and 20mM methyllycaconitine on respiratory neurons. Dihydro-beta-erythroidine induces hyperpolarization and decreases intraburst firing frequency of inspiratory and preinspiratory neurons. In contrast, methyllycaconitine has no effect on the membrane potential of inspiratory neurons, but does decrease their intraburst firing frequency while inducing hyperpolarization and decreasing intraburst firing frequency in preinspiratory neurons. These findings indicate that alpha4beta2 nAChR is involved in both inspiratory and preinspiratory neurons, whereas alpha7 nAChR functions only in preinspiratory neurons to modulate C4 respiratory rate.     

Opioid-induced quantal slowing reveals dual networks for respiratory rhythm generation

Current consensus holds that a single medullary network generates respiratory rhythm in mammals. Pre-B?tzinger Complex inspiratory (I) neurons, isolated in transverse slices, and preinspiratory (pre-I) neurons, found only in more intact en bloc preparations and in vivo, are each proposed as necessary for rhythm generation. Opioids slow I, but not pre-I, neuronal burst periods. In slices, opioids gradually lengthened respiratory periods, whereas in more intact preparations, periods jumped nondeterministically to integer multiples of the control period (quantal slowing). These findings suggest that opioid-induced quantal slowing results from transmission failure of rhythmic drive from pre-I neurons to preB?tC I networks, depressed below threshold for spontaneous rhythmic activity. Thus, both I (in the slice), and pre-I neurons are sufficient for respiratory rhythmogenesis.     

Dual oscillator model of the respiratory neuronal network generating quantal slowing of respiratory rhythm

We developed a dual oscillator model to facilitate the understanding of dynamic interactions between the parafacial respiratory group (pFRG) and the preBötzinger complex (preBötC) neurons in the respiratory rhythm generation. Both neuronal groups were modeled as groups of 81 interconnected pacemaker neurons; the bursting cell model described by Butera and others [model 1 in Butera et al. (J Neurophysiol 81:382–397, 1999a)] were used to model the pacemaker neurons. We assumed (1) both pFRG and preBötC networks are rhythm generators, (2) preBötC receives excitatory inputs from pFRG, and pFRG receives inhibitory inputs from preBötC, and (3) persistent Na⁺ current conductance and synaptic current conductances are randomly distributed within each population. Our model could reproduce 1:1 coupling of bursting rhythms between pFRG and preBötC with the characteristic biphasic firing pattern of pFRG neurons, i.e., firings during pre-inspiratory and post-inspiratory phases. Compatible with experimental results, the model predicted the changes in firing pattern of pFRG neurons from biphasic expiratory to monophasic inspiratory, synchronous with preBötC neurons. Quantal slowing, a phenomena of prolonged respiratory period that jumps non-deterministically to integer multiples of the control period, was observed when the excitability of preBötC network decreased while strengths of synaptic connections between the two groups remained unchanged, suggesting that, in contrast to the earlier suggestions (Mellen et al., Neuron 37:821–826, 2003; Wittmeier et al., Proc Natl Acad Sci USA 105(46):18000–18005, 2008), quantal slowing could occur without suppressed or stochastic excitatory synaptic transmission. With a reduced excitability of preBötC network, the breakdown of synchronous bursting of preBötC neurons was predicted by simulation. We suggest that quantal slowing could result from a breakdown of synchronized bursting within the preBötC.     

The role of spiking and bursting pacemakers in the neuronal control of breathing

Breathing is controlled by a distributed network involving areas in the neocortex, cerebellum, pons, medulla, spinal cord, and various other subcortical regions. However, only one area seems to be essential and sufficient for generating the respiratory rhythm: the preBötzinger complex (preBötC). Lesioning this area abolishes breathing and following isolation in a brain slice the preBötC continues to generate different forms of respiratory activities. The use of slice preparations led to a thorough understanding of the cellular mechanisms that underlie the generation of inspiratory activity within this network. Two types of inward currents, the persistent sodium current (I_NaP) and the calcium-activated non-specific cation current (I_CAN), play important roles in respiratory rhythm generation. These currents give rise to autonomous pacemaker activity within respiratory neurons, leading to the generation of intrinsic spiking and bursting activity. These membrane properties amplify as well as activate synaptic mechanisms that are critical for the initiation and maintenance of inspiratory activity. In this review, we describe the dynamic interplay between synaptic and intrinsic membrane properties in the generation of the respiratory rhythm and we relate these mechanisms to rhythm generating networks involved in other behaviors.     

Effects of neuromuscular blocking agents on central respiratory chemosensitivity in newborn rats

Neuromuscular blocking agents suppress central respiratory activity through their inhibitory effects on preinspiratory neurons and the synaptic drive from preinspiratory neurons to inspiratory neurons. Central CO2-chemosensitive areas, which partly consist of CO2-excited neurons, in the rostral ventrolateral medulla are thought to provide tonic drive to the central respiratory network and involve cholinergic mechanisms, which led us to hypothesize that neuromuscular blocking agents can inhibit CO2-excited neurons and attenuate respiratory CO2 responsiveness. To test this hypothesis, we used isolated brainstem-spinal cord preparations from newborn rats. The increase of C4 burst frequency induced by a hypercapnic superfusate, i.e. respiratory CO2 responsiveness, was suppressed by the application of neuromuscular blocking agents, either d-tubocurarine (10, 100 microM) or vecuronium (100 microM). These agents (40 microM) also induced hyperpolarization and decreases in firing frequency of CO2-excited neurons in the rostral ventrolateral medulla. Our results demonstrate that neuromuscular blocking agents inhibit CO2-excited tonic firing neurons and attenuate respiratory CO2 responsiveness.     

Respiratory pattern generator model using Ca++-induced Ca++ release in neurons shows both pacemaker and reciprocal network properties

There are two contradictory explanations for central respiratory rhythmogenesis. One suggests that respiratory rhythm emerges from interaction between inspiratory and expiratory neural semicenters that inhibit each other and thereby provide reciprocal rhythmic activity (Brown 1914). The other uses bursting pacemaker activity of individual neurons to produce the rhythm (Feldman and Cleland 1982). Hybrid models have been developed to reconcile these two seemingly conflicting mechanisms (Smith et al. 2000; Rybak et al. 2001). Here we report computer simulations that demonstrate a unified mechanism of the two types of oscillator. In the model, we use the interaction of Ca⁺⁺-dependent K⁺ channels (Mifflin et al. 1985) with Ca⁺⁺-induced Ca⁺⁺ release from intracellular stores (McPherson and Campbell 1993), which was recently revealed in neurons (Hernandez-Cruz et al. 1997; Mitra and Slaughter 2002a,b; Scornik et al. 2001). Our computations demonstrate that uncoupled neurons with these intracellular mechanisms show conditional pacemaker properties (Butera et al. 1999) when exposed to steady excitatory inputs. Adding weak inhibitory synapses (based on increased K⁺ conductivity) between two model neural pools surprisingly synchronizes the activity of both neural pools. As inhibitory synaptic connections between the two pools increase from zero to higher values, the model produces first dissociated pacemaker activity of individual neurons, then periodic synchronous bursts of all neurons (inspiratory and expiratory), and finally reciprocal rhythmic activity of the neural pools.     

Two types of independent bursting mechanisms in inspiratory neurons: an integrative model

The network of coupled neurons in the pre-Bötzinger complex (pBC) of the medulla generates a bursting rhythm, which underlies the inspiratory phase of respiration. In some of these neurons, bursting persists even when synaptic coupling in the network is blocked and respiratory rhythmic discharge stops. Bursting in inspiratory neurons has been extensively studied, and two classes of bursting neurons have been identified, with bursting mechanism depends on either persistent sodium current or changes in intracellular Ca²⁺, respectively. Motivated by experimental evidence from these intrinsically bursting neurons, we present a two-compartment mathematical model of an isolated pBC neuron with two independent bursting mechanisms. Bursting in the somatic compartment is modeled via inactivation of a persistent sodium current, whereas bursting in the dendritic compartment relies on Ca²⁺ oscillations, which are determined by the neuromodulatory tone. The model explains a number of conflicting experimental results and is able to generate a robust bursting rhythm, over a large range of parameters, with a frequency adjusted by neuromodulators.