Rhythm Generation

In amphibians, there is some evidence that (1) anatomically separate brainstem respiratory oscillators are involved in rhythm generation, one for the buccal rhythm and another for the lung rhythm and (2) they become functionally coupled during metamorphosis. The present analysis, performed on neurograms recorded using brainstem preparations from Lithobates catesbeianus, aims to investigate the temporal organisation of lung and buccal burst types. Continuous Wavelet Transfom applied to the separated buccal and lung signals of a neurogram revealed that both buccal and lung frequency profiles exhibited the same low frequency peak around 1 Hz. This suggests that a common 'clock' organises both rhythms within an animal. A cross-correlation analysis applied to the buccal and lung burst signals revealed their similar intrinsic oscillation features, occurring at approximately 25 Hz. These observations suggest that a coupling between the lung and buccal oscillators emerges at metamorphosis. This coupling may be related to inter-connectivity between the two oscillators, and to a putative common drive.

How mammalian neural circuits generate rhythmic activity in motor behaviors, such as breathing, walking, and chewing, remains elusive. For breathing, rhythm generation is localized to a brainstem nucleus, the preBötzinger Complex (preBötC). Rhythmic preBötC population activity consists of strong inspiratory bursts, which drive motoneuronal activity, and weaker burstlets, which we hypothesize reflect an emergent rhythmogenic process. If burstlets underlie inspiratory rhythmogenesis, respiratory depressants, such as opioids, should reduce burstlet frequency. Indeed, in medullary slices from neonatal mice, the μ-opioid receptor (μOR) agonist DAMGO slowed burstlet generation. Genetic deletion of μORs in a glutamatergic preBötC subpopulation abolished opioid-mediated depression, and the neuropeptide Substance P, but not blockade of inhibitory synaptic transmission, reduced opioidergic effects. We conclude that inspiratory rhythmogenesis is an emergent process, modulated by opioids, that ...

verse slice and en bloc preparations. Inspiratory burst timing should be dominated by I neurons in transverse University of California, Los Angeles P.O. Box 951763 slices, whereas burst timing should depend on the interaction of I and pre-I neurons in en bloc preparations. Los Angeles, California 90095 Results Summary Continuous Slowing in Slices Consistent with earlier observations (Gray et al., 1999), Current consensus holds that a single medullary netin the transverse slice at 9 mM [K ϩ ] o , respiratory period work generates respiratory rhythm in mammals. Pregradually slowed following bath application of the Bö tzinger Complex inspiratory (I) neurons, isolated in-opiate agonist DAMGO (200-300 nM; n ϭ 5) (Figure transverse slices, and preinspiratory (pre-I) neurons, 1A) and returned to control values following bath applifound only in more intact en bloc preparations and cation of the-opiate antagonist naloxone (10 M; data in vivo, are each proposed as necessary for rhythm not shown). In normalized period histograms (Figure 1A, generation. Opioids slow I, but not pre-I, neuronal right), the graded nature of the opioid-induced slowing burst periods. In slices, opioids gradually lengthened is apparent as a long tail to the right of the peak associrespiratory periods, whereas in more intact preparaated with control periods. Matching results were obtions, periods jumped nondeterministically to integer tained in slices from P6 rats (n ϭ 3, data not shown). multiples of the control period (quantal slowing). These findings suggest that opioid-induced quantal slowing Quantal Slowing En Bloc and In Vivo results from transmission failure of rhythmic drive In the en bloc preparation (n ϭ 8), bath-applied DAMGO from pre-I neurons to preBö tC I networks, depressed (200-400 nM) initially caused a graded slowing of respibelow threshold for spontaneous rhythmic activity. ratory period over 20-40 cycles (Figure 1B, left, black Thus, both I (in the slice), and pre-I neurons are suffibar). This abruptly gave way to quantal, i.e., step-like, cient for respiratory rhythmogenesis. slowing of respiratory period. In normalized period histograms, this quantal slowing is apparent as separate Introduction peaks at integer multiples of the control period (Figure 1B, right, blue histograms; Figure 3B). When this proto-Two distinct neural networks in the ventrolateral medulla col was repeated with [K ϩ ] o elevated to concentrations are separately proposed to be the essential substrate matching the transverse slice (9 mM), periods were disfor respiratory rhythm generation (Onimaru et al., 1997; tributed continuously (Figure 1B, right, red histograms), Rekling and Feldman, 1998): (i) I neurons in the preand respiratory rhythm persisted at DAMGO concentra-Bö tzinger Complex (preBö tC) (Gray et al., 2001; Smith tions (Ͼ400 nM) that completely blocked respiratory et al., 1991) and (ii) pre-I neurons within and rostral to rhythm at standard en bloc [K ϩ ] o (3 mM). the preBö tC (Onimaru et al., 1995). In neonatal rodent If quantal slowing is an intrinsic property of the respitransverse medullary slices that isolate the preBö tC and ratory oscillator rather than an artifact of in vitro condirequire elevated [K ϩ ] o to generate a respiratory-related tions, a similar response should be inducible in vivo. We rhythm, neurons with pre-I firing pattern (Smith et al., tested this by systemic administration of the selective 1991) have not been described, either because they are-opiate receptor agonist fentanyl citrate (Chen et al., scarce, because their pattern of activity is transformed 1996) to otherwise intact, unanesthetized juvenile rats by removal of synaptic inputs, or because elevated [K ϩ ] o (P5-P7). Low-order quantal slowing with a high variabildisrupts their activity. The more inclusive en bloc brainity of respiratory period was observed (Figure 1C, n ϭ stem-spinal cord preparation, which generates an es-6), with more pronounced initial continuous slowing sentially identical rhythm at normal (3 mM) [K ϩ ] o (Smith compared to that seen in vitro. We speculated that this and Feldman, 1987; Suzue, 1984), has both preBö tC and response was more dispersed than that seen en bloc pre-I networks. The connectivity and synaptic interacdue to both sensory feedback from the lungs (Bruce, tions between these populations have been inferred 1996) and the effects of wakefulness. This was borne out (Ballanyi et al., 1999; Mellen and Feldman, 2001), but by the more clearly observable opioid-induced quantal the functional significance of their interactions has reslowing obtained in anesthetized, vagotomized juvenile mained unresolved. rats (P5-P7), (Figure 1D, n ϭ 4). Opiates slow I neuron, but have no effect on pre-I neuron, burst period (Takeda et al., 2001). We exploited this differential sensitivity to test the roles of these popu-Constraints on Quantal Slowing Mechanisms Both the pre-I and preBö tC networks are rhythmically active in the en bloc preparation; thus quantal slowing

How mammalian neural circuits generate rhythmic activity in motor behaviors, such as breathing, walking, and chewing, remains elusive. For breathing, rhythm generation is localized to a brainstem nucleus, the preBötzinger Complex (preBötC). Rhythmic preBötC population activity consists of strong inspiratory bursts, which drive motoneuronal activity, and weaker burstlets, which we hypothesize reflect an emergent rhythmogenic process. If burstlets underlie inspiratory rhythmogenesis, respiratory depressants, such as opioids, should reduce burstlet frequency. Indeed, in medullary slices from neonatal mice, the μ-opioid receptor (μOR) agonist DAMGO slowed burstlet generation. Genetic deletion of μORs in a glutamatergic preBötC subpopulation abolished opioid-mediated depression, and the neuropeptide Substance P, but not blockade of inhibitory synaptic transmission, reduced opioidergic effects. We conclude that inspiratory rhythmogenesis is an emergent process, modulated by opioids, that ...

This paper explores musical, psychological and philosophical ideas about how humans and machines interact in creative processes. It argues that creativity is a function of both generator and receiver, and that these roles can be amorphous in the creation and experience of electronic music. It offers an approach to structuring temporal spaces for rhythmic composition, which leads to the idea of machine rhythms, which are proposed as a promising area for creative expression.

Our goal was to determine whether time-dependent changes in respiratory motor output in vitro could be minimized by altering bath solution composition. Adult turtle brainstems were bathed in standard solution, nutrient-rich Dulbecco's Eagle media (100 or 25% concentration), or standard solution with phenylbiguanide (PBG, 5-HT 3 agonist which increases respiratory drive). Except for a 63% frequency increase in PBG solution, hypoglossal bursts were unaltered within 100 min of observation. Respiratory activity was abolished within 7 h in 100% Dulbecco's compared with a mean of 24 Á/31 h in other test solutions. At 12 h, burst frequency decreased faster in standard solution and 25% Dulbecco's ((/0.289/0.07 and (/0.139/0.05 bursts/h, respectively) compared with PBG solution ((/0.099/0.04 bursts/ h); amplitude declined at Â/2%/h in all solutions. The tendency for episodic discharge decreased gradually in standard solution, but was eliminated in 25% Dulbecco's and PBG solution. Certain bath solutions may minimize timedependent frequency reductions but may also cause breathing pattern changes.

SYNOPSIS. The episodic, or intermittent, breathing of frogs and many ectothermic vertebrates results in important fluctuations of arterial blood gases. This pattern of breathing differs from the rhythmic and continuous alternation of inspiration observed in most homeotherms, which maintain O 2 and CO 2 levels within narrow ranges. These differences in pattern of breathing indicate that the respiratory control systems of ectotherms and homeotherms differ substantially. The results of recent studies using in vitro brainstemspinal cord preparations of adult frogs and premetamorphic tadpoles {Rana catesbeiana and Rana pipiens) demonstrate, however, that the mechanisms for rhythm generation and pattern formation described previously for mammals are also key features of the respiratory control system of frogs. These findings therefore support the hypothesis that the respiratory control system is highly conserved amongst air breathing vertebrates, whether they breathe continuously or episodically.

  Este artículo analiza ciertos aspectos evolutivos en el intercambio gaseoso, el desa- rrollo pulmonar, la bomba respiratoria, el estado ácido-base y el control de la ventila- ción en relación con un evento trascendente: el pasaje de la vida acuática a la terres- tre. Su estudio puede permitir comprender ciertos aspectos con los que lidiamos en la práctica clínica: ¿Por qué las personas con debilidad muscular respiratoria extremarespiran como ranas (respiración frog)?, ¿Por qué los recién nacidos con difcultad respiratoria tienen aleteo nasal y quejido espiratorio?, ¿cómo es posible que los mús-culos abdominales, típicamente espiratorios, asistan a la inspiración en casos de la parálisis diafragmática?, ¿por qué en la insufciencia respiratoria el patrón respiratoriotiene menos variabilidad y se torna más rígido? y, por último, ¿es posible imaginar un pH neutro que no tenga el valor de 7,0, para qué sirve este conocimiento y como se deben interpretar los gases en hipotermia?La trans...

In central respiratory circuitry, synaptic excitation is responsible for synchronizing neuronal activity in the different respiratory rhythm phases, whereas chloride-mediated inhibition is important for shaping the respiratory pattern itself. The potassium chloride cotransporter KCC2, which serves to maintain low intraneuronal Clconcentration and thus render chloride-mediated synaptic signaling inhibitory, exists in two isoforms, KCC2a and KCC2b. KCC2 is essential for functional breathing motor control at birth, but the specific contribution of the KCC2a isoform remains unknown. Here, to address this issue, we investigated the respiratory phenotype of mice deficient for KCC2a. In vivo plethysmographic recordings revealed that KCC2a-deficient pups at P0 transiently express an abnormally low breathing rate and a high occurrence of apneas. Immunostainings confirmed that KCC2a is normally expressed in the brainstem neuronal groups involved in breathing (pre-Bötzinger complex, parafacial respiratory group, hypoglossus nucleus) and is absent in these regions in the KCC2a-/mutant. However, in variously reduced in vitro medullary preparations, spontaneous rhythmic respiratory activity is similar to that expressed in wild-type preparations, as is hypoglossal motor output, and no respiratory pauses are detected, suggesting that the rhythm-generating networks are not intrinsically affected in mutants at P0. In contrast, inhibitory neuromodulatory influences exerted by the pons on respiratory rhythmogenesis are stronger in the mutant, thereby explaining the breathing anomalies observed in vivo. Thus, our results indicate that the KCC2a isoform is important for establishing proper breathing behavior at the time of birth, but by acting at sites that are extrinsic to the central respiratory networks themselves.

In amphibians, there is some evidence that (1) anatomically separate brainstem respiratory oscillators are involved in rhythm generation, one for the buccal rhythm and another for the lung rhythm and (2) they become functionally coupled during metamorphosis. The present analysis, performed on neurograms recorded using brainstem preparations from Lithobates catesbeianus, aims to investigate the temporal organisation of lung and buccal burst types. Continuous Wavelet Transfom applied to the separated buccal and lung signals of a neurogram revealed that both buccal and lung frequency profiles exhibited the same low frequency peak around 1 Hz. This suggests that a common 'clock' organises both rhythms within an animal. A cross-correlation analysis applied to the buccal and lung burst signals revealed their similar intrinsic oscillation features, occurring at approximately 25 Hz. These observations suggest that a coupling between the lung and buccal oscillators emerges at metamorphosis. This coupling may be related to inter-connectivity between the two oscillators, and to a putative common drive.

; 10.1152/japplphysiol.00558. 2002.-Central CO2 chemoreception and the role of carbonic anhydrase were assessed in brain stems from Rana catesbeiana tadpoles and frogs. Buccal and lung rhythms were recorded from cranial nerve VII and spinal nerve II during normocapnia and hypercapnia before and after treatment with 25 M acetazolamide. The lung response to acetazolamide mimicked the hypercapnic response in early-stage and midstage metamorphic tadpoles and frogs. In late-stage tadpoles, acetazolamide actually inhibited hypercapnic responses. Acetazolamide and hypercapnia decreased the buccal frequency but had no effect on the buccal duty cycle. Carbonic anhydrase activity was present in the brain stem in every developmental stage. Thus more frequent lung ventilation and concomitantly less frequent buccal ventilation comprised the hypercapnic response, but the response to acetazolamide was not consistent during metamorphosis. Therefore, acetazolamide is not a useful tool for central CO 2 chemoreceptor studies in this species. The reversal of the effect of acetazolamide in late-stage metamorphosis may reflect reorganization of central chemosensory processes during the final transition from aquatic to aerial respiration. isolated brain stem preparation; carbonic anhydrase; acetazolamide; hypercapnia

The occurrence of hiccoughs (hiccups) is very widespread and yet their neuronal origin and physiological significance are still unresolved. Several hypotheses have been proposed. Here we consider a phylogenetic perspective, starting from the concept that the ventilatory central pattern generator of lower vertebrates provides the base upon which central pattern generators of higher vertebrates develop. Hiccoughs are characterized by glottal closure during inspiration and by early development in relation to lung ventilation. They are inhibited when the concentration of inhaled CO 2 is increased and they can be abolished by the drug baclofen (an agonist of the GABA B receptor). These properties are shared by ventilatory motor patterns of lower vertebrates, leading to the hypothesis that hiccough is the expression of archaic motor patterns and particularly the motor pattern of gill ventilation in bimodal breathers such as most frogs. A circuit that can generate hiccoughs may persist in mammals because it has permitted the development of pattern generators for other useful functions of the pharynx and chest wall muscles, such as suckling or eupneic breathing.

SUMMARY This study was designed to determine whether lung inflation stimulates or inhibits breathing in frogs by examining the effect of tonic lung inflation on the ‘fictive’ breathing pattern of decerebrate, unidirectionally ventilated bullfrogs. Neural discharge was monitored in the trigeminal nerve as an indication of the frequency and force of contraction of the buccal pump, and in the laryngeal branch of the vagus nerve as an indication of glottal opening, and hence fictive lung ventilation. Based on the temporal coordination of discharge in the trigeminal and vagus nerves during naturally occurring breaths it was possible to characterize the fictive breaths as inflation, deflation or balanced breaths. Increasing lung inflation increased absolute breathing frequency by reducing the duration of apnea between breaths and promoting a change in breathing pattern from no breathing to single breaths, breathing episodes and, finally, to continuous breathing. Associated with this was a...

The nucleus isthmi (NI) is a mesencephalic structure of the amphibian brain. It has been reported that NI plays an important role in integration of CO 2 chemoreceptor information and glutamate is probably involved in this function. However, very little is known about the mechanisms involved. Recently, it has been shown that nitric oxide synthase (NOS) is expressed in the brain of the frog. Thus the gas nitric oxide (NO) may be involved in different functions in the brain of amphibians and may act as a neurotransmitter or neuromodulator. We tested the hypothesis that NO plays a role in CO 2-drive to breathing, specifically in the NI comparing pulmonary ventilation, breathing frequency and tidal volume, after microinjecting 100 nmol/0.5 µl of L-NAME (a nonselective NO synthase inhibitor) into the NI of toads (Bufo paracnemis) exposed to normocapnia and hypercapnia. Control animals received microinjections of vehicle of the same volume. Under normocapnia no significant changes were observed between control and L-NAME-treated toads. Hypercapnia caused a significant (P<0.01) increase in ventilation only after intracerebral microinjection of L-NAME. Exposure to hypercapnia caused a significant increase in breathing frequency both in control and L-NAME-treated toads (P<0.01 for the control group and P<0.001 for the L-NAME group). The tidal volume of the L-NAME group tended to be higher than in the control group under hypercapnia, but the increase was not statistically significant. The data indicate that NO in the NI has an inhibitory effect only when the respiratory drive is high (hypercapnia), probably acting on tidal volume. The observations reported in the present investigation, together with other studies on the presence of NOS in amphibians, indicate a considerable degree of phylogenetic conservation of the NO pathway amongst vertebrates.

The isolated neonatal rat medulla generates respiratory-related rhythms recorded from cervical spinal cord ventral roots. When lungs and their vagal innervation are retained, respiratory activity is modulated by lung mechanoreceptor feedback: transient lung inflation triggered off inspiratory onset (phasic inflation) shortens inspiration and increases respiratory frequency. In this study, the activity of six respiratory neuron classes before and during phasic inflation was studied. Type 1 and 2 inspiratory neurons, identified in the transverse slice, were distinguished by the presence of a transient outward current or a hyperpolarization-activated inward current, respectively. Cell types only identified in the en bloc medulla included type II and III inspiratory neurons, distinguished by delayed onset and peri-inspiratory inhibition, respectively, and preinspiratory neurons, active before and after but silent during inspiration. Biphasic neurons, identified in the preparation used here, fired briskly during lung inflation but are otherwise quiescent. During phasic inflation, biphasic neurons showed a decrementing expiratory pattern of activity, matched by augmented postinspiratory hyperpolarization in type 1 neurons only, suggesting that biphasic neurons inhibit type 1 neurons, removing drive to other inspiratory neurons and terminating the inspiratory burst. This mechanism could account for a phasic inflation-induced increase in respiratory frequency via resetting effects. Alternatively, the phasic inflation-induced respiratory frequency increase may be attributable to slow facilitation. Slow modulation consistent with facilitation was apparent in the earlier onset of pre-I firing before inspiration and loss of postinspiratory firing and in the earlier onset of depolarization in type 2 neurons. On the basis of relative onset times and responses to phasic inflation, connectivity between these cell types is proposed.

The chuckwalla, Sauromalus obesus, avoids predation by wedging itself in a rock crevice and inflating its lungs beyond their normal inspiratory volume. Buccal and pulmonary pressures were recorded in S. obesus during defensive inflation and wedging. Maximum pulmonary pressure generated during defensive wedging was significantly higher and was achieved faster than that of nonwedging inflation. During inflation and wedging, S. obesus forces air into the lungs by pulsatile contraction of the buccal cavity. Buccal pulse pumping is an ancestral ventilation behavior of vertebrates that S. obesus uses for defensive inflation.

There is an urgent need to develop novel compounds that prevent the deleterious effects of opioids such as fentanyl on minute ventilation while, if possible, preserving the analgesic actions of the opioids. We report that L-glutathione ethyl ester (GSHee) may be such a novel compound. In this study, we measured tail flick latency (TFL), arterial blood gas (ABG) chemistry, Alveolar-arterial gradient, and ventilatory parameters by whole body plethysmography to determine the responses elicited by bolus injections of fentanyl (75 μg/kg, IV) in male adult Sprague–Dawley rats that had received a bolus injection of GSHee (100 μmol/kg, IV) 15 min previously. GSHee given alone had minimal effects on TFL, ABG chemistry and A-a gradient whereas it elicited changes in some ventilatory parameters such as an increase in breathing frequency. In vehicle-treated rats, fentanyl elicited (1) an increase in TFL, (2) decreases in pH, pO2 and sO2 and increases in pCO2 (all indicative of ventilatory depre...

The emergence of air breathing during amphibian metamorphosis requires significant changes to the brainstem circuits that generate and regulate breathing. However, the mechanisms controlling this developmental process are unknown. Because corticosterone plays an important role in the neuroendocrine regulation of amphibian metamorphosis, we tested the hypothesis that corticosterone augments fictive air breathing frequency in Xenopus laevis. To do so, we compared the fictive air breathing frequency produced by in vitro brainstem preparations from pre-metamorphic tadpoles and adult frogs before and after 1 h application of corticosterone (100 nmol l(-1)). Fictive breathing measurements related to gill and lung ventilation were recorded extracellularly from cranial nerve rootlets V and X. Corticosterone application had no immediate effect on respiratory-related motor output produced by brainstems from either developmental stage. One hour after corticosterone wash out, fictive lung venti...

Chronic ethanol exposure early in development is deleterious to neural development and may impair responses to ventilatory stimuli (ventilatory drive) that maintain homeostasis. Central hypercapnic ventilatory drive (CHVD) increases ventilation to ensure pH homeostasis and accommodate the metabolic production of CO 2. We tested the hypothesis that chronic ethanol exposure impairs CHVD in bullfrog tadpoles. Early and late metamorphic tadpoles were exposed in vivo to 0.12-0.06 g/dL ethanol for either 3or 10-wk durations. Brainstems from these animals were isolated and the neural correlates of ventilation were recorded in vitro during superfusion with normocapnic (1.5% CO 2 :98.5% O 2) and hypercapnic (5.0% CO 2 :95.0% O 2) artificial cerebral spinal fluid. Normocapnic neuroventilation was unaffected by chronic ethanol exposure. The typical response to hypercapnia, an increase in lung burst frequency, was lost following 10 but not 3 wk of ethanol exposure in both early and late metamorphic tadpoles. The neuroventilatory effects of chronic ethanol exposure were distinguishable from those of acute central ethanol (0.08 g/dL) exposure, which attenuated early metamorphic tadpole normocapnic neuroventilation, but had no effect on tadpole CHVD. Thus, 10 wk of ethanol exposure both early and late in metamorphosis impairs CHVD in bullfrog tadpoles.

Opioids are perhaps the most effective analgesics in medicine. However, between 1999 and 2018, over 400,000 people in the United States died from opioid overdose. Excessive opioids make breathing lethally slow and shallow, a side-effect called opioid-induced respiratory depression. This doubled-edged sword has sparked the desire to develop novel therapeutics that provide opioid-like analgesia without depressing breathing. One such approach has been the design of so-called ‘biased agonists’ that signal through some, but not all pathways downstream of the µ-opioid receptor (MOR), the target of morphine and other opioid analgesics. This rationale stems from a study suggesting that MOR-induced ß-arrestin 2 dependent signaling is responsible for opioid respiratory depression, whereas adenylyl cyclase inhibition produces analgesia. To verify this important result that motivated the ‘biased agonist’ approach, we re-examined breathing in ß-arrestin 2-deficient mice and instead find no conne...

Most vertebrate rhythmic motor networks require synaptic inhibition to regulate timing and pattern. For example, network models of respiratory rhythm generation generally include groups of neurons that are reciprocally coupled via inhibitory synaptic connections, thereby establishing exclusive bursts of activity associated with the phases of respiration (reviewed in Richter et al.

Spatiotemporal activity patterns during respiratory rhythmogenesis in the rat ventrolateral medulla. . One of the most important brain rhythms is that which generates involuntary breathing movements. The lower brain stem contains neural circuitry for respiratory rhythm generation in mammals. To date, microsectioning and selective lesioning studies have revealed anatomical regions necessary for respiratory rhythmogenesis. Although respiratory neurons distributed within these regions can be identified by their firing patterns in different phases of the respiratory cycle, conventional electrophysiology techniques have limited the study of spatial organization within this network. Optical imaging techniques offer the potential for monitoring the spatiotemporal activity of large groups of neurons simultaneously. Using high-speed voltage-sensitive dye imaging and spatial correlation analysis in an arterially perfused in situ preparation of the juvenile rat, we determined the spatial distribution of respiratory neuronal activity in a region of the ventrolateral respiratory group containing the pre-Bötzinger complex (pBC) during spontaneous eupneic breathing. While distinctly pre-and postinspiratory-related responses were spatially localizable on length scales less than 100 m, we found the studied area on whole exhibited a spatial mixture of phasespanning and postinspiratory-related activity. Additionally, optical recordings revealed significant widespread hyperpolarization, suggesting inhibition in the same region during expiration. This finding is consistent with the hypothesis that inhibitory neurons play a crucial role in the inspiration-expiration phase transition in the pBC. To our knowledge this is the first optical imaging of a near fully intact in situ preparation that exhibits both eupneic respiratory activity and functional reflexes.

Inspiratory and expiratory rhythms in mammals are thought to be generated by pacemaker-like neurons in 2 discrete brainstem regions: pre-Bö tzinger complex (preBö tC) and parafacial respiratory group (pFRG). How these putative pacemakers or pacemaker networks may interact to set the overall respiratory rhythm in synchrony remains unclear. Here, we show that a pacemakers 2-way ''handshake'' process comprising pFRG excitation of the preBö tC, followed by reverse inhibition and postinhibitory rebound (PIR) excitation of the pFRG and postinspiratory feedback inhibition of the preBö tC, can provide a phase-locked mechanism that sequentially resets and, hence, synchronizes the inspiratory and expiratory rhythms in neonates. The order of this handshake sequence and its progression vary depending on the relative excitabilities of the preBö tC vs. the pFRG and resultant modulations of the PIR in various excited and depressed states, leading to complex inspiratory and expiratory phase-resetting behaviors in neonates and adults. This parsimonious model of pacemakers synchronization and mutual entrainment replicates key experimental data in vitro and in vivo that delineate the developmental changes in respiratory rhythm from neonates to maturity, elucidating their underlying mechanisms and suggesting hypotheses for further experimental testing. Such a pacemakers handshake process with conjugate excitation-inhibition and PIR provides a reinforcing and evolutionarily advantageous fail-safe mechanism for respiratory rhythmogenesis in mammals. entrainment ͉ parafacial respiratory group ͉ postinhibitory rebound ͉ preBö tzinger complex ͉ rhythm

Intermittent lung ventilation is a respiratory pattern wherein breaths occur in clusters. Intermittent lung ventilation is common in amphibians and can occur in mammals. Isolated brainstems from postmetamorphic tadpoles exhibiting episodic lung ventilatory bursts were superfused with baclofen, a potent g-aminobutyric acid (GABA) B receptor agonist. At moderate concentrations (0.125 to 0.5 mM), the number of lung bursts per episode decreased but their overall frequency was unchanged. At 0.5 mM, only 1:22^0:24 lung bursts occurred per episode, indicating virtually no clustering. Only at higher concentrations was overall breathing frequency decreased. Therefore, at moderate concentrations of baclofen continuous ventilation replaced episodic ventilation, suggesting that a GABA B receptor-dependent pathway may regulate the clustering of lung breaths. q

The pre-Bötzinger Complex (preBötC) inspiratory centre remains active in isolated brainstem-spinal cords and brainstem slices. The extent to which findings in these models depend on their dimensions or superfusate [K + ] and [Ca 2+ ] (both of which determine neuronal excitability) is not clear. We report here that inspiratory-related rhythms in newborn rat slices and brainstem-spinal cords with defined boundaries were basically similar in physiological Ca 2+ (1.2 mM) and K + (3 mM). Hypoglossal nerve rhythm was 1 : 1-coupled to preBötC activity in slices and to cervical nerve bursts in en bloc preparations lacking the facial motonucleus (VII). Hypoglossal rhythm was depressed in brainstems containing (portions of) VII, while pre/postinspiratory lumbar nerve bursting was present only in preparations with > 79% VII. preBötC-related slice rhythms were inhibited in 1.5 mM Ca 2+ solution, whereas their longevity and burst rate were substantially augmented in 1 mM Ca 2+ . Ca 2+ depression of slice rhythms was antagonized by raising superfusate K + to 8-10 mM. This strong extracellular Ca 2+ /K + antagonism of inspiratory (motor) rhythms was also revealed in brainstem-spinal cords without VII, while the inhibition was progressively attenuated with increasing amount of rostral tissue. We hypothesize that depression of hypoglossal rhythm and decreased Ca 2+ sensitivity of preBötC rhythm are probably not related to an increased content of rostral respiratory structures, but rather to larger brainstem dimensions resulting in interstitial gradients for neuromodulator(s) and K + , respectively. We discuss whether block of pre/postinspiratory activity in preparations with < 79% VII is due to impairment of the pathway from preinspiratory interneurons to abdominal muscles

1. The isolated brainstem of larval Rana catesbeiana maintained in vitro generates neural bursts that correspond to the lung and gill ventilatory activity generated in the intact specimen. To investigate the role of chloride channel-dependent inhibitory mechanisms mediated by GABA(A) and/or glycine receptors on fictive lung and gill ventilation, we superfused the isolated brainstems with agonists, antagonists (bicuculline and/or strychnine) or a chloride-free solution while recording multi-unit activity from the facial motor nucleus. 2. Superfusion with the agonists (GABA or glycine) produced differential effects on frequency, amplitude and duration of the neural bursts related to lung and gill ventilation. At a GABA or glycine concentration of 1.0 mM, fictive gill bursts were abolished while fictive lung bursts persisted, albeit with reduced amplitude and frequency. 3. At the lowest concentrations used (1.0-2.5 microM), the GABA(A) receptor antagonist bicuculline produced an increa...

The following letters are in response to the Point:Counterpoint series "The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBotC) is the primary site of respiratory rhythm generation in the mammal" that appears in this issue. 2. Monnier A, Alheid GF, and McCrimmon DR. Defining ventral medullary respiratory compartments with a glutamate receptor agonist in the rat. J Physiol 548: 859 -874, 2003. 3. Nattie EE and Li A. Related Articles, Substance P-saporin lesion of neurons with NK1 receptors in one chemoreceptor site in rats decreases ventilation and chemosensitivity. J Physiol 544: 603-616, 2002. 4. Onimaru H and Homma I; Feldman JL and Janczewski WA. Point: Counterpoint: The parafacial respiratory group (pFRG)/ pre-Botzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal. Fluorescent tagging of rhythmically active respiratory neurons within the pre-Botzinger complex of rat medullary slice preparations. J Neurosci 25: 2591-2596, 2005. 2. Gray PA, Janczewski WA, Mellen N, McCrimmon DR, and Feldman JL. Normal breathing requires preBotzinger complex neurokinin-1 receptor-expressing neurons. Nat Neurosci 4: 927-930, 2001. 3. Guyenet PG, Mulkey DK, Stornetta RL, and Bayliss DA. Regulation of ventral surface chemoreceptors by the central respiratory pattern generator. J Neurosci 25: 8938 -8947, 2005. 4. Janczewski WA and Feldman JL. Distinct rhythm generators for inspiration and expiration in the juvenile rat. J Physiol 570: 407-420, 2006. 5. Onimaru H and Homma I; Feldman JL and Janczewski WA. Point: Counterpoint: The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBotC) is the primary site of respiratory rhythm generation in the mammal.

The occurrence of hiccoughs (hiccups) is very widespread and yet their neuronal origin and physiological significance are still unresolved. Several hypotheses have been proposed. Here we consider a phylogenetic perspective, starting from the concept that the ventilatory central pattern generator of lower vertebrates provides the base upon which central pattern generators of higher vertebrates develop. Hiccoughs are characterized by glottal closure during inspiration and by early development in relation to lung ventilation. They are inhibited when the concentration of inhaled CO 2 is increased and they can be abolished by the drug baclofen (an agonist of the GABA B receptor). These properties are shared by ventilatory motor patterns of lower vertebrates, leading to the hypothesis that hiccough is the expression of archaic motor patterns and particularly the motor pattern of gill ventilation in bimodal breathers such as most frogs. A circuit that can generate hiccoughs may persist in mammals because it has permitted the development of pattern generators for other useful functions of the pharynx and chest wall muscles, such as suckling or eupneic breathing.

The frog, with two distinct ventilatory acts, provides a useful model to investigate the prospective interaction of two oscillators in generating the respiratory rhythm. Building on evidence supporting the existence of separate oscillators generating buccal and lung ventilation, we have attempted to uncouple the two rhythms in the isolated brain stem preparation. Opioid preferentially inhibits the lung rhythm, suggesting an uncoupling of the lung from the buccal oscillator. Reduction of the superfusate chloride concentration alters both the buccal and the lung rhythms. Joint application of opioid and reducedchloride superfusate leads to an increase in the variability of the buccal burst-to-lung burst intervals. This increase in variability suggests that chloride-mediated mechanisms are involved in coupling the buccal oscillator to the lung oscillator. Given the results from these interventions, we propose a simple schematic model of the frog respiratory rhythm generator, outlining the coupling of the lung and buccal oscillators.

Evolution of central respiratory chemosensitivity has been linked traditionally to the need for carbon dioxide regulation that accompanied the evolution of air breathing in terresterial animals. We examined the validity of this linkage by investigating the possibility of central chemoreception in air breathing fish that diverged from the amphibian lineage long before the appearance of terrestriality. We showed that the isolated brainstem preparation of the long nose gar (Lepisosteus osseus) produces a putative motor pattern for lung ventilation, which is responsive to CO 2 . These findings, together with more inferential evidence, suggest an association between air breathing and central chemosensitivity in aquatic animals that spans the major branches in vertebrate phylogeny. Furthermore, developmental observations in tadpoles suggest that the neural substrates for central chemoreception exist in proximity to that for rhythm generation. We postulate that a primitive ancestral CPG, sensitive to CO 2 is conserved and is evidenced in the intrinsic coupling of respiratory CPG and central chemoreception in modern tetrapods.

Though the mechanics of breathing differ fundamentally between amniotes and "lower" vertebrates, homologous rhythm generators may drive air breathing in all lunged vertebrates. In both frogs and rats, two coupled oscillators, one active during the inspiratory (I) phase and the other active during the preinspiratory (PreI) phase, have been hypothesized to generate the respiratory rhythm. We used opioids to uncouple these oscillators. In the intact rat, complete arrest of the external rhythm by opioid-induced suppression of the putative I oscillator, that is, pre-Bötzinger complex (PBC) oscillator, did not arrest the putative PreI oscillator. In the unanesthetized frog, the comparable PreI oscillator, that is, the putative buccal/ gill oscillator, was refractory to opioids even though the comparable I oscillator, the putative lung oscillator, was arrested. Studies in en bloc brainstem preparations derived from both juvenile frogs and metamorphic tadpoles confirmed these results and suggested that opioids may play a role in the clustering of lung bursts into episodes. As the frog and rat respiratory circuitry produce functionally equivalent motor outputs during lung inflation, these data argue for a close homology between the frog and rat oscillators. We suggest that the respiratory rhythm of all lunged vertebrates is generated by paired coupled oscillators. These may have originated from the gill and lung oscillators of the earliest air breathers.