Genetic factors determining the functional organization of neural circuits controlling rhythmic movements the murine embryonic parafacial rhythm generator.

In mammals, fetal movements governed by central pattern generators are essential for the development of adaptive behaviors such as breathing, walking, and chewing, which are vital after birth. Combining targeted mutations and genetic fate mapping can help to define the molecular determinants that control the development of these central pattern generators. In this chapter, recent results are presented on the embryonic parafacial (e-pF) rhythm generator, one of the two oscillators involved in controlling the breathing behavior and chemosensitive responsiveness.

Developmental basis of the rostro-caudal organization of the brainstem respiratory rhythm generator.

The Hox genetic network plays a key role in the anteroposterior patterning of the rhombencephalon at pre- and early-segmental stages of development of the neural tube. In the mouse, it controls development of the entire brainstem respiratory neuronal network, including the pons, the parafacial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC). Inactivation of Krox20/Egr2 eliminates the pFRG activity, thereby causing life-threatening neonatal apnoeas alternating with respiration at low frequency. Another respiratory abnormality, the complete absence of breathing, is induced when neuronal synchronization fails to develop in the preBötC. The present paper summarizes data on a third type of respiratory deficits induced by altering Hox function at pontine levels. Inactivation of Hoxa2, the most rostrally expressed Hox gene in the hindbrain, disturbs embryonic development of the pons and alters neonatal inspiratory shaping without affecting respiratory frequency and apnoeas. The same result is obtained by the Phox2a(+/-) mutation modifying the number of petrosal chemoafferent neurons, by eliminating acetylcholinesterase and by altering Hox-dependent development of the pons with retinoic acid administration at embryonic day 7.5. In addition, embryos treated with retinoic acid provide a mouse model for hyperpnoeic episodic breathing, widely reported in pre-term neonates, young girls with Rett's syndrome, patients with Joubert syndrome and adults with Cheyne-Stokes respiration. We conclude that specific respiratory deficits in vivo are assignable to anteroposterior segments of the brainstem, suggesting that the adult respiratory neuronal network is functionally organized according to the rhombomeric, Hox-dependent segmentation of the brainstem in embryos.

Abnormal inspiratory depth in Phox2a haploinsufficient mice.

Mutations of genes encoding Phox2a or Phox2b transcription factors induce modifications of different brainstem neuronal networks. Such modifications are associated with defects in breathing behavior at birth. In particular, an abnormal breathing frequency is observed in Phox2a-/- mutant mice, resulting from abnormal development of the locus coeruleus (LC) nucleus. However, the role of Phox2a proteins in the establishment of respiratory neuronal pathways is unknown, largely because mutants die shortly after birth. In the present study, we examined the effects of a haploinsufficiency of the Phox2a gene. Phox2a heterozygotes survive and exhibit a significantly larger inspiratory volume both during normoxic breathing and in response to hypoxia and a delayed maturation of inspiratory duration compared to wild-type animals. This phenotype accompanied by an unaltered frequency is evident at birth and persists until at least postnatal day 10. Morphological analyses of Phox2a+/- animals revealed no anomaly in the LC region, but highlighted an increase in the number of cells expressing tyrosine hydroxylase enzyme, a marker of chemoafferent neurons, in the petrosal sensory ganglion. These data indicate that Phox2a plays a critical role in the ontogeny of the reflex control of inspiration.

Ontogeny of central rhythm generation in chicks and rodents.

Recent studies help in understanding how the basic organization of brainstem neuronal circuits along the anterior-posterior (AP) axis is set by the Hox-dependent segmentation of the neural tube in vertebrate embryos. Neonatal respiratory abnormalities in Krox20(-/-), Hoxa1(-/-) and kreisler mutant mice indicate the vital role of a para-facial (Krox20-dependent, rhombomere 4-derived) respiratory group, that is distinct from the more caudal rhythm generator called Pre-Bötzinger complex. Embryological studies in the chick suggest homology and conservation of this Krox20-dependent induction of parafacial rhythms in birds and mammals. Calcium imaging in embryo indicate that rhythm generators may derive from different cell lineages within rhombomeres. In mice, the Pre-Bötzinger complex is found to be distinct from oscillators producing the earliest neuronal activity, a primordial low-frequency rhythm. In contrast, in chicks, maturation of the parafacial generator is tightly linked to the evolution of this primordial rhythm. It seems therefore that ontogeny of brainstem rhythm generation involves conserved processes specifying distinct AP domains in the neural tube, followed by diverse, lineage-specific regulations allowing the emergence of organized rhythm generators at a given AP level.

The pre-Bötzinger oscillator in the mouse embryo.

Studies of the sites and mechanisms involved in mammalian respiratory rhythm generation point to two clusters of rhythmic neurons forming a coupled oscillator network within the brainstem. The location of these oscillators, the pre-Bötzinger complex (preBötC) at vagal level, and the para-facial respiratory group at facial level, probably result from regional patterning schemes specifying neural types in the hindbrain during embryogenesis. Here, we report evidence that the preBötC oscillator (i) is first active at embryonic stages, (ii) originates in the post-otic hindbrain neural tube and (iii) requires the glutamate vesicular transporter 2 for rhythm generation.

Neural tube patterning by Krox20 and emergence of a respiratory control.

Recent data begin to bridge the gap between developmental events controlling hindbrain neural tube regional patterning and the emergence of breathing behaviour in the fetus and its vital adaptive function after birth. In vertebrates, Hox paralogs and Hox-regulating genes orchestrate, in a conserved manner, the transient formation of developmental compartments in the hindbrain, the rhombomeres, in which rhythmic neuronal networks of the brainstem develop. Genetic inactivation of some of these genes in mice leads to pathological breathing at birth pointing to the vital importance of rhombomere 3 and 4 derived territories for maintenance of the breathing frequency. In chick embryo at E7, we investigated neuronal activities generated in neural tube islands deriving from combinations of rhombomeres isolated at embryonic day E1.5. Using a gain of function approach, we reveal a role of the transcription factor Krox20, specifying rhombomeres 3 and 5, in inducing a rhythm generator at the parafacial level of the hindbrain. The developmental genes selecting and regionally coordinating the fate of CNS progenitors may hold further clues to conserved aspects of neuronal network formation and function. However, the most immediate concern is to take advantage of early generated rhythmic activities in the hindbrain to pursue their downstream cellular and molecular targets, for it seems likely that it will be here that rhythmogenic properties will eventually take on a vital role at birth.

Identification of two types of inspiratory pacemaker neurons in the isolated respiratory neural network of mice.

In the respiratory network of mice, we characterized with the whole cell patch-clamp technique pacemaker properties in neurons discharging in phase with inspiration. The respiratory network was isolated in a transverse brain stem slice containing the pre-Bötzinger complex (PBC), the presumed site for respiratory rhythm generation. After blockade of respiratory network activity with 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), 18 of 52 inspiratory neurons exhibited endogenous pacemaker activity, which was voltage dependent, could be reset by brief current injections and could be entrained by repetitive stimuli. In the pacemaker group (n = 18), eight neurons generated brief bursts (0.43 +/- 0.03 s) at a relatively high frequency (1.05 +/- 0.12 Hz) in CNQX. These bursts resembled the bursts that these neurons generated in the intact network during the interval between two inspiratory bursts. Cadmium (200 microM) altered but did not eliminate this bursting activity, while 0.5 microM tetrodotoxin suppressed bursting activity. Another set of pacemaker neurons (10 of 18) generated in CNQX longer bursts (1.57 +/- 0.07 s) at a lower frequency (0.35 +/- 0.01 Hz). These bursts resembled the inspiratory bursts generated in the intact network in phase with the population activity. This bursting activity was blocked by 50-100 microM cadmium or 0.5 microM tetrodotoxin. We conclude that the respiratory neural network contains pacemaker neurons with two types of bursting properties. The two types of pacemaker activities might have different functions within the respiratory network.

Role of inspiratory pacemaker neurons in mediating the hypoxic response of the respiratory network in vitro.

In severe hypoxia the breathing frequency is modulated in a biphasic manner: an initial increase (augmentation) is followed by a depression and cessation of breathing (apnea). Using a mouse slice preparation that contains the functional respiratory network, we aimed at identifying the neurons responsible for this frequency modulation. Whole-cell patch recordings revealed that expiratory neurons become tonically active during anoxia, indicating that these neurons cannot be responsible for the respiratory frequency modulation. Inspiratory neurons tended to depolarize (by 6.9 mV; n = 9), and the frequency of rhythmic activity was significantly increased during anoxia (from 0.16 to 0.4 Hz; n = 9). After the blockade of network activity with 6-cyano-7-nitroquinoxaline-2, 3-dione, most inspiratory neurons became tonically active (72%; n = 25, non-pacemaker). In anoxia, the membrane potential of these non-pacemaker neurons did not change (-0.26 mV; n = 6), and their tonic activity ceased. Only a subpopulation of inspiratory neurons remained rhythmically active in the absence of network activity (pacemaker neurons, 28%, 7 of 25 inspiratory neurons). In anoxia two subgroups of pacemaker neurons were differentiated; one group showed a transient increase in the bursting activity, followed by a decrease and cessation of rhythmic activity. These neurons tended to depolarize (by 10.3 mV) during anoxia. The second group remained rhythmic during the entire anoxic exposure and exhibited no depolarization. The time course of the frequency modulation in all pacemaker neurons resembled that of the intact network. We conclude that pacemaker neurons are primarily responsible for the frequency modulation in anoxia and that in the respiratory network there is a strict separation between rhythm- and pattern-generating mechanisms.

Transition to endogenous bursting after long-term decentralization requires De novo transcription in a critical time window.

Rhythmic motor pattern generation by the pyloric network in the lobster stomatogastric ganglion (STG) requires neuromodulatory inputs from adjacent ganglia. However, although suppression of these inputs by cutting the stomatogastric nerve (stn) causes the pyloric network to fall silent, network output similar to that expressed when the stn is intact returns after 3-4 days in organ culture. Intracellular recordings from identified pyloric dilator (PD) neurons indicate that the fundamental change underlying rhythm recovery resides with the intrinsic excitability of pyloric neurons themselves, since the prolonged absence of extrinsic modulatory inputs allows the expression of an endogenous oscillatory capability that is maintained in a strictly conditional state when these inputs are present. To examine whether gene transcription was involved in this change in neuronal behavior, we performed in vitro experiments in which the STG was exposed to the RNA-synthesis inhibitor actinomycin D (ACD). ACD (50 microM) incubation at the time of decentralization prevented subsequent reacquisition of PD neuron bursting, but the inhibitor was much less effective when applied at later postdecentralization times, suggesting that the recovery process arises from new protein synthesis triggered when modulatory inputs are first removed. Moreover, in the nondecentralized STG, trans-synaptic modulatory instruction may sustain the conditional pyloric network phenotype by continuously regulating expression of genes responsible for intrinsic neuronal rhythmogenesis.

Reconfiguration of the neural network controlling multiple breathing patterns: eupnea, sighs and gasps [see comment].

Are different forms of breathing derived from one or multiple neural networks? We demonstrate that brainstem slices containing the pre-Bötzinger complex generated two rhythms when normally oxygenated, with striking similarities to eupneic ('normal') respiration and sighs. Sighs were triggered by eupneic bursts under control conditions, but not in the presence of strychnine (1 microM). Although all neurons received synaptic inputs during both activities, the calcium channel blocker cadmium (4 microM) selectively abolished sighs. In anoxia, sighs ceased, and eupneic activity was reconfigured into gasping, which like eupnea was insensitive to 4 microM cadmium. This reconfiguration was accompanied by suppression of synaptic inhibition. We conclude that a single medullary network underlies multiple breathing patterns.

The role of the hyperpolarization-activated current in modulating rhythmic activity in the isolated respiratory network of mice.

We examined the role of the hyperpolarization-activated current (I(h)) in the generation of the respiratory rhythm using a spontaneously active brainstem slice of mice. This preparation contains the hypoglossus (XII) nucleus, which is activated in-phase with inspiration and the pre-Bötzinger complex (PBC), the presumed site for respiratory rhythm generation. Voltage-clamp recordings (n = 90) indicate that cesium (Cs) (5 mM) blocked 77.2% of the I(h) current, and ZD 7288 (100 microM) blocked 85.8% of the I(h) current. This blockade increased the respiratory frequency by 161% in Cs and by 150% in ZD 7288 and increased the amplitude of integrated population activity in the XII by 97% in Cs and by 162% in ZD 7288, but not in the PBC (Cs, by 19%; ZD 7288, by -4.56%). All inspiratory PBC neurons (n = 44) recorded in current clamp within the active network revealed a significantly decreased frequency of action potentials during the interburst interval and an earlier onset of inspiratory bursts after I(h) current blockade. However, hyperpolarizing current pulses evoked only in a small proportion of inspiratory neurons (0% of type I; 29% of type II neurons) a depolarizing sag. Most of the neurons expressing an I(h) current (86%) were pacemaker neurons, which continued to generate rhythmic bursts after inactivating the respiratory network pharmacologically with CNQX alone or with CNQX, AP-5, strychnine, bicuculline, and carbenoxolone. Cs and ZD 7288 increased the frequency of pacemaker bursts and decreased the frequency of action potentials between pacemaker bursts. Our findings suggest that the I(h) current plays an important role in modulating respiratory frequency, which is presumably mediated by pacemaker neurons.

Neuromodulatory inputs maintain expression of a lobster motor pattern-generating network in a modulation-dependent state: evidence from long-term decentralization in vitro.

Neuromodulatory inputs play a critical role in governing the expression of rhythmic motor output by the pyloric network in the crustacean stomatogastric ganglion (STG). When these inputs are removed by cutting the primarily afferent stomatogastric nerve (stn) to the STG, pyloric neurons rapidly lose their ability to burst spontaneously, and the network falls silent. By using extracellular motor nerve recordings from long-term organotypic preparations of the stomatogastric nervous system of the lobster Jasus lalandii, we are investigating whether modulatory inputs exert long-term regulatory influences on the pyloric network operation in addition to relatively short-term neuromodulation. When decentralized (stn cut), quiescent STGs are maintained in organ culture, pyloric rhythmicity gradually returns within 3-5 d and is similar to, albeit slower than, the triphasic motor pattern expressed when the stn is intact. This recovery of network activity still occurred after photoinactivation of axotomized input terminals in the isolated STG after migration of Lucifer yellow. The recovery does not depend on action potential generation, because it also occurred in STGs maintained in TTX-containing saline after decentralization. Resumption of rhythmicity was also not activity-dependent, because recovery still occurred in STGs that were chronically depolarized with elevated K+ saline or were maintained continuously active with the muscarinic agonist oxotremorine after decentralization. We conclude that the prolonged absence of extraganglionic modulatory inputs to the pyloric network allows expression of an inherent rhythmogenic capability that is normally maintained in a strictly conditional state when these extrinsic influences are present.