Developmental anomalies of central respiratory neural control contribute to newborn mortality and morbidity. Elucidation of the cellular, molecular, trophic, and genetic mechanisms involved in the formation and function of respiratory nuclei during prenatal development will provide a foundation for understanding pathologies. The pre-Bötzinger Complex (pre-BötC) is a specific group of neurons located in the ventrolateral medulla that is critical for respiratory rhythmogenesis. Thus it has become a major focus of research. Here, we provide an overview of current knowledge regarding the anatomical and functional emergence of the rodent pre-BötC during the prenatal period.
- respiratory rhythm
- neural network
breathing is a spontaneous rhythmic motor behavior that is vital at birth and throughout life. Data from studies using in vitro and in vivo models support the hypothesis that the basic rhythm underlying breathing arises from a specific region of the ventrolateral medulla called the pre-Bötzinger complex (pre-BötC) (12, 22, 26, 48). Figure 1 shows the location of the pre-BötC relative to surrounding medullary nuclei. It is located ventral to the semi-compact division of the nucleus ambiguus (NA), caudal to the compact division of the NA and rostral to the lateral reticular formation. In addition, recent data suggest that there is a region surrounding the facial nucleus (parafacial respiratory group; pFRG) that may be involved in the generation of rhythmic drive to expiratory motoneurons (22, 33). The possibility of there being two coupled rhythm generating centers requires further study. Nevertheless, it is generally accepted that the pre-BötC is responsible for generating the respiratory rhythm necessary for driving inspiratory musculature.
The pre-BötC network contains a heterogeneous population of rhythmically active neurons that are interconnected through glutamate-mediated synaptic inputs. Distinct complements of membrane conductances differentiate classes of pre-BötC neurons. This includes two types of neurons that express bursting-pacemaker properties (23, 25, 48). The pacemaker properties of one is dependent on activation of a persistent sodium current (INap) and the other on the activation of a calcium-activated nonspecific and voltage-insensitive cation current (ICAN) (7, 39, 52). It has been hypothesized that these two types of pacemaker neurons differ in their involvement in respiratory rhythm generation depending on the environmental conditions and age (25, 38, 39, 52). However, there is debate regarding whether the collective data support the “pacemaker hypothesis” in which the rhythm relies exclusively on bursting-pacemaker neurons or the “group pacemaker/network hypothesis” in which both synaptic connections and pacemaker membrane properties contribute to rhythm generation (12, 47).
The development of the respiratory rhythm generator must be well established and functionally robust by birth. Indeed, its prenatal function of generating fetal breathing movements (FBMs) are necessary for the proper maturation of the lungs and fetal inspiratory drive transmission influences the development of respiratory motoneurons and muscle in utero (reviewed in Ref. 17). Respiration-related movements are present prenatally in all mammalian models studied. The present review will focus on current knowledge regarding the anatomical and functional development of the pre-BötC in prenatal rodents. There are limited data regarding the ontogeny of the pFRG and thus it will not be discussed.
ANATOMICAL EMERGENCE OF THE PRE-BÖTC NETWORK
Spatiotemporal expression of anatomical markers for pre-BötC neurons.
In adult and juvenile rodents, the pre-BötC can be anatomically defined as a region containing neurons expressing the neurokinin-1 receptor (NK1R), μ-opioid receptor, tyrosine kinase B receptor (TrkB), somatostatin (SST), and the type 2 vesicular glutamate transporter VGlut2 (15, 49, 51). Correspondingly, respiratory rhythm is modulated by a large number of substances including substance P, opioids, brain-derived neurotrophic factor (BDNF), and somatostatin (5, 15, 27, 29, 41, 50, 51). There is evidence from several studies suggesting that the subset of neurons within the pre-BötC that express NK1R are particularly important for rhythm generation. These data include the following: 1) immunohistochemical labeling for NK1R delineates a population of glutamatergic respiratory neurons within the pre-BötC, 2) local application of the ligand of NK1R, Substance P, in the pre-BötC leads to a marked increase in respiratory frequency and, 3) targeted destruction of NK1R-positive pre-BötC neurons with toxic Substance P-saporin profoundly disrupts breathing rhythm in vivo (14, 15, 27, 28, 49).
Thus to discern when the pre-BötC forms during prenatal development, the spatiotemporal pattern of NK1R expression has been examined throughout the prenatal period in both rat and mouse embryos (34, 54). NK1R labeling has been performed in conjunction with labeling for other putative pre-BötC markers, SST and TrkB, and markers for motoneuronal populations, Islet1,2 (11) and choline acetyl-transferase (ChAT). At embryonic day (E) 14 in the mouse and E16 in the rat (two comparable developmental stages given the longer gestational period in the rat), weak NK1R labeling is detected in a region ventral to the NA (recognized by its immunoreactivity for Islet1,2 or ChAT) that corresponds to the pre-BötC. At E15 (mouse)/E17 (rat), NK1R is more strongly expressed in that same region (Fig. 1). From these stages onward, the intensity and spatial extension of NK1R immunostaining increases to conform to the spatial extension of NK1R-positive areas described in the newborn. Expression profiles for SST and TrkB in the region follow a similar time course of development as NK1R, with inception at E15/E17 and extension onward (34, and Ogier, personal communication). The complex heterogeneity of neuronal labeling profiles for NK1R, SST, and TrkB (i.e., degree of overlap among neurons) observed in the newborn ventrolateral medulla is present from the inception of the pre-BötC in the fetus.
Birth dating for NK1R-positive pre-BötC neurons.
The date of birth and inception of migration of pre-BötC neurons was analyzed in two series of experiments performed in rats using 5-bromo-2′-deoxyuridine (BrdU) injections. BrdU is a thymidine analog that incorporates within actively dividing cells and is revealed using immunolabeling with a specific antibody. Thus the time at which cells of interest are born can be determined by injecting pregnant females with BrdU at distinct gestational stages, followed by BrdU immunodetection at developmental stages when the pre-BötC network has formed. In the first series of experiments, BrdU was injected at different times of gestation (E10–E16) and neurons in the pre-BötC region immunoreactive for both NK1R and BrdU were detected in tissues obtained from postnatal preparations (Fig. 2). Those data demonstrated that the majority (71%) of pre-BötC neurons expressing NK1R were born at E12–E13, 2 days later than NK1R-positive neurons in the adjacent NA (34). A second series of experiments confirmed this birth date and defined the timing of migration of NK1R-positive pre-BötC neurons. BrdU was injected at one embryonic age between E10 and E13, and embryos were subsequently analyzed at different gestational stages (E15–E18). Neurons coexpressing BrdU and NK1R were found in the pre-BötC region at E17 and E18 when BrdU was injected at E12 and E13, respectively (34). Collectively those data indicate that NK1R-expressing pre-BötC neurons are born on E12–E13 and reach their final location between E17 and E18. However, pre-BötC neurons express NK1R only after settlement at their final ventrolateral locations and thus defining their site of origin and migratory pathway is not possible using BrdU/NK1R detection. Using transgenic mouse lines for which progenitor domains or neuronal migration are perturbed may be a more suitable approach.
Despite the fact that neurons expressing NK1R are important for the generation of respiratory rhythmic activity, they constitute only a subpopulation of all pre-BötC neurons. It is possible that other respiratory neurons (those that do not express NK1R) could be settled and functional at earlier or later times of development. If that were the case, then the emergence of respiratory-like rhythmic activity in the embryo and the inception of anatomical markers in the pre-BötC region may be unrelated. To test this, anatomical studies have been systematically associated with functional examination of spontaneous activity in the pre-BötC network through calcium imaging, electrophysiological recordings, and pharmacological treatments.
FUNCTIONAL EMERGENCE OF THE PRE-BÖTC NETWORK
Respiratory-like activity in prenatal rodents.
Recordings of spontaneous respiratory activity performed in vivo (plethysmographic recordings), in utero (ultrasound recordings), and in vitro (calcium imaging and electrophysiological recordings performed on isolated brain stem-spinal cord and medullary slice preparations) have demonstrated that respiratory-related neuronal activity emerges at E15 (Fig. 3) and E17 in mouse and rat, respectively (1, 8, 18, 24, 34, 54, 55). Thus, there is a solid correlation between the anatomical emergences of the pre-BötC, based on immunohistochemical labeling criteria, and the onset of rhythmic respiratory motor discharge. At the inception of rhythmic respiratory activity, burst frequency is low (2–6 inspiratory bursts/min). The frequency and stability of respiratory rhythm increase toward that observed in the newborn late in gestation (10, 18, 32, 55). Prior to the inception of respiratory activity, respiratory neurons are recruited as part of a robust, regular rhythmic motor pattern that is generated at very low frequency along the full extent of the developing spinal cord and medulla. The spontaneous embryonic rhythmic motor pattern is postulated to play a key role in regulating the events involved in the early development of neuronal circuits and establishing motoneuronal phenotype, but is not related to respiratory activity (20, 30, 31, 35, 43, 59). This rhythmic activity commences at early stages when axons are migrating to the primordial muscle targets (by E13). These early rhythms subside as more spatially restricted respiratory and locomotor rhythms emerge at E15.5 and E17 in mouse and rat, respectively. Furthermore, there is a clear distinction between the widespread embryonic and prenatal respiratory rhythmic activity based on frequency, motor burst duration, site of initiation, spatial extension, and the sensitivity to gap junction blockers.
Membrane properties of pre-BötC neurons during the prenatal period.
In vivo and in situ recordings from pontine and medullary nuclei have demonstrated a rich complement of respiratory neuronal types based on their firing discharge patterns in relation to the respiratory cycle (2, 37). Comparably, the complexity of firing patterns present in reduced, deafferented, in vitro preparations is less (44). Calcium imaging and patch-clamp recordings have been used to examine neuronal activity within the pre-BötC prenatally. The following three types of pre-BötC neurons have been characterized based on discharge pattern (Fig. 4). 1) Neurons that receive chloride-mediated synaptic drive during the inspiratory phase. These likely constitute pre-BötC neurons that will fire during the expiratory phase at birth after maturation of the transmembrane chloride gradient (discussed below) and therefore classified as expiratory-like neurons (Fig. 4A). 2) Neurons that receive excitatory synaptic drive during inspiration that no longer burst after block of glutamatergic excitatory neurotransmission (inspiratory-like neurons; Fig. 4B). 3) Neurons that are rhythmically active in phase with inspiration and also exhibit voltage-dependent intrinsic bursting properties (pacemaker-like neuron; Fig. 4C) that persist after block of glutamatergic synaptic connections (54). Further studies characterizing membrane properties of embryonic pacemaker neurons are required to determine if there is heterogeneity among pacemaker neuronal populations prenatally or whether the inception of additional pacemaker types occurs during postnatal development, as suggested by Pena et al. (39).
The current/voltage profiles obtained from embryonic pre-BötC neurons show an inward rectification at hyperpolarized potentials, suggesting the activation of voltage-dependent conductances. To date, only one specific membrane conductance has been described in embryonic respiratory neurons: the hyperpolarization-activated non-specific cationic Ih current (Fig. 4D; 54). This current is present in 70% of the neurons tested, starts to be activated at approximately −80 mV, and has a mean voltage for half activation (V1/2) of 102.5 ± 0.8 mV (when the current is evoked by voltage pulses ranging from −50 mV to −120 mV). Postnatally, this current plays an important role in regulating respiratory rhythmogenesis (40, 53) and is modulated by BDNF (51). Prenatally, the role of this current and its possible modulation remains to be defined. Furthermore, analyses of ionic currents at prenatal stages need to be extended to other membrane conductances involved in respiratory rhythmogenesis, including those required for bursting-pacemaker properties dependent on INap and ICAN (6, 7, 38, 39). A combination of maturational changes in neuromodulatory conditioning drive and pre-BötC electrophysiological properties likely underlie the increased stability and frequency of respiratory rhythmogenesis prior to birth (17, 21).
Neurotransmission in the pre-BötC network prenatally.
Postnatally, respiratory rhythmogenesis depends on synchronization between pre-BötC neurons through activation of non-NMDA glutamatergic receptors (13, 19, 25, 48). Pre-BötC neurons expressing NK1R also contain the vesicular glutamate transporter VGlu2 mRNA and receive VGlut2-positive terminals, providing additional data implicating the importance of glutamate neurotransmission for respiratory rhythmogenesis (49, 57, 58). Prenatally, rhythmic pre-BötC neurons receive phasic and non-phasic glutamate-mediated synaptic inputs (Fig. 4E). Bath application of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to in vitro embryonic preparations suppresses the respiratory network output (54). Furthermore, respiration-related rhythmic activity fails to develop in VGlut2 null mouse embryos, despite the presence of pacemaker neurons in the pre-BötC region (56). Thus, similar to postnatal rodents, communication between fetal respiratory neurons necessary for rhythmic network activity relies on glutamatergic synaptic drive.
In the mammalian nervous system, fast inhibitory transmission is primarily mediated by the neurotransmitters GABA and glycine. Although not essential for respiratory rhythmogenesis in neonatal rodents, chloride-mediated inhibitory inputs play an important role in modulating motor output patterning and respiratory rhythm generation (4, 36, 45, 46). Patch-clamp recordings from embryonic pre-BötC neurons demonstrate the presence of inspiratory phasic and non-phasic chloride-mediated synaptic inputs (unpublished observation and Fig. 4E). Due to an immature transmembrane chloride gradient at early embryonic stages, activation of chloride-mediated transmission induces respiratory neuron depolarization and an increase in respiratory frequency at the time of emergence of pre-BötC activity (42). The transition from excitatory to inhibitory action of chloride-mediated synaptic inputs on respiratory rhythmogenesis occurs before birth in rats (E19) and is associated with the ontogenesis of chloride cotransporters (Fig. 5; 42). By birth, activation of GABA and glycine receptors results in depression of respiratory rhythm. This transition likely constitutes one of the major events contributing to the functional maturation of the pre-BötC network needed for the generation of a multi-phase motor pattern.
Neuromodulatory control of respiratory activity prenatally.
The neuromodulatory control of the pre-BötC network prenatally is likely an important contributor to episodic FBMs. Furthermore, studies of prenatal modulation are pertinent toward determining if the receptors used as pre-BötC anatomical markers (NK1R for Substance P, TrkB for BDNF) are functional at the inception of their expression. Pharmacological experiments using prenatal in vitro rodent preparations have demonstrated that Substance P, BDNF, thyrotropin-releasing hormone, noradrenaline, and serotonin increase, while opioid agonists decrease, respiratory rhythm from the time of initial pre-BötC formation (3, 9, 16, 34, 54, 55). Thus, in addition to being functional, the prenatal pre-BötC network is susceptible to modulation, ensuring adaptability of FBMs to varying in utero environmental conditions (e.g., hypoxia, state-dependent changes).
An understanding of the cellular and molecular mechanisms underlying respiratory rhythmogenesis during the prenatal period has progressed considerably in the past decade. Data from rodent models have determined when the pre-BötC forms as an anatomical entity and commences generating rhythmic respiratory neural activity. From its inception, the pre-BötC depends on non-NMDA receptor-mediated glutamatergic drive for rhythm generation. Furthermore, its rhythmic discharge is influenced by many of the same neuromodulators acting postnatally. Our understanding of the electrophysiological properties of fetal pre-BötC neurons is just beginning to emerge.
Basic knowledge of pre-BötC ontogeny provides the foundation for identifying the embryonic origins and the transcriptional and trophic control mechanisms that define their specific phenotype (13b). It also provides the basis for understanding the anatomical and physiological defects associated with mutant mouse models with central respiratory disorders (Ref. 13a). Ultimately, the emerging data could contribute to a mechanistic understanding of the pathogenesis associated with breathing abnormalities such as central apneas associated with prematurity, Rett Syndrome, Prader-Willi syndrome, congenital central hypoventilation syndrome, and Sudden Infant Death Syndrome.
M. Thoby-Brisson is supported by the Centre National de la Recherche Scientifique and the Institut National de la Santé et de la Recherche Médicale. J. J. Greer is a Scientist of the Alberta Heritage Foundation for Medical Research and is supported by Canadian Institutes for Health Research and Rett Foundation.
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