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The location of neurons generating the rhythm of breathing in mammals is unknown. By microsection of the neonatal rat brainstem in vitro, a limited region of the ventral medulla (the pre-Bötzinger Complex) that contains neurons essential for rhythmogenesis was identified. Rhythm generation was eliminated by removal of only this region. Medullary slices containing the pre-Bötzinger Complex generated respiratory-related oscillations similar to those generated by the whole brainstem in vitro, and neurons with voltage-dependent pacemaker-like properties were identified in this region. Thus, the respiratory rhythm in the mammalian neonatal nervous system may result from a population of conditional bursting pacemaker neurons in the pre-Bötzinger Complex.
The Rhythm of Breathing Animates mammalian life, and the source of this rhythm, the noeud vital (1), is unknown. The basic oscillator lies within the brainstem, but technical limitations of experiments in the mammalian nervous system in vivo have hindered localization of the neurons generating the rhythm (2). An in vitro preparation of neonatal mammalian brainstem and spinal cord that spontaneously generates respiratory rhythm (3, 4) allows application of a broader range of techniques and has provided insights into neural mechanisms controlling breathing (5). Analysis of synaptic mechanisms in this preparation has led to the hypothesis that conditional pacemaker neurons in the medulla are the kernel for rhythm generation (6). Further tests of this hypothesis and network-based models (2, 7) of rhythmogenesis require identification of the sites and specific cells producing the rhythm. In our experiments we have systematically microsectioned the in vitro neonatal rat brainstem and precisely localized regions with neurons critical for rhythmogenesis (8).
Serial transverse microsections (50 to 75 μm thick) were made with a Vibratome, either caudally along the brainstem, starting from the pontomedullary junction, or rostrally from the spinomedullary junction, and perturbations of rhythmogenesis were analyzed (9). Rostral to caudal sectioning (n = 20 experiments) did not perturb the frequency of inspiratory phase motor discharge (of phrenic and other respiratory motoneurons) until the level of caudal retrofacial nucleus; further sectioning induced instabilities in the rhythm and then eliminated rhythmic motor output (10) (Fig. 1). Perturbations of rhythmogenesis occurred only with the removal of sections at this level of the medulla. More caudal medullary regions were not essential for rhythm generation, because sectioning rostrally from the spinomedullary junction (n = 11) did not disrupt respiratory motor output on cranial nerves (11) until a level rostral to the obex, within 200 μm of the level causing cessation of respiratory output in the rostral to caudal sectioning experiments. Transections in horizontal planes (n = 11), which removed regions dorsal to nucleus ambiguus (Fig. 1) but left intact motor circuits of the ventral medulla (2), did not alter the rhythmic discharge of medullary motoneurons (12).
Neurons essential for respiratory rhythmogenesis thus appear localized in the ventral medulla just caudal to the level of retrofacial nucleus. Rhythmically active neurons are localized within this critical area (2, 4, 13) just caudal to the Bötzinger Complex of respiratory neurons (Fig. 1) (14). We refer to this region with rhythmically active cells as the pre-Bötzinger Complex (8, 15) and propose that it contains neurons that generate respiratory rhythm. This region has not been previously identified as a site for rhythm generation. These results exclude more rostral areas as primary sites for rhythmogenesis (16).
To establish whether neurons in the pre-Bötzinger Complex could generate respiratory rhythm, we prepared transverse medullary slices (350 to 600 μm thick) that included this region (Fig. 2), local circuits for motor output generation, and hypoglossal respiratory motoneurons and nerves (17). The thinner slices (350 μm thick) just enclosed the boundaries of the pre-Bötzinger Complex. These slices generated rhythmic motor output on hypoglossal nerves (Fig. 2) (18), confirming that neurons at the level of the pre-Bötzinger Complex could generate rhythm. The motoneuron population discharge, as well as synaptic drive potentials and currents of medullary neurons, were similar in frequency, duration, and temporal pattern (Fig. 2, A and B) to that generated in the en bloc brainstem (4, 18); thus, the oscillatory neuronal activity of the slice represented respiratory activity. To verify that pre-Bötzinger Complex neurons in these slices could affect the rhythm, we produced local perturbations of neuronal excitability. Transient neuronal depolarization by microinjection (20 to 40 nl) of physiological solutions with high K+ concentrations (25 mM) reversibly increased the motor burst frequency three- to fourfold (Fig. 2C). Reduction of local excitatory synaptic transmission by microinjection of 6-cyano-7-nitroquinoxaline-2,3-di-one (CNQX) (250 fmol), a non–N-methyl-D-aspartate (NMDA) receptor antagonist, transiently reduced the frequency and reversibly eliminated the oscillatory output (Fig. 2C). This result is consistent with findings that excitatory neurotransmission mediated by endogenously released excitatory amino acids is necessary for rhythmogenesis (19).
Oscillatory properties of neurons in the pre-Bötzinger Complex in the slices were investigated with whole-cell patch-clamp recording techniques (20). Several populations of neurons exhibited periodic membrane potential depolarization synchronous with the motor output (Figs. 2 and and3)3) (20). To test for voltage-dependent pacemaker properties (19), we depolarized neurons under current-clamp recording conditions. In ~25% (4 of 15) of the rhythmically active cells, small membrane currents (30 to 100 pA) sufficient to depolarize the baseline potential to −55 to −45 mV resulted in oscillatory bursting at a higher frequency than that of the motor output (Fig. 3B). This type of voltage-dependent bursting property is characteristic of conditional pacemaker neurons. These bursting pacemaker-like cells were a distinct class, because other rhythmically active neurons in the region, as well as in adjacent regions, responded to membrane depolarization by generating a continuous stream of action potentials but not bursting oscillations. The rhythmic depolarization of these neurons without intrinsic bursting properties was due to periodic synaptic inputs with 300- to 600-pA peak synaptic currents (Fig. 2B) and reversal potentials near 0 mV.
Neurons with voltage-dependent oscillatory properties were also identified in the pre-Bötzinger Complex in thinner slices (250 to 350 μm thick) that did not generate rhythmic motor activity (21). These cells (n = 8) generated large-amplitude (10- to 15-mV) membrane potential oscillations and bursts of action potentials with depolarization into the −55 to −45 mV range. The frequency of these oscillations increased as the holding potential was elevated in this range (Fig. 3A). The periods of the oscillations covered the range of respiratory cycle periods (~3 to 15 s) observed in rhythmically active slices and en bloc brainstem-spinal cord preparations.
To determine if there are unique features of the cellular organization of the pre-Bötzinger Complex, we examined the anatomy of the homologous region in adult rats (Fig. 4), where we could label neurons on the basis of their connections within the respiratory network (22). We established several distinct cytoarchitectonic features. In the pre-Bötzinger Complex there was a marked absence of brainstem output neurons (that is, bulbospinal neurons) compared to adjacent regions. The highest number of bulbospinal neurons was in the ventrolateral reticular formation near the obex, caudal to the pre-Bötzinger Complex (Fig. 4A). The pre-Bötzinger Complex contained the highest percentage of propriobulbar interneurons (23) (Fig. 4B). This distribution of neuron types suggests that the pre-Bötzinger Complex has a distinct neuronal organization. Direct intracellular recordings from respiratory neurons in the pre-Bötzinger Complex in the adult cat (24) also indicate a dense concentration of interneurons. The pre-Bötzinger Complex interneurons have direct connections to the more caudal areas containing bulbospinal neurons (22). These interneurons may represent the substrate for transmission of the locally generated rhythm to the premotoneurons that transmit the oscillatory drive to spinal respiratory motoneurons.
We have identified a very limited region of the ventrolateral medulla containing neurons capable of generating respiratory rhythm in the neonatal nervous system. This region may contain the minimal neuronal substrate for rhythmogenesis. The presence of neurons with voltage-dependent oscillatory properties in this region is consistent with the hypothesis that conditional bursting pacemaker neurons are the kernel for rhythm generation in the neonate (6). However, before it can be concluded that the conditionally bursting neurons found in the pre-Bötzinger Complex are in fact pacemaker neurons generating the rhythm, the identified cells must be shown to produce the oscillatory drive to the network. Furthermore, because a transformation of rhythm generation mechanisms could occur during postnatal development, a hybrid of network and pacemaker properties may be essential for rhythmogenesis in the adult mammal (25). The slice preparations developed in our studies contain an isolated, functionally active circuit in vitro and will facilitate further analysis of mechanisms underlying the generation and transmission of respiratory oscillations in neonatal mammals.
Supported by NIH grants HL4095, NS24742, Research Career Development Award HL02204 (J.C.S.), DFG Ba 1095/1-1, and an Alexander von Humboldt Foundation Research Fellowship (J.C.S.).
Jeffrey C. Smith, Systems Neurobiology Laboratory, Department of Kinesiology, University of California, Los Angeles, CA 90024–1527.
Howard H. Ellenberger, Systems Neurobiology Laboratory, Department of Kinesiology, University of California, Los Angeles, CA 90024–1527.
Klaus Ballanyi, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-3400 Göttingen, Federal Republic of Germany.
Diethelm W. Richter, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-3400 Göttingen, Federal Republic of Germany.
Jack L. Feldman, Systems Neurobiology Laboratory, Department of Kinesiology, University of California, Los Angeles, CA 90024–1527.