The preBötzinger complex, often abbreviated as preBötC, is a functionally and anatomically specialized site in the ventral-lateral region of the lower medulla oblongata (i.e., lower brainstem). The preBötC is part of the ventral respiratory group of respiratory related interneurons. Its foremost function is to generate the inspiratory breathing rhythm in mammals. In addition, the preBötC is widely and paucisynaptically connected to higher brain centers that regulate arousal and excitability more generally such that respiratory brain function is intimately connected with many other rhythmic and cognitive functions of the brain and central nervous system. Further, the preBötC receives mechanical sensory information from the airways that encode lung volume as well as pH, oxygen, and carbon dioxide content of circulating blood and the cerebrospinal fluid.
The preBötC is approximately colocated with the hypoglossal (XII) cranial motor nucleus as well as the 'loop' portion of the inferior olive in the anterior-posterior axis. The caudal border of the preBötC is slightly caudal to the obex, where the brainstem merges with the cervical spinal cord.
Discovery The initial description of the preBötC was widely disseminated in a 1991 paper in Science, but its discovery predates that paper by one year. The team was led by Jack L. Feldman and Jeffrey C. Smith at the University of California, Los Angeles (UCLA), but the Science paper also included UCLA coauthor Howard Ellenberger, as well as Klaus Ballanyi and Diethelm W. Richter from Göttingen University in Germany. The region derives its name from a neighboring medullary region involved in expiratory breathing rhythm dubbed Bötzinger complex, which was named after the Silvaner (Bötzinger) variety of wine, featured at the conference at which that region was named (click here to hear a BBC interview with Jack Feldman on the topic of Bötzinger / preBötzinger nomenclature).
Functional definition of the preBötC The first definition of the preBötC was based largely on functional criteria. If the central neuraxis from pons to lumbar spinal cord is removed from a newborn rodent, then basic neural motor patterns can be generated and recorded using microelectrodes in vitro. The breathing rhythm emerges spontaneously with robust and continuous motor activity measurable on any cranial or spinal motor nerve that innervates breathing related musculature.
By isolating a rhythmically active newborn rat brainstem-spinal cord in a microsectioning vibratome, Smith and colleagues performed a series of 75 μm-thick transverse sections while monitoring inspiratory-related motor rhythms. The preBötC represented the portion of the ventral-lateral lower brainstem that was necessary and sufficient to generate inspiratory related rhythm and motor output in vitro. Surprisingly, if microsections were applied from the anterior and posterior regions of the neuraxis simultaneously, a transverse section of thickness ~500 μm – which retained the preBötC and XII motoneurons – generated a rhythm and motor pattern that was almost identical to the rhythm and pattern in the full brainstem-spinal cord preparation. Perturbations that elevated excitability in preBötC sped up respiratory rhythm, whereas perturbations that depressed its excitability slowed the rhythm down. The authors concluded that these preBötC-retaining slice preparations preserved the core network generating inspiratory rhythm as well as premotor and motor neurons that define a minimal breathing-related circuit suitable for studies under controlled conditions in vitro. Breathing slices became a widely exploited preparation for such studies that continue to be used by laboratories worldwide to the present day.
Anatomic definition of the preBötC Anatomical observations advanced understanding of the preBötC by providing specific markers expressed by its constituent neurons, which helped understand its approximate borders. The superset of markers is based largely on neuropeptides and peptide receptors, whose expression patterns have come to define the borders of preBötC and its constituent rhythm-generating and output pattern-related interneurons . preBötC neurons selectively express neurokinin-1 receptors (NK1Rs), μ-opioid receptors (μORs), as well as somatostatin (SST) and SST2a-type receptors. Of course, selectively does not mean exclusively or entirely. Each marker has limitations as a defining feature of the preBötC core, but generally speaking, the neuropeptide-related markers below have proved to be both reliable and of great utility in the quest to define preBötC structure and function.
Peptide markers have been used to probe preBötC function. Substance P (SP) accelerated inspiratory rhythms in vitro by depolarizing putatively rhythmogenic preBötC neurons. SP also depolarized preBötC neurons whose function is premotor-related, i.e., those neurons transmit the nascent inspiratory rhythm to motoneurons outside the preBötC. The net result was that SP sped up the rhythm and elevated the baseline level neural activity in XII nerve recordings in vitro.
The expression of NK1Rs by preBötC neurons was used to test its inspiratory rhythm-generating, role. SP, conjugated to the ribosomal toxin saporin, was injected into the preBötC of adult rats. Over the course of a week, this intervention caused progressive breathing deficits that ultimately resulted in severely pathological (i.e., ataxic) breathing. SP-saporin-injected rats also experienced sleep deficits and extraordinary sensitivity to anesthesia.
Expression of μORs appear to be less widespread than NK1Rs among constituent preBötC neurons. Although expressed somewhat more sparsely, the application of μOR agonists like [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (i.e., DAMGO) potently slowed the inspiratory rhythm. Note, this observation in vitro presaged the 2010-2020's crisis of opioid-drug related deaths by respiratory failure, which are attributable in large part to depression of rhythm-generating function in the preBötC (but also see:).
In the late 1980s and early 1990s, following discovery of the preBötC, in vitro preparations from neonates were not yet widely accepted as experimental models of the respiratory neural control system in adults. Some groups argued that in vitro rhythms reflected gasping rather than breathing, despite the fact that in vitro preparations, show physiological levels of oxygen and pH even several hundred micrometers below the surface of the tissue. Thus, the SP-saporin experiments were critical for showing that the preBötC was necessary for normal breathing in un-anesthetized adult animals.
Nevertheless, one is confronted with a disparity of motor patterns. The pattern of phrenic or XII nerve activity in vitro shows an abrupt onset followed by a decremental pattern, whereas in vivo the inspiratory motor nerves typically show an incremental onset followed by a more precipitous offset. The differences in the motor patterns measured in adults in vivo and those of in vitro preparations can be explained age- and development-related differences, the loss of mechanical sensory feedback in vitro, and the temperature (in vitro preparations are typically maintained ~10 °C lower than physiological temperature).
SST and SST2a receptors are expressed by neurons in the preBötC. Unlike NK1R expression, which remains rather strong in regions caudal to the preBötC within the cervical spinal cord, SST expression appears to peak in the anterior-posterior axis at the region recognized as the preBötC. Could SST-expressing preBötC neurons be markers for the preBötC core? Investigators installed in the preBötC a peptide receptor from fruit fly, adapted for expression in mammals, that activates potassium channels. Whether awake or anesthetized, activation of those potassium channel-linked receptors in SST-expressing neurons of the preBötC reduced breathing movements, both their amplitude and frequency, and ultimately caused apnea, i.e., a lack of breathing. The exogenous peptide that activates the fly receptor was ultimately cleared from the central nervous system: injected rats nonetheless needed mechanical ventilation until they recovered from the experiment. Subsequent studies examined the underlying cellular mechanisms and have come to the conclusion that preBötC neurons expressing SST are related to transmission of the rhythm from core rhythmogenic neurons to premotor neurons inspiratory neurons. The SST "output" neurons are intermingled in the preBötC with rhythm-generating neurons, and their function is to coactivate and pass on inspiratory rhythm to dedicated premotor populations outside of the preBötC.
Other markers for the preBötC include peptide hormone thyrotropin releasing hormone (TRH) and the glycoprotein reelin.
In summary, the preBötC is the source of rhythmic activity that – once distributed to premotor and motoneurons of respiratory muscles – produces inspiratory breathing movements. The neurons that comprise the preBötC express NK1Rs, μORs, SST2a receptors, and SST. Each of these markers holds functional significance for modulation of preBötC rhythmicity, and their expression delineates the borders of the preBötC. SP accelerated inspiratory rhythms measured in vitro and ablation of NK1R-expressing preBötC neurons caused severe pathologies of breathing that were ultimately fatal. The μORs also map the preBötC and opioid drugs depress breathing rhythms, which is further evidence of the preeminent rhythmogenic role of the preBötC. SST is a peptide transmitter rather than a receptor, but its expression also maps the preBötC. SST-expressing neurons are breathing essential, but their role is linked to the production of motor output rather than generation of rhythm per se.
Cellular composition of the preBötC
Excitatory (glutamatergic) neurons The rhythm-generating core of preBötC incorporates glutamatergic interneurons that express the gene Slc17a6 (i.e., Vglut2). preBötC glutamatergic neurons also express NK1Rs and μORs, but probably not SST. Pharmacological studies showed that excitatory transmission, predominantly via AMPA- and kainate-type ionotropic glutamate receptors were essential for rhythm generation as well as transmission to premotor neurons and ultimately motor output. Furthermore, Vglut2-knockout mice fail to breathe at birth. Transverse slices from late-stage embryos of Vglut2-knockout mice fail to generate rhythmic activity in the preBötC. Nevertheless, the cellular composition of the preBötC appears relatively unperturbed and constituent neurons express electrical properties associated with the preBötC in early postnatal mice, which emphasizes the importance of excitatory synaptic interactions for rhythm generation.
Dbx1-derived neurons A subset of preBötC glutamatergic neurons are derived from progenitor cells that express transcription factor Dbx1 (developing brain homeobox 1) during embryonic development. In slices from early postnatal Dbx1 reporter mice, Dbx1-derived preBötC neurons are rhythmically active in vitro in sync with inspiratory rhythm and motor output. Examined histologically, Dbx1-derived preBötC neurons express NK1Rs, μORs, SST2a receptors, as well as SST. Also in slices from postnatal Dbx1 reporter mice, the selective photonic ablation of Dbx1-derived preBötC neurons diminishes XII motor output magnitude and decelerates then irreversibly stops the XII rhythm. In adult mice that express light-sensitive cation channels (channelrhodopsin 2) in Dbx1-derived neurons, optogenetic photostimulation speeds up breathing and increases tidal volume of the breaths. Mice expressing proton pumps (archaerhodopsin) in Dbx1-derived preBötC neurons slows or stops breathing movements. When the breathing is slowed via photoinhibition of Dbx1-derived preBötC neurons, the tidal volume of the breaths is diminished.