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1.  Reelin Demarcates a Subset of Pre-Bötzinger Complex Neurons in Adult Rat 
Identification of two markers of neurons in the pre- Bötzinger complex (pre-BötC), the neurokinin 1 receptor (NK1R) and somatostatin (Sst) peptide, has been of great utility in understanding the essential role of the pre-BötC in breathing. Recently, the transcription factor dbx1 was identified as a critical, but transient, determinant of glutamatergic pre-BötC neurons. Here, to identify additional markers, we constructed and screened a single-cell subtractive cDNA library from pre-BötC inspiratory neurons. We identified the glycoprotein reelin as a potentially useful marker, because it is expressed in distinct populations of pre-BötC and inspiratory bulbospinal ventral respiratory group (ibsVRG) neurons. Reelin ibsVRG neurons were larger (27.1 ± 3.8 µm in diameter) and located more caudally (>12.8 mm caudal to Bregma) than reelin pre-BötC neurons (15.5 ± 2.4 µm in diameter, <12.8 mm rostral to Bregma). Pre-BötC reelin neurons coexpress NK1R and Sst. Reelin neurons were also found in the parahypoglossal and dorsal parafacial regions, pontine respiratory group, and ventromedial medulla. Reelin-deficient (Reeler) mice exhibited impaired respones to hypoxia compared with littermate controls. We suggest that reelin is a useful molecular marker for pre-BötC neurons in adult rodents and may play a functional role in pre- BötC microcircuits.
PMCID: PMC3751584  PMID: 21858819
breathing; hypoxia; reelin; Ahi1; latexin; PRMC1
2.  Nicotine Delivery to Rats via Lung Alveolar Region-Targeted Aerosol Technology Produces Blood Pharmacokinetics Resembling Human Smoking 
Nicotine & Tobacco Research  2012;15(7):1248-1258.
Nicotine is a heavily used addictive drug acquired through smoking tobacco. Nicotine in cigarette smoke is deposited and absorbed in the lungs, which results in a rapidly peaked slowly declining arterial concentration. This pattern plays an important role in initiation of nicotine addiction.
A method and device were developed for delivering nicotine to rodents with lung alveolar region-targeted aerosol technology. The dose of delivery can be controlled by the nicotine aerosol concentration and duration of exposure.
Our data showed that, in the breathing zone of the nose-only exposure chamber, the aerosol droplet size distribution was within the respirable diameter range. Rats were exposed to nicotine aerosol for 2min. The arterial blood nicotine concentration reached 43.2±15.7ng/ml (mean ± SD) within 1–4min and declined over the next 20min, closely resembling the magnitude and early pharmacokinetics of a human smoking a cigarette. The acute inhalation toxicity of nicotine: LC50 = 2.3mg/L was determined; it was affected by pH, suggesting that acidification decreases nicotine absorption and/or bioavailability.
A noninvasive method and toolkit were developed for delivering nicotine to rodents that enable rapid delivery of a controllable amount of nicotine into the systemic circulation and brain-inducing dose-dependent pharmacological effects, even a lethal dose. Aerosol inhalation can produce nicotine kinetics in both arterial and venous blood resembling human smoking. This method can be applied to studies of the effects of chronic intermittent nicotine exposure, nicotine addiction, toxicology, tobacco-related diseases, teratogenicity, and for discovery of pharmacological therapeutics.
PMCID: PMC3682843  PMID: 23239844
3.  Active Expiration Induced by Excitation of Ventral Medulla in Adult Anesthetized Rats 
Data from perinatal and juvenile rodents support our hypothesis that the preBötzinger complex generates inspiratory rhythm and the retrotrapezoid nucleus–parafacial respiratory group (RTN/pFRG) generates active expiration (AE). Although the role of the RTN/pFRG in adulthood is disputed, we hypothesized that its rhythmogenicity persists but is typically silenced by synaptic inhibition. We show in adult anesthetized rats that local pharmacological disinhibition or optogenetic excitation of the RTN/pFRG can generate AE and transforms previously silent RTN/pFRG neurons into rhythmically active cells whose firing is correlated with late-phase active expiration. Brief excitatory stimuli also reset the respiratory rhythm, indicating strong coupling of AE to inspiration. The AE network location in adult rats overlaps with the perinatal pFRG and appears lateral to the chemosensitive region of adult RTN. We suggest that (1) the RTN/pFRG contains a conditional oscillator that generates AE, and (2) at rest and in anesthesia, synaptic inhibition of RTN/pFRG suppresses AE.
PMCID: PMC3142740  PMID: 21414911
4.  Unilateral Ablation of Pre-Bötzinger Complex Disrupts Breathing during Sleep but Not Wakefulness 
Rationale: In adult rats, bilateral ablation of pre-Bötzinger complex (preBötC) neurokinin 1–expressing (NK1R) neurons leads to a progressive and irreversible disruption in breathing pattern, initially during sleep, eventually resulting in an ataxic breathing pattern during wakefulness.
Objectives: Here we determine whether ablation of fewer preBötC NK1R neurons leads to a persistent pattern of disordered breathing during sleep but not during wakefulness.
Methods: Adult male Sprague-Dawley rats (n = 12) were instrumented to record diaphragmatic, abdominal, and neck EMG, and EEG. Fourteen days later, a second surgery was performed to stereotaxically microinject into the preBötC on one side the toxin saporin conjugated to substance P (SP-SAP), which selectively ablates NK1R neurons.
Measurements and Main Results: Postinjection, rats were monitored within a plethysmograph until they were killed (Days 21–51). At Days 6–9 post–unilateral SP-SAP injection, respiratory pattern during sleep, particularly REM sleep, became increasingly disordered, characterized by an increase in frequency of central sleep apnea and hypopneas (36.8 ± 7.4 episodes/h of REM vs. 6 ± 2.0 episodes/h in preinjection controls; P < 0.05), whereas breathing during resting wakefulness remained stable. Unlike bilateral SP-SAP–injected rats, an ataxic breathing pattern did not develop during wakefulness. Rats that were monitored up to 51 days post–SP-SAP injection continued to have sleep-disordered breathing; breathing during wakefulness remained relatively stable. Histologic analysis of the ventrolateral medulla confirmed that NK1R neurons within the preBötC on the injected but not on the contralateral side of the medulla were ablated.
Conclusions: Gradual loss of preBötC NK1R neurons may be an underlying factor of sleep-disordered breathing, in particular of central sleep apnea.
PMCID: PMC2441928  PMID: 18420958
respiratory control; apnea; ventrolateral medulla; saporin; neurokinin 1
5.  Understanding the rhythm of breathing: so near yet so far 
Annual review of physiology  2012;75:423-452.
Understanding the mechanisms leading from DNA to molecules to neurons to networks to behavior is a major goal for neuroscience, but largely out of reach for many fundamental and interesting behaviors. The neural control of breathing may be a rare exception, presenting a unique opportunity to understand how the nervous system functions normally, how it balances inherent robustness with a highly regulated lability, how it adapts to rapidly and slowly changing conditions, and how particular dysfunctions result in disease. Why can we assert this? First and foremost, the functions of breathing are clearly definable, starting with its regulatory job of maintaining blood (and brain) O2, CO2 and pH; failure is not an option. Breathing is also an essential component of many vocal and emotive behaviors including, e.g., crying, laughing, singing, and sniffing, and must be coordinated with such vital behaviors as suckling and swallowing, even at birth. Second, the regulated variables, O2, CO2 and pH (and temperature in non-primate mammals), are continuous and are readily and precisely quantifiable, as is ventilation itself along with the underlying rhythmic motor activity, i.e., respiratory muscle EMGs. Third, we breathe all the time, except for short breaks as during breath-holding (which can be especially long in diving or hibernating mammals) or sleep apnea. Mammals (including humans) breathe in all behavioral states, e.g., sleep-wake, rest, exercise, panic, or fear, during anesthesia and even following decerebration. Moreover, essential aspects of the neural mechanisms driving breathing, including rhythmicity, are present at levels of reduction down to a medullary slice. Fourth, the relevant circuits exhibit a remarkable combination of extraordinary reliability, starting ex utero with the first air breath – intermittent breathing movements actually start in utero during the third trimester – and continuing for as many as ~109 breaths, as well as considerable lability, responding rapidly (in less than one second) and with considerable precision, over an order of magnitude in metabolic demand for O2 (~0.25 to ~5 liters of O2/min). Breathing does indeed persist! Finally, breathing is genetically determined to work at birth, with a well-defined developmental program underlying a neuroanatomical organization with apparent segregation of function, i.e., rhythmogenesis is separate from motor pattern (burst shape and coordination) generation. Importantly, single human gene mutations can affect breathing, and several neurodegenerative disorders compromise breathing by direct effects on brainstem respiratory circuits (See below).
PMCID: PMC3671763  PMID: 23121137
6.  Distinct inspiratory rhythm and pattern generating mechanisms in the preBötzinger Complex 
In the mammalian respiratory central pattern generator, the preBötzinger Complex (preBötC) produces rhythmic bursts that drive inspiratory motor output. Cellular mechanisms initiated by each burst are hypothesized to be necessary to determine the timing of the subsequent burst, playing a critical role in rhythmogenesis. To explore mechanisms relating inspiratory burst generation to rhythmogenesis, we compared preBötC and hypoglossal (XII) nerve motor activity in medullary slices from neonatal mice in conditions where periods between successive inspiratory XII bursts were highly variable and distributed multimodally. This pattern resulted from rhythmic preBötC neural population activity that consisted of bursts, concurrent with XII bursts, intermingled with significantly smaller “burstlets”. Burstlets occurred at regular intervals during significantly longer XII interburst intervals, at times when a XII burst was expected. When a preBötC burst occurred, its high amplitude inspiratory component (I-burst) was preceded by a preinspiratory component that closely resembled the rising phase of burstlets. Cadmium (8 μM) eliminated preBötC and XII bursts, but rhythmic preBötC burstlets persisted. Burstlets and preinspiratory activity were observed in ~90% of preBötC neurons that were active during I-bursts. When preBötC excitability was raised significantly, burstlets could leak through to motor output in medullary slices and in vivo in adult anesthetized rats. Thus, rhythmic bursting, a fundamental mode of nervous system activity and an essential element of breathing, can be deconstructed into a rhythmogenic process producing low amplitude burstlets and preinspiratory activity that determine timing, and a pattern-generating process producing suprathreshold I-bursts essential for motor output.
PMCID: PMC3737080  PMID: 23719793
Postsynaptic inhibition is a key element of neural circuits underlying behavior, with 20-50% of all mammalian (non-granule) neurons considered inhibitory. For rhythmic movements in mammals, e.g., walking, swimming, suckling, chewing, breathing, inhibition is often hypothesized to play an essential rhythmogenic role. Here we study the role of fast synaptic inhibitory neurotransmission in the generation of breathing pattern by blocking GABAA and glycine receptors in the preBötzinger Complex (preBötC), a site essential for generation of normal breathing pattern, and in the neighboring Bötzinger Complex (BötC). The breathing rhythm continued following this blockade, but the lung inflation-induced Breuer-Hering inspiratory-inhibitory reflex was suppressed. The antagonists were efficacious, as this blockade abolished the profound effects of the exogenously applied GABAA receptor agonist muscimol or glycine, either of which under control conditions stopped breathing in vagus-intact or vagotomized, anesthetized, spontaneously breathing adult rats. In vagotomized rats, GABAAergic and glycinergic antagonists had little, if any, effect on rhythm. The effect in vagus intact rats was to slow the rhythm to a pace equivalent to that seen after suppression of the aforementioned Breuer-Hering inflation reflex. We conclude that postsynaptic inhibition within the preBötC and BötC is not essential for generation of normal respiratory rhythm in intact mammals. We suggest the primary role of inhibition is in shaping the pattern of respiratory motor output, assuring its stability, and in mediating reflex or volitional apnea, but not in the generation of rhythm per se.
PMCID: PMC3724454  PMID: 23536061
breathing; respiratory rhythm; inhibition; preBötzinger Complex; Bötzinger Complex; Breuer-Hering reflex
8.  Emergence of Population Bursts from Simultaneous Activation of Small Subsets of preBötzinger Complex Inspiratory Neurons 
During rhythmic movements, central pattern generators (CPGs) trigger bursts of motor activity with precise timing. However, the number of neurons that must be activated within CPGs to generate motor output is unknown. In the mammalian breathing rhythm, a fundamentally important motor behavior, the preBötzinger Complex (preBötC) produces synchronous population-wide bursts of activity to control inspiratory movements. We probed mechanisms underlying inspiratory burst generation in the preBötC using holographic photolysis of caged glutamate in medullary slices from neonatal mice. With stimulation parameters determined to confine photoactivation to targeted neurons, simultaneous excitation of 4–9 targeted neurons could initiate ectopic, endogenous-like bursts with delays averaging 255 ms, placing a critical and novel boundary condition on the microcircuit undelying respiratory rhythmogenesis.
PMCID: PMC3708687  PMID: 23426661
9.  Projections of PreBötzinger Complex Neurons in Adult Rats 
The Journal of comparative neurology  2010;518(10):1862-1878.
The preBötzinger Complex (preBötC) contains neural microcircuitry essential for normal respiratory rhythm generation in rodents. A subpopulation of preBötC neurons expresses somatostatin, a neuropeptide with a modulatory action on breathing. Acute silencing of a subpopulation of preBötC neurons transfected by a virus driving protein expression under the somatostatin promoter results in persistent apnea in awake adult rats. Given the profound effect of silencing these neurons, their projections are of interest. We used an adeno-associated virus to overexpress enhanced green fluorescent protein driven by the somatostatin promoter in preBötC neurons to label their axons and terminal fields. These neurons send brainstem projections to: 1) contralateral preBötC; 2) ipsi- and contralateral Bötzinger Complex; 3) ventral respiratory column caudal to preBötC; 4) parafacial respiratory group / retrotrapezoid nucleus; 5) parahypoglossal nucleus/nucleus of the solitary tract; 6) parabrachial/Kölliker-Fuse nuclei; and 7) periaqueductal gray. We did not find major projections to either cerebellum or spinal cord. We conclude that there are widespread projections from preBötC somatostatin-expressing neurons specifically targeted to brainstem regions implicated in control of breathing, and provide a network basis for the profound effects and the essential role of the preBötC in breathing.
PMCID: PMC3494295  PMID: 20235095
preBötzinger Complex; breathing; respiratory rhythm; AAV2
10.  α4* Nicotinic Receptors in preBötzinger Complex Mediate Cholinergic/Nicotinic Modulation of Respiratory Rhythm 
Acetylcholine and nicotine can modulate respiratory patterns by acting on nicotinic acetylcholine receptors (nAChRs) in the preBötzinger complex (preBötC). To further explore the molecular composition of these nAChRs, we studied a knock-in mouse strain with a leucine-to-alanine mutation in the M2 pore-lining region (L9′A) of the nAChR α4 subunit; this mutation renders α4-containing receptors hypersensitive to agonists. We recorded respiratory-related rhythmic motor activity from hypoglossal nerve (XIIn) and patch-clamped preBötC inspiratory neurons in an in vitro medullary slice preparation from neonatal mice. Nicotine affected respiratory rhythm at concentrations ~100-fold lower in the homozygous L9′A knock-in mice compared with wild-type mice. Bath application of 5 nm nicotine increased the excitability of preBötC inspiratory neurons, increased respiratory frequency, and induced tonic/seizure-like activities in XIIn in L9′A mice, effects similar to those induced by 1 μm nicotine in wild-type mice. In L9′A mice, microinjection of low nanomolar concentrations of nicotine into the preBötC increased respiratory frequency, whereas injection into the ipsilateral hypoglossal (XII) nucleus induced tonic/seizure-like activity. The α4*-selective nAChR antagonist dihydro-β-erythroidine produced opposite effects and blocked the nicotinic responses. These data, showing that nAChRs in the preBötC and XII nucleus in L9'A mice are hypersensitive to nicotine and endogenous ACh, suggest that functional α4* nAChRs are present in the preBötC. They mediate cholinergic/nicotinic modulation of the excitability of preBötC inspiratory neurons and of respiratory rhythm. Furthermore, functional α4* nAChRs are present in XII nucleus and mediate cholinergic/nicotinic modulation of tonic activity in XIIn.
PMCID: PMC3477875  PMID: 18184794
L9′A knock-in mice; α4 subunit; preBötzinger complex; inspiratory neurons; hypoglossal nucleus; nicotine
11.  Rhythmogenic neuronal networks, emergent leaders, and k-cores 
Neuronal network behavior results from a combination of the dynamics of individual neurons and the connectivity of the network that links them together. We study a simplified model, based on the proposal of Feldman and Del Negro (FDN) [Nat. Rev. Neurosci. 7, 232 (2006)], of the preBötzinger Complex, a small neuronal network that participates in the control of the mammalian breathing rhythm through periodic firing bursts. The dynamics of this randomly connected network of identical excitatory neurons differ from those of a uniformly connected one. Specifically, network connectivity determines the identity of emergent leader neurons that trigger the firing bursts. When neuronal desensitization is controlled by the number of input signals to the neurons (as proposed by FDN), the network's collective desensitization—required for successful burst termination—is mediated by k -core clusters of neurons.
PMCID: PMC3477876  PMID: 21230504
12.  Synaptically Activated Burst-Generating Conductances Underlie a Group-Pacemaker Mechanism for Respiratory Rhythm Generation in Mammals 
Progress in Brain Research  2010;187:111-136.
Breathing, chewing and walking are critical life-sustaining behaviors in mammals that consist essentially of simple rhythmic movements. Breathing movements in particular involve the diaphragm, thorax, and airways but emanate from a network in the lower brain stem. This network can be studied in reduced preparations in vitro and using simplified mathematical models that make testable predictions. An iterative approach that employs both in vitro and in silico models has ruled out canonical mechanisms for respiratory rhythm that involve reciprocal inhibition and pacemaker properties. We present an alternative model in which emergent network properties play the key rhythmogenic role. Specifically, we show evidence that synaptically activated burst-generating conductances – which are only available in the context of network activity – engender robust periodic bursts in respiratory neurons. Because the cellular burst-generating mechanism is linked to network synaptic drive we dub this type of system a group pacemaker.
PMCID: PMC3370336  PMID: 21111204
preBötzinger Complex; pre-Bötzinger Complex; central pattern generator (CPG); metabotropic glutamate receptors; calcium-activated nonspecific cation current; mathematical models; emergent network properties; breathing
13.  Pre-Bötzinger Complex: A Brainstem Region That May Generate Respiratory Rhythm in Mammals 
Science (New York, N.Y.)  1991;254(5032):726-729.
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.
PMCID: PMC3209964  PMID: 1683005
14.  Opioid-Induced Quantal Slowing Reveals Dual Networks for Respiratory Rhythm Generation 
Neuron  2003;37(5):821-826.
Current consensus holds that a single medullary network generates respiratory rhythm in mammals. Pre-Bötzinger Complex inspiratory (I) neurons, isolated in transverse slices, and preinspiratory (pre-I) neurons, found only in more intact en bloc preparations and in vivo, are each proposed as necessary for rhythm generation. Opioids slow I, but not pre-I, neuronal burst periods. In slices, opioids gradually lengthened respiratory periods, whereas in more intact preparations, periods jumped nondeterministically to integer multiples of the control period (quantal slowing). These findings suggest that opioid-induced quantal slowing results from transmission failure of rhythmic drive from pre-I neurons to preBötC I networks, depressed below threshold for spontaneous rhythmic activity. Thus, both I (in the slice), and pre-I neurons are sufficient for respiratory rhythmogenesis.
PMCID: PMC3210017  PMID: 12628172
15.  Glycinergic pacemaker neurons in preBötzinger Complex of neonatal mouse 
The preBötzinger Complex (preBötC) is essential for normal respiratory rhythm generation in rodents, for which the underlying mechanisms remain unknown. Excitatory preBötC pacemaker neurons are proposed to be necessary for rhythm generation. Here we report the presence of a population of preBötC glycinergic pacemaker neurons. We used rhythmic in vitro transverse slice preparations from transgenic mice where neurons expressing the glycine transporter 2 (GlyT2) gene co-express enhanced green fluorescent protein (EGFP). We combined epifluorescence and whole-cell patch-clamp recording to study preBötC EGFP-labeled, i.e., glycinergic, inspiratory-modulated neurons with pacemaker properties. We defined glycinergic pacemaker neurons as those preBötC EGFP neurons that exhibited: 1) ectopic bursting in rhythmic slices when depolarized during their normally silent period, and; 2) bursting when depolarized in non-rhythmic slices (following AMPA receptor blockade). 42% of EGFP-labeled neurons were inspiratory (n=48 of 115), of which 23% (n=11 of 48 inspiratory; 10% of the total recorded) were pacemakers. We conclude that there is a population of preBötC inspiratory-modulated glycinergic, presumably inhibitory, pacemaker neurons that constitute a substantial fraction of all preBötC pacemaker neurons. These findings challenge contemporary models for respiratory rhythmogenesis that assume the excitatory nature of preBötC pacemaker neurons. Testable and non-trivial predictions of the functional role of excitatory and inhibitory pacemaker neurons need to be proposed and the necessary experiments performed.
PMCID: PMC2947441  PMID: 20219997
respiratory rhythm generation; breathing; GlyT2-EGFP mice; glycine; inhibition; Pacemaker
16.  Sleep-disordered breathing after targeted ablation of preBötzinger complex neurons 
Nature neuroscience  2005;8(9):1142.
Ablation of preBötzinger complex (preBötC) neurons, critical for respiratory rhythm generation, resulted in a progressive, increasingly severe disruption of respiratory pattern, initially during sleep and then also during wakefulness in adult rats. Sleep-disordered breathing is highly prevalent in elderly humans and in some patients with neurodegenerative disease. We propose that sleep-disordered breathing results from loss of preBötC neurons and could underlie death during sleep in these populations.
PMCID: PMC2819071  PMID: 16116455
17.  Looking for inspiration: new perspectives on respiratory rhythm 
Recent experiments in vivo and in vitro have advanced our understanding of the sites and mechanisms involved in mammalian respiratory rhythm generation. Here we evaluate and interpret the new evidence for two separate brainstem respiratory oscillators and for the essential role of emergent network properties in rhythm generation. Lesion studies suggest that respiratory cell death might explain morbidity and mortality associated with neurodegenerative disorders and ageing.
PMCID: PMC2819067  PMID: 16495944
18.  Practice makes perfect, even for breathing 
Nature neuroscience  2009;12(8):961-963.
Breathing relies on a respiratory rhythm generator. A study characterizes an early emerging oscillatory group of Phox2b-expressing parafacial cells that entrain and couple with the preBötzinger Complex at the onset of fetal breathing.
PMCID: PMC2810466  PMID: 19636348
19.  Breathing: Rhythmicity, Plasticity, Chemosensitivity 
Annual review of neuroscience  2003;26:239-266.
Breathing is a vital behavior that is particularly amenable to experimental investigation. We review recent progress on three problems of broad interest. (i) Where and how is respiratory rhythm generated? The preBötzinger Complex is a critical site, whereas pacemaker neurons may not be essential. The possibility that coupled oscillators are involved is considered. (ii) What are the mechanisms that underlie the plasticity necessary for adaptive changes in breathing? Serotonin-dependent long-term facilitation following intermittent hypoxia is an important example of such plasticity, and a model that can account for this adaptive behavior is discussed. (iii) Where and how are the regulated variables CO2 and pH sensed? These sensors are essential if breathing is to be appropriate for metabolism. Neurons with appropriate chemosensitivity are spread throughout the brainstem; their individual properties and collective role are just beginning to be understood.
PMCID: PMC2811316  PMID: 12598679
preBötzinger; pacemaker; neurokinin; serotonin; raphe
20.  Modulation of Respiratory Frequency by Peptidergic Input to Rhythmogenic Neurons in the PreBötzinger Complex 
Science (New York, N.Y.)  1999;286(5444):1566-1568.
Neurokinin-1 receptor (NK1R) and μ-opioid receptor (μOR) agonists affected respiratory rhythm when injected directly into the preBötzinger Complex (preBötC), the hypothesized site for respiratory rhythmogenesis in mammals. These effects were mediated by actions on preBötC rhythmogenic neurons. The distribution of NK1R+ neurons anatomically defined the preBötC. Type 1 neurons in the preBötC, which have rhythmogenic properties, expressed both NK1Rs and μORs, whereas type 2 neurons expressed only NK1Rs. These findings suggest that the preBötC is a definable anatomic structure with unique physiological function and that a subpopulation of neurons expressing both NK1Rs and μORs generate respiratory rhythm and modulate respiratory frequency.
PMCID: PMC2811082  PMID: 10567264
21.  Normal breathing requires preBötzinger complex neurokinin-1 receptor-expressing neurons 
Nature neuroscience  2001;4(9):927-930.
The normal breathing rhythm in mammals is hypothesized to be generated by neurokinin-1 receptor (NK1R)-expressing neurons in the preBötzinger complex (preBötC), a medullary region proposed to contain the kernel of the circuits generating respiration. If this hypothesis is correct, then complete destruction of preBötC NK1R neurons should severely perturb and perhaps even fatally arrest breathing. Here we show that specific and near complete bilateral (but not unilateral) destruction of preBötC NK1R neurons results in both an ataxic breathing pattern with markedly altered blood gases and pH, and pathological responses to challenges such as hyperoxia, hypoxia and anesthesia. Thus, these ~600 neurons seem necessary for the generation of normal breathing in rats.
PMCID: PMC2810393  PMID: 11528424
22.  Central cholinergic regulation of respiration: nicotinic receptors 
Acta Pharmacologica Sinica  2009;30(6):761-770.
Nicotinic acetylcholine receptors (nAChRs) are expressed in brainstem and spinal cord regions involved in the control of breathing. These receptors mediate central cholinergic regulation of respiration and effects of the exogenous ligand nicotine on respiratory pattern. Activation of α4* nAChRs in the preBötzinger Complex (preBötC), an essential site for normal respiratory rhythm generation in mammals, modulates excitatory glutamatergic neurotransmission and depolarizes preBötC inspiratory neurons, leading to increases in respiratory frequency. nAChRs are also present in motor nuclei innervating respiratory muscles. Activation of post- and/or extra-synaptic α4* nAChRs on hypoglossal (XII) motoneurons depolarizes these neurons, potentiating tonic and respiratory-related rhythmic activity. As perinatal nicotine exposure may contribute to the pathogenesis of sudden infant death syndrome (SIDS), we discuss the effects of perinatal nicotine exposure on development of the cholinergic and other neurotransmitter systems involved in control of breathing. Advances in understanding of the mechanisms underlying central cholinergic/nicotinic modulation of respiration provide a pharmacological basis for exploiting nAChRs as therapeutic targets for neurological disorders related to neural control of breathing such as sleep apnea and SIDS.
PMCID: PMC4002383  PMID: 19498418
respiratory control; nicotinic acetylcholine receptors; preBötzinger complex; inspiratory neuron; hypoglossal nucleus; phrenic nucleus; motoneuron; perinatal nicotine exposure
23.  Silencing preBötzinger Complex somatostatin-expressing neurons induces persistent apnea in awake rat 
Nature neuroscience  2008;11(5):538-540.
Delineating neurons that underlie complex behaviors is of fundamental interest. Using adeno-associated virus 2, we expressed the Drosophila allatostatin receptor in somatostatin (Sst)-expressing neurons in the preBötzinger Complex (preBötC). Rapid silencing of these neurons in awake rats induced a persistent apnea without any respiratory movements to rescue their breathing. We hypothesize that breathing requires preBötC Sst neurons and that their sudden depression can lead to serious, even fatal, respiratory failure.
PMCID: PMC2515565  PMID: 18391943
24.  Efficient measurement of endogenous neurotransmitters in small localized regions of central nervous systems in vitro with HPLC 
Journal of neuroscience methods  2006;160(2):256-263.
High performance liquid chromatography (HPLC) is widely used to determine neurotransmitter concentrations in the central nervous system (CNS). Finding the optimal methods to sample from CNS tissue poses a challenge for neuroscientists. Here we describe a method that allows assay of neurotransmitters (or other chemicals) in small regions (down to 180 μm in diameter) in in vitro preparations concurrently with electrophysiological recordings. The efficiency for measuring small amounts of chemicals is enhanced by a sample collecting pipette with filter paper at the tip that makes close contact with the target region in CNS tissue. With a wire plunger in the calibrated pipette controlled by a microsyringe pump, there is virtually no dead volume. Samples in a volume of 10 μl (taken, e.g., at 2 μL/min over 5 minutes) can be injected into a HPLC machine with microbore columns. We demonstrate the effectiveness of this method by measuring acetylcholine (ACh) in the ventral horn and its surrounding areas of the spinal cord in en bloc brainstem-spinal cord preparations. In control conditions, endogenous ACh levels in these regions were detectable. Application of neostigmine (an inhibitor of acetylcholinesterases (AChEs)) increased ACh concentrations, and at the same time, induced tonic/seizure-like activity in efferent motor output recorded from cervical ventral nerve roots. Higher ACh concentrations in the ventral horn were differentiated from nearby regions: the lateral and midline aspects of the ventral spinal cord. In addition, ACh in the preBötzinger Complex (preBötC) and the hypoglossal nucleus in medullary slice preparations can also be measured. Our results indicate that the method proposed in this study can be used to measure neurotransmitters in small and localized CNS regions. Correlation between changes in neurotransmitters in target regions and the neuronal activities can be revealed in vitro. Our data also suggest that there is endogenous ACh release in spinal ventral motor columns at 4th cervical (C4) level that regulates the respiratory-related motor activity.
PMCID: PMC2441908  PMID: 17092561
Neurotransmitter measurement; HPLC; Acetylcholine; ventral motor columns of the spinal cord; preBötzinger Complex; brain slice; in vitro; respiratory modulation

Results 1-24 (24)