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.

Delineating neurons that underlie complex behaviors is of fundamental interest. Using adenoassociated 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.The preBötC in the ventrolateral medulla is hypothesized to be a kernel for the generation of respiratory rhythm in vitro and in vivo 1-4 . Adult rats with slow (~days), toxin-induced neurodegeneration of > 80% of neurokinin 1 receptor (NK1R)-expressing preBötC neurons survive with an ataxic rhythm during wakefulness and apnea during sleep 1,5 . Whether this pathological breathing pattern is driven by neurons that normally control respiratory-related muscles, including those underlying volitional or emotional behaviors, or by a compensatory reorganization in response to the slow neurodegeneration is unknown. To eliminate adaptation resulting from slow lesions, we rapidly (~minutes) decreased excitability in a glutamatergic subpopulation of preBötC neurons that express Sst 6 . This population overlaps with neurons expressing NK1R; 28 ± 2% of preBötC Sst neurons expressed NK1R and 41 ± 1% of preBötC NK1R neurons expressed Sst (5-6-week-old rats, n = 3; Supplementary Fig. 1 online), consistent with their overlap in neonatal rat preBötC 7 . We hypothesized that preBötC Sst neurons are essential for normal breathing and predicted that silencing these neurons would cause acute apnea in awake adult rats; as hypoxia and hypercapnea worsened with apnea, we presumed that other mechanisms, particularly in relation to volitional or emotional drives, would restore breathing, as appears to be the case in central congenital hypoventilation syndrome 8 . Rapid, reversible silencing of genotypic neuronal subpopulations can illuminate their role in behavior. This can be done by targeted expression of allatostatin receptor (AlstR), a G protein-coupled receptor that is neither expressed nor activated by any endogenous ligand in mammals 9-11 . Mammalian cortical and spinal cord neurons that are made to express AlstR can be rapidly and reversibly inactivated in vitro 12,13 and in vivo under anesthesia 10 by administration of allatostatin, which opens K + channels 9,13 to hyperpolarize them. WeCorrespondence should be addressed to J.L.F. (feldman@ucla.edu). 3 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Neuroscience website. expressed AlstR and enhanced green fluorescent protein (EGFP) in targeted preBötC neurons and studied the effect of allatostatin application on breathing in adult rats. NIH Public AccessTo obtain stable and reliable expression of exogenous genes in adult rat preBötC neurons, we used adeno-associated virus 2 (AAV2) to ensure high infecti...

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.

Acetylcholine and nicotine can modulate respiratory patterns by acting on nicotinic acetylcholine receptors (nAChRs) in the preBötz-inger 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 (L9ЈA) of the nAChR ␣4 subunit; this mutation renders ␣4-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 ϳ100-fold lower in the homozygous L9ЈA 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 L9ЈA mice, effects similar to those induced by 1 M nicotine in wild-type mice. In L9ЈA 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 ␣4*-selective nAChR antagonist dihydro-␤-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 ␣4* nAChRs are present in the preBötC. They mediate cholinergic/nicotinic modulation of the excitability of preBötC inspiratory neurons and of respiratory rhythm. Furthermore, functional ␣4* nAChRs are present in XII nucleus and mediate cholinergic/nicotinic modulation of tonic activity in XIIn.

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.