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.

Physiological homeostasis is essential for organism survival. Highly responsive neuronal networks are involved but constituent neurons are just beginning to be resolved. To query brain serotonergic neurons in homeostasis, we used a synthetic GPCR (Di)-based neuronal silencing tool, mouse RC∷FPDi, designed for cell type-specific, ligand (clozapine-N-oxide, CNO)-inducible and reversible suppression of action potential firing. In mice harboring Di-expressing serotonergic neurons, CNO administration by systemic injection attenuated the chemoreflex that normally increases respiration in response to tissue CO2 elevation and acidosis. At the cellular level, CNO suppressed firing rate increases evoked by CO2/acidosis. Body thermoregulation at room temperature was also disrupted following CNO triggering of Di; core temperatures plummeted, then recovered. This work establishes that serotonergic neurons regulate life-sustaining respiratory and thermoregulatory networks, and demonstrates a noninvasive tool for mapping neuron function.

The sudden infant death syndrome (SIDS) is the sudden death of an infant under one year of age that is typically associated with sleep and that remains unexplained after a complete autopsy and death scene investigation. A leading hypothesis about its pathogenesis is that many cases result from defects in brainstem-mediated protective responses to homeostatic stressors occurring during sleep in a critical developmental period. Here we review the evidence for the brainstem hypothesis in SIDS with a focus upon abnormalities related to the neurotransmitter serotonin in the medulla oblongata, as these are the most robust pathologic findings to date. In this context, we synthesize the human autopsy data with genetic, whole-animal, and cellular data concerning the function and development of the medullary serotonergic system. These emerging data suggest an important underlying mechanism in SIDS that may help lead to identification of infants at risk and specific interventions to prevent death.

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Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN), located in the ventrolateral brainstem, are sensitive to changes in PCO 2 /pH, have excitatory projections to the central respiratory rhythm/pattern generator, and their activation enhances central respiratory drive. Using in vivo (conscious and anesthetized rats) and in situ (arterially perfused rat brainstem-spinal cord preparations) models, we evaluated the functional significance of this neuronal population for both resting respiratory activity and the CO 2 -evoked respiratory responses by reversibly inhibiting these neurons using the insect peptide allatostatin following transduction with a lentiviral construct to express the G-protein-coupled Drosophila allatostatin receptor. Selective inhibition of the Phox2b-expressing neurons in the ventrolateral brainstem, including the RTN, using allatostatin was without effect on resting respiratory activity in conscious rats, but decreased the amplitude of the phrenic nerve discharge in anesthetized rats and the in situ rat preparations. Postinspiratory activity was also reduced in situ. In the absence or presence of the peripheral chemoreceptor input, inhibiting the Phox2b-expressing neurons during hypercapnia abolished the CO 2 -evoked abdominal expiratory activity in anesthetized rats and in situ preparations. Inspiratory responses evoked by rising levels of CO 2 in the breathing air were also reduced in anesthetized rats with denervated carotid bodies and conscious rats with peripheral chemoreceptors intact (by 28% and 60%, respectively). These data indicate a crucial dependence of central expiratory drive upon Phox2b-expressing neurons of the ventrolateral brainstem and support the hypothesis that these neurons contribute in a significant manner to CO 2 -evoked increases of inspiratory activity.

We produced local tissue acidosis in various brain stem regions with 1-nl injections of acetazolamide (AZ) to locate the sites of central chemoreception. To determine whether the local acidosis resulted in a stimulation of breathing, we performed the experiment in chloralose-urethan anesthetized vagotomized carotid-denervated (cats) paralyzed servo-ventilated cats and rats and measured phrenic nerve activity (PNA) as the response index. Measurements of extracellular brain tissue pH by glass microelectrodes showed that AZ injections induced a change in pH at the injection center equivalent to that produced by an increase in end-tidal PCO2 of approximately 36 Torr and that the change in brain pH was limited to a tissue volume with a radius of < 350 microns. We found AZ injections sites that caused a significant increase in PNA to be located 1) within 800 microns of the ventrolateral medullary surface at locations within traditional rostral and caudal chemosensitive areas and the intermediate area, 2) within the vicinity of the nucleus tractus solitarii, and 3) within the vicinity of the locus coeruleus. Single AZ injections produced increases in PNA that were < or = 69% of the maximum value observed with an increase in end-tidal PCO2. We conclude that central chemoreceptors are distributed at many locations within the brain stem, all within 1.5 mm of the surface, and that stimulation of a small fraction of all central chemoreceptors can result in a large ventilatory response.

Neurokinin-1 receptor (NK1R)-expressing neurones that are involved in chemoreception at the retrotrapezoid nucleus (Nattie & Li, 2002b) are also prominent at locations that contain medullary serotonergic neurones, which are chemosensitive in vitro. In medullary regions containing both types, we evaluated their role in central chemoreception by specific cell killing. We injected (2 × 100 nl) (a) substance P-saporin (SP-SAP; 1 µM) to kill NK1R-expressing neurones, (b) a novel conjugate of a monoclonal antibody to the serotonin transporter (SERT) and saporin (anti-SERT-SAP; 1 µM) to kill serotonergic neurones, or (c) SP-SAP and anti-SERT-SAP together to kill both types. Controls received IgG-SAP injections (1 µM). There was no double-labelling of NK1R-immunoreactive (ir) and tryptophan-hydroxylase (TPOH)-ir neurones. Cell (somatic profile) counts showed that NK1R-ir neurones in the SP-SAP group were reduced by 31%; TPOH-ir neurones in the anti-SERT-SAP group by 28%; and NK1R-ir and TPOH-ir neurones, respectively, in the combined lesion group by 55% and 31% (P < 0.001; two-way ANOVA; P < 0.05, Tukey's post hoc test). The treatments had no significant effect on sleep/wake time, body temperature, or oxygen consumption but all three reduced the ventilatory response to 7% inspired CO 2 in wakefulness and sleep by a similar amount. SP-SAP treatment decreased the averaged CO 2 responses (3, 7 and 14 days after lesions) in wakefulness and sleep by 21% and 16%, anti-SERT-SAP decreased the responses by 15% and 18%, and the combinedtreatmentdecreasedtheresponsesby12%and12%(P <0.001;two-wayANOVA;P < 0.05, Tukey's post hoc test). We conclude that separate populations of serotonergic and adjacent NK1R-expressing neurones in the medulla are both involved in central chemoreception in vivo. An increase in CO 2 or H + in blood or brain stimulates breathing. This occurs largely by means of central chemoreceptors, which are widely distributed within the brainstem (Loeschcke, 1982;Coates et al. 1993;Forster et al. 1997;Li et al. 1999;Nattie, 1998Nattie, , 1999Nattie, , 2000Nattie, , 2001Ballantyne & Scheid, 2001;H. Wang et al. 2001;, 2002aRicherson et al. 2001;Okada et al. 2002;Ribas-Salgueiro et al. 2003;Feldman et al. 2003). Central chemoreceptor sites have been identified by breathing responses to focal acidic stimulation in vivo. They include; (a) the caudal nucleus tractus solitarius (NTS) (b) the locus coeruleus (LC) (c) the rostral aspect of the ventral respiratory group (d) regions lying just beneath the ventral medullary surface in rostral (the RTN and adjacent parapyramidal and marginal glial regions) and caudal locations, and (e) the medullary serotonergic cell group, which is the subject of this study.To study the function of single chemoreceptor sites in an unanaesthetized in vivo model we have applied a dual strategy. We examine the effects of (1) focal acidosis on breathing, or (2) focal cell specific lesions on the response to systemically applied CO 2 , the CO 2 response. Focal CO 2 stimulation at the retrotrapezoid nu...

All medullary central chemoreceptor sites contain neurokinin‐1 receptor immunoreactivity (NK1R‐ir). We ask if NK1R‐ir neurons and processes are involved in chemoreception. At one site, the retrotrapezoid nucleus/parapyramidal region (RTN/Ppy), we injected a substance P‐saporin conjugate (SP‐SAP; 0.1 pmol in 100 nl) to kill NK1R‐ir neurons specifically, or SAP alone as a control. We made measurements for 15 days after the injections in two groups of rats. In group 1, with unilateral injections made in the awake state via a pre‐implanted guide cannula, we compared responses within rats using initial baseline data. In group 2, with bilateral injections made under anaesthesia at surgery, we compared responses between SP‐SAP‐ and SAP‐treated rats. SP‐SAP treatment reduced the volume of the RTN/Ppy region that contained NK1R‐ir neuronal somata and processes by 44 % (group 1) and by 47 and 40 % on each side, respectively (group 2). Ventilation (V̇E) and tidal volume (VT) were decreased during air breathing in sleep and wakefulness (group 2; P < 0.001; two‐way ANOVA) and Pa,CO2 was increased (group 2; P < 0.05; Student's t test). When rats breathed an air mixture containing 7 % CO2 during sleep and wakefulness, V̇E and VT were lower (groups 1 and 2; P < 0.001; ANOVA) and the ΔV̇E in air containing 7 % CO2 compared to air was decreased by 28‐30 % (group 1) and 17‐22 % (group 2). SP‐SAP‐treated rats also slept less during air breathing. We conclude that neurons with NK1R‐ir somata or processes in the RTN/Ppy region are either chemosensitive or they modulate chemosensitivity. They also provide a tonic drive to breathe and may affect arousal.