Melissa B. Coates

Control of breathing by raphe obscurus serotonergic neurons in mice

We used optogenetics to determine the global respiratory effects produced by selectively stimulating raphe obscurus (RO) serotonergic neurons in anesthetized mice and to test whether these neurons detect changes in the partial pressure of CO2, and hence function as central respiratory chemoreceptors. Channelrhodopsin-2 (ChR2) was selectively (∼97%) incorporated into ∼50% of RO serotonergic neurons by injecting AAV2 DIO ChR2-mCherry (adeno-associated viral vector double-floxed inverse open reading frame of ChR2-mCherry) into the RO of ePet-Cre mice. The transfected neurons heavily innervated lower brainstem and spinal cord regions involved in autonomic and somatic motor control plus breathing but eschewed sensory related regions. Pulsed laser photostimulation of ChR2-transfected serotonergic neurons increased respiratory frequency (fR) and diaphragmatic EMG (dEMG) amplitude in relation to the duration and frequency of the light pulses (half saturation, 1 ms; 5–10 Hz). dEMG amplitude and fR increased slowly (half saturation after 10–15 s) and relaxed monoexponentially (tau, 13–15 s). The breathing stimulation was reduced ∼55% by methysergide (broad spectrum serotonin antagonist) and potentiated (∼16%) at elevated levels of inspired CO2 (8%). RO serotonergic neurons, identified by their entrainment to short light pulses (threshold, 0.1–1 ms) were silent (nine cells) or had a low and regular level of activity (2.1 ± 0.4 Hz; 11 cells) that was not synchronized with respiration. These and nine surrounding neurons with similar characteristics were unaffected by adding up to 10% CO2 to the breathing mixture. In conclusion, RO serotonergic neurons activate breathing frequency and amplitude and potentiate the central respiratory chemoreflex but do not appear to have a central respiratory chemoreceptor function.

Phox2b-Expressing Neurons of the Parafacial Region Regulate Breathing Rate, Inspiration, and Expiration in Conscious Rats

The retrotrapezoid nucleus contains Phox2b-expressing glutamatergic neurons (RTN-Phox2b neurons) that regulate breathing in a CO2-dependent manner. Here we use channelrhodopsin-based optogenetics to explore how these neurons control breathing in conscious and anesthetized adult rats. Respiratory entrainment (pacing) of breathing frequency (fR) was produced over 57% (anesthetized) and 28% (conscious) of the natural frequency range by burst activation of RTN-Phox2b neurons (3–8 × 0.5–20 ms pulses at 20 Hz). In conscious rats, pacing under normocapnic conditions increased tidal volume (VT) and each inspiration was preceded by active expiration, denoting abdominal muscle contraction. During long-term pacing VT returned to prestimulation levels, suggesting that central chemoreceptors such as RTN-Phox2b neurons regulate VT partly independently of their effect on fR. Randomly applied light trains reset the respiratory rhythm and shortened the expiratory phase when the stimulus coincided with late-inspiration or early-expiration. Importantly, continuous (20 Hz) photostimulation of the RTN-Phox2b neurons and a saturating CO2 concentration produced similar effects on breathing that were much larger than those elicited by phasic RTN stimulation. In sum, consistent with their anatomical projections, RTN-Phox2b neurons regulate lung ventilation by controlling breathing frequency, inspiration, and active expiration. Adult RTN-Phox2b neurons can entrain the respiratory rhythm if their discharge is artificially synchronized, but continuous activation of these neurons is much more effective at increasing lung ventilation. These results suggest that RTN-Phox2b neurons are no longer rhythmogenic in adulthood and that their average discharge rate may be far more important than their discharge pattern in driving lung ventilation.

Selective Optogenetic Activation of Rostral Ventrolateral Medullary Catecholaminergic Neurons Produces Cardiorespiratory Stimulation in Conscious Mice

Activation of rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons e.g., by hypoxia is thought to increase sympathetic outflow thereby raising blood pressure (BP). Here we test whether these neurons also regulate breathing and cardiovascular variables other than BP. Selective expression of ChR2-mCherry by RVLM-CA neurons was achieved by injecting Cre-dependent vector AAV2-EF1α-DIO-ChR2-mCherry unilaterally into the brainstem of dopamine-β-hydroxylaseCre/0 mice. Photostimulation of RVLM-CA neurons increased breathing in anesthetized and conscious mice. In conscious mice, photostimulation primarily increased breathing frequency and this effect was fully occluded by hypoxia (10% O2). In contrast, the effects of photostimulation were largely unaffected by hypercapnia (3 and 6% CO2). The associated cardiovascular effects were complex (slight bradycardia and hypotension) and, using selective autonomic blockers, could be explained by coactivation of the sympathetic and cardiovagal outflows. ChR2-positive RVLM-CA neurons expressed VGLUT2 and their projections were mapped. Their complex cardiorespiratory effects are presumably mediated by their extensive projections to supraspinal sites such as the ventrolateral medulla, the dorsal vagal complex, the dorsolateral pons, and selected hypothalamic nuclei (dorsomedial, lateral, and paraventricular nuclei). In sum, selective optogenetic activation of RVLM-CA neurons in conscious mice revealed two important novel functions of these neurons, namely breathing stimulation and cardiovagal outflow control, effects that are attenuated or absent under anesthesia and are presumably mediated by the numerous supraspinal projections of these neurons. The results also suggest that RVLM-CA neurons may underlie some of the acute respiratory response elicited by carotid body stimulation but contribute little to the central respiratory chemoreflex.

Pre-Botzinger Complex Receives Glutamatergic Innervation From Galaninergic and Other Retrotrapezoid Nucleus Neurons

The retrotrapezoid nucleus (RTN) contains CO2‐responsive neurons that regulate breathing frequency and amplitude. These neurons (RTN‐Phox2b neurons) contain the transcription factor Phox2b, vesicular glutamate transporter 2 (VGLUT2) mRNA, and a subset contains preprogalanin mRNA. We wished to determine whether the terminals of RTN‐Phox2b neurons contain galanin and VGLUT2 proteins, to identify the specific projections of the galaninergic subset, to test whether RTN‐Phox2b neurons contact neurons in the pre‐Bötzinger complex, and to identify the ultrastructure of these synapses. The axonal projections of RTN‐Phox2b neurons were traced by using biotinylated dextran amine (BDA), and many BDA‐ir boutons were found to contain galanin immunoreactivity. RTN galaninergic neurons had ipsilateral projections that were identical with those of this nucleus at large: the ventral respiratory column, the caudolateral nucleus of the solitary tract, and the pontine Kölliker‐Fuse, intertrigeminal region, and lateral parabrachial nucleus. For ultrastructural studies, RTN‐Phox2b neurons (galaninergic and others) were transfected with a lentiviral vector that expresses mCherry almost exclusively in Phox2b‐ir neurons. After spinal cord injections of a catecholamine neuron‐selective toxin, there was a depletion of C1 neurons in the RTN area; thus it was determined that the mCherry‐positive terminals located in the pre‐Bötzinger complex originated almost exclusively from the RTN‐Phox2b (non‐C1) neurons. These terminals were generally VGLUT2‐immunoreactive and formed numerous close appositions with neurokinin‐1 receptor‐ir pre‐Bötzinger complex neurons. Their boutons (n = 48) formed asymmetric synapses filled with small clear vesicles. In summary, RTN‐Phox2b neurons, including the galaninergic subset, selectively innervate the respiratory pattern generator plus a portion of the dorsolateral pons. RTN‐Phox2b neurons establish classic excitatory glutamatergic synapses with pre‐Bötzinger complex neurons presumed to generate the respiratory rhythm. J. Comp. Neurol. 520:1047–1061, 2012.

C1 neurons excite locus coeruleus and A5 noradrenergic neurons along with sympathetic outflow in rats

•  C1 neurons activate sympathetic tone and stimulate the hypothalamic–pituitary–adrenal axis in circumstances such as pain, hypoxia or hypotension. •  C1 neurons innervate pontine noradrenergic cell groups, including the locus coeruleus (LC) and A5. •  In this study, using an optogenetic approach in anaesthetized rats, we show that C1 neurons form excitatory synapses with LC neurons and that selective stimulation of C1 neurons activates LC and A5 neurons. •  These results show that the C1 neurons activate pontine noradrenergic neurons through the release of glutamate. This effect may be important in the arousal‐promoting effects of hypoxia and pain.

Optogenetic Stimulation of Adrenergic C1 Neurons Causes Sleep State–Dependent Cardiorespiratory Stimulation and Arousal with Sighs in Rats

RATIONALE The rostral ventrolateral medulla (RVLM) contains central respiratory chemoreceptors (retrotrapezoid nucleus, RTN) and the sympathoexcitatory, hypoxia-responsive C1 neurons. Simultaneous optogenetic stimulation of these neurons produces vigorous cardiorespiratory stimulation, sighing, and arousal from non-REM sleep. OBJECTIVES To identify the effects that result from selectively stimulating C1 cells. METHODS A Cre-dependent vector expressing channelrhodopsin 2 (ChR2) fused with enhanced yellow fluorescent protein or mCherry was injected into the RVLM of tyrosine hydroxylase (TH)-Cre rats. The response of ChR2-transduced neurons to light was examined in anesthetized rats. ChR2-transduced C1 neurons were photoactivated in conscious rats while EEG, neck muscle EMG, blood pressure (BP), and breathing were recorded. MEASUREMENTS AND MAIN RESULTS Most ChR2-expressing neurons (95%) contained C1 neuron markers and innervated the spinal cord. RTN neurons were not transduced. While the rats were under anesthesia, the C1 cells were faithfully activated by each light pulse up to 40 Hz. During quiet resting and non-REM sleep, C1 cell stimulation (20 s, 2-20 Hz) increased BP and respiratory frequency and produced sighs and arousal from non-REM sleep. Arousal was frequency-dependent (85% probability at 20 Hz). Stimulation during REM sleep increased BP, but had no effect on EEG or breathing. C1 cell-mediated breathing stimulation was occluded by hypoxia (12% FIO2), but was unchanged by 6% FiCO2. CONCLUSIONS C1 cell stimulation reproduces most effects of acute hypoxia, specifically cardiorespiratory stimulation, sighs, and arousal. C1 cell activation likely contributes to the sleep disruption and adverse autonomic consequences of sleep apnea. During hypoxia (awake) or REM sleep, C1 cell stimulation increases BP but no longer stimulates breathing.

Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus

The C1 neurons are a nodal point for blood pressure control and other autonomic responses. Here we test whether these rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons use glutamate as a transmitter in the dorsal motor nucleus of the vagus (DMV). After injecting Cre-dependent adeno-associated virus (AAV2) DIO-Ef1α-channelrhodopsin2(ChR2)-mCherry (AAV2) into the RVLM of dopamine-β-hydroxylase Cre transgenic mice (DβHCre/0), mCherry was detected exclusively in RVLM-CA neurons. Within the DMV >95% mCherry-immunoreactive(ir) axonal varicosities were tyrosine hydroxylase (TH)-ir and the same proportion were vesicular glutamate transporter 2 (VGLUT2)-ir. VGLUT2-mCherry colocalization was virtually absent when AAV2 was injected into the RVLM of DβHCre/0;VGLUT2flox/flox mice, into the caudal VLM (A1 noradrenergic neuron-rich region) of DβHCre/0 mice or into the raphe of ePetCre/0 mice. Following injection of AAV2 into RVLM of TH-Cre rats, phenylethanolamine N-methyl transferase and VGLUT2 immunoreactivities were highly colocalized in DMV within EYFP-positive or EYFP-negative axonal varicosities. Ultrastructurally, mCherry terminals from RVLM-CA neurons in DβHCre/0 mice made predominantly asymmetric synapses with choline acetyl-transferase-ir DMV neurons. Photostimulation of ChR2-positive axons in DβHCre/0 mouse brain slices produced EPSCs in 71% of tested DMV preganglionic neurons (PGNs) but no IPSCs. Photostimulation (20 Hz) activated PGNs up to 8 spikes/s (current-clamp). EPSCs were eliminated by tetrodotoxin, reinstated by 4-aminopyridine, and blocked by ionotropic glutamate receptor blockers. In conclusion, VGLUT2 is expressed by RVLM-CA (C1) neurons in rats and mice regardless of the presence of AAV2, the C1 neurons activate DMV parasympathetic PGNs monosynaptically and this connection uses glutamate as an ionotropic transmitter.

Optogenetic Stimulation of C1 and Retrotrapezoid Nucleus Neurons Causes Sleep State–Dependent Cardiorespiratory Stimulation and Arousal in Rats

C1 catecholaminergic neurons and neurons of the retrotrapezoid nucleus are integrative nodes within the brain stem network regulating cardiorespiratory reflexes elicited by hypoxia and hypercapnia, stimuli that also produce arousal from sleep. In the present study, Channelrhodopsin-2 was selectively introduced into these neurons with a lentiviral vector to determine whether their selective activation also produces arousal in sleeping rats. Sleep stages were identified from electroencephalographic and neck muscle electromyographic recordings. Breathing was measured using unrestrained whole body plethysmography and blood pressure by telemetry. During nonrapid eye movement sleep, unilateral photostimulation of the C1 region caused arousal in 83.0±14.7% of trials and immediate and intense cardiorespiratory activation. Arousal during photostimulation was also observed during rapid eye movement sleep (41.9±5.6% of trials), but less reliably than during nonrapid eye movement sleep. The cardiorespiratory responses elicited by photostimulation were dramatically smaller during rapid eye movement sleep than nonrapid eye movement sleep or wakefulness. Systemic &agr;1-adrenoreceptor blockade reduced the cardiorespiratory effects of photostimulation but had no effect on the arousal caused by photostimulation during nonrapid eye movement sleep. Postmortem histology showed that neurons expressing Channelrhodopsin 2–mCherry were predominantly catecholaminergic (81%). These results show that selective activation of C1 and retrotrapezoid nucleus neurons produces state-dependent arousal and cardiorespiratory stimulation. These neurons, which are powerfully activated by chemoreceptor stimulation, may contribute to the sleep disruption associated with obstructive sleep apnea.

The orexinergic neurons receive synaptic input from C1 cells in rats.

The C1 cells, located in the rostral ventrolateral medulla (RVLM), are activated by pain, hypoxia, hypoglycemia, infection, and hypotension and elicit cardiorespiratory stimulation, adrenaline and adrenocorticotropic hormone (ACTH) release, and arousal. The orexin neurons contribute to the autonomic responses to acute psychological stress. Here, using an anatomical approach, we consider whether the orexin neurons could also be contributing to the autonomic effects elicited by C1 neuron activation. Phenylethanolamine N‐methyl transferase‐immunoreactive (PNMT‐ir) axons were detected among orexin‐ir somata, and close appositions between PNMT‐ir axonal varicosities and orexin‐ir profiles were observed. The existence of synapses between PNMT‐ir boutons labeled with diaminobenzidine and orexinergic neurons labeled with immunogold was confirmed by electron microscopy. We labeled RVLM neurons with a lentiviral vector that expresses the fusion protein ChR2‐mCherry under the control of the catecholaminergic neuron‐selective promoter PRSx8 and obtained light and ultrastructural evidence that these neurons innervate the orexin cells. By using a Cre‐dependent adeno‐associated vector and TH‐Cre rats, we confirmed that the projection from RVLM catecholaminergic neurons to the orexinergic neurons originates predominantly from PNMT‐ir catecholaminergic (i.e., C1 cells). The C1 neurons were found to establish predominantly asymmetric synapses with orexin‐ir cell bodies or dendrites. These synapses were packed with small clear vesicles and also contained dense‐core vesicles. In summary, the orexin neurons are among the hypothalamic neurons contacted and presumably excited by the C1 cells. The C1–orexin neuronal connection is probably one of several suprabulbar pathways through which the C1 neurons activate breathing and the circulation, raise blood glucose, and facilitate arousal from sleep. J. Comp. Neurol. 522:3834–3846, 2014.