Publications by authors named "Stephen B G Abbott"

33 Publications

TRPM4 mediates a subthreshold membrane potential oscillation in respiratory chemoreceptor neurons that drives pacemaker firing and breathing.

Cell Rep 2021 Feb;34(5):108714

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA. Electronic address:

Brainstem networks that control regular tidal breathing depend on excitatory drive, including from tonically active, CO/H-sensitive neurons of the retrotrapezoid nucleus (RTN). Here, we examine intrinsic ionic mechanisms underlying the metronomic firing activity characteristic of RTN neurons. In mouse brainstem slices, large-amplitude membrane potential oscillations are evident in synaptically isolated RTN neurons after blocking action potentials. The voltage-dependent oscillations are abolished by sodium replacement; blocking calcium channels (primarily L-type); chelating intracellular Ca; and inhibiting TRPM4, a Ca-dependent cationic channel. Likewise, oscillation voltage waveform currents are sensitive to calcium and TRPM4 channel blockers. Extracellular acidification and serotonin (5-HT) evoke membrane depolarization that augments TRPM4-dependent oscillatory activity and action potential discharge. Finally, inhibition of TRPM4 channels in the RTN of anesthetized mice reduces central respiratory output. These data implicate TRPM4 in a subthreshold oscillation that supports the pacemaker-like firing of RTN neurons required for basal, CO-stimulated, and state-dependent breathing.
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http://dx.doi.org/10.1016/j.celrep.2021.108714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888550PMC
February 2021

Differential Contribution of the Retrotrapezoid Nucleus and C1 Neurons to Active Expiration and Arousal in Rats.

J Neurosci 2020 11 24;40(45):8683-8697. Epub 2020 Sep 24.

Department of Pharmacology, University of Virginia, Charlottesville, Virginia 29908

Collectively, the retrotrapezoid nucleus (RTN) and adjacent C1 neurons regulate breathing, circulation and the state of vigilance, but previous methods to manipulate the activity of these neurons have been insufficiently selective to parse out their relative roles. We hypothesize that RTN and C1 neurons regulate distinct aspects of breathing (e.g., frequency, amplitude, active expiration, sighing) and differ in their ability to produce arousal from sleep. Here we use optogenetics and a combination of viral vectors in adult male and female -Cre rats to transduce selectively RTN (Phox2b) or C1 neurons (Phox2b/) with Channelrhodopsin-2. RTN photostimulation modestly increased the probability of arousal. RTN stimulation robustly increased breathing frequency and amplitude; it also triggered strong active expiration but not sighs. Consistent with these responses, RTN innervates the entire pontomedullary respiratory network, including expiratory premotor neurons in the caudal ventral respiratory group, but RTN has very limited projections to brainstem regions that regulate arousal (locus ceruleus, CGRP parabrachial neurons). C1 neuron stimulation produced robust arousals and similar increases in breathing frequency and amplitude compared with RTN stimulation, but sighs were elicited and active expiration was absent. Unlike RTN, C1 neurons innervate the locus ceruleus, CGRP processes within the parabrachial complex, and lack projections to caudal ventral respiratory group. In sum, stimulating C1 or RTN activates breathing robustly, but only RTN neuron stimulation produces active expiration, consistent with their role as central respiratory chemoreceptors. Conversely, C1 stimulation strongly stimulates ascending arousal systems and sighs, consistent with their postulated role in acute stress responses. The C1 neurons and the retrotrapezoid nucleus (RTN) reside in the rostral ventrolateral medulla. Both regulate breathing and the cardiovascular system but in ways that are unclear because of technical limitations (anesthesia, nonselective neuronal actuators). Using optogenetics in unanesthetized rats, we found that selective stimulation of either RTN or C1 neurons activates breathing. However, only RTN triggers active expiration, presumably because RTN, unlike C1, has direct excitatory projections to abdominal premotor neurons. The arousal potential of the C1 neurons is far greater than that of the RTN, however, consistent with C1's projections to brainstem wake-promoting structures. In short, C1 neurons orchestrate cardiorespiratory and arousal responses to somatic stresses, whereas RTN selectively controls lung ventilation and arterial Pco stability.
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http://dx.doi.org/10.1523/JNEUROSCI.1006-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643293PMC
November 2020

Neuronal Networks in Hypertension: Recent Advances.

Hypertension 2020 Aug 29;76(2):300-311. Epub 2020 Jun 29.

Department of Chemical Physiology and Biochemistry, Oregon Health & Sciences University, Portland (V.L.B.).

Neurogenic hypertension is associated with excessive sympathetic nerve activity to the kidneys and portions of the cardiovascular system. Here we examine the brain regions that cause heightened sympathetic nerve activity in animal models of neurogenic hypertension, and we discuss the triggers responsible for the changes in neuronal activity within these regions. We highlight the limitations of the evidence and, whenever possible, we briefly address the pertinence of the findings to human hypertension. The arterial baroreflex reduces arterial blood pressure variability and contributes to the arterial blood pressure set point. This set point can also be elevated by a newly described cerebral blood flow-dependent and astrocyte-mediated sympathetic reflex. Both reflexes converge on the presympathetic neurons of the rostral medulla oblongata, and both are plausible causes of neurogenic hypertension. Sensory afferent dysfunction (reduced baroreceptor activity, increased renal, or carotid body afferent) contributes to many forms of neurogenic hypertension. Neurogenic hypertension can also result from activation of brain nuclei or sensory afferents by excess circulating hormones (leptin, insulin, Ang II [angiotensin II]) or sodium. Leptin raises blood vessel sympathetic nerve activity by activating the carotid bodies and subsets of arcuate neurons. Ang II works in the lamina terminalis and probably throughout the brain stem and hypothalamus. Sodium is sensed primarily in the lamina terminalis. Regardless of its cause, the excess sympathetic nerve activity is mediated to some extent by activation of presympathetic neurons located in the rostral ventrolateral medulla or the paraventricular nucleus of the hypothalamus. Increased activity of the orexinergic neurons also contributes to hypertension in selected models.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.14521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347452PMC
August 2020

EP3R-Expressing Glutamatergic Preoptic Neurons Mediate Inflammatory Fever.

J Neurosci 2020 03 20;40(12):2573-2588. Epub 2020 Feb 20.

Department of Neurology, Division of Sleep Medicine, and Program in Neuroscience, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, and

Fever is a common phenomenon during infection or inflammatory conditions. This stereotypic rise in body temperature (Tb) in response to inflammatory stimuli is a result of autonomic responses triggered by prostaglandin E2 action on EP3 receptors expressed by neurons in the median preoptic nucleus (MnPO neurons). To investigate the identity of MnPO neurons, we first used hybridization to show coexpression of EP3R and the VGluT2 transporter in MnPO neurons. Retrograde tracing showed extensive direct projections from MnPO but few from MnPO neurons to a key site for fever production, the raphe pallidus. Ablation of MnPO but not MnPO neurons abolished fever responses but not changes in Tb induced by behavioral stress or thermal challenges. Finally, we crossed EP3R conditional knock-out mice with either VGluT2-IRES-cre or Vgat-IRES-cre mice and used both male and female mice to confirm that the neurons that express EP3R and mediate fever are glutamatergic, not GABAergic. This finding will require rethinking current concepts concerning the central thermoregulatory pathways based on the MnPO neurons being GABAergic. Body temperature is regulated by the CNS. The rise of the body temperature, or fever, is an important brain-orchestrated mechanism for fighting against infectious or inflammatory disease, and is tightly regulated by the neurons located in the median preoptic nucleus (MnPO). Here we demonstrate that excitatory MnPO neurons mediate fever and examine a potential central circuit underlying the development of fever responses.
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http://dx.doi.org/10.1523/JNEUROSCI.2887-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083539PMC
March 2020

Contribution of the Retrotrapezoid Nucleus and Carotid Bodies to Hypercapnia- and Hypoxia-induced Arousal from Sleep.

J Neurosci 2019 12 22;39(49):9725-9737. Epub 2019 Oct 22.

Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908.

The combination of hypoxia and hypercapnia during sleep produces arousal, which helps restore breathing and normalizes blood gases. Hypercapnia and hypoxia produce arousal in mammals by activating central (pH-sensitive) and peripheral (primarily O-sensitive) chemoreceptors. The relevant chemoreceptors and the neuronal circuits responsible for arousal are largely unknown. Here we examined the contribution of two lower brainstem nuclei that could be implicated in CO and hypoxia-induced arousal: the retrotrapezoid nucleus (RTN), a CO-responsive nucleus, which mediates the central respiratory chemoreflex; and the C1 neurons, which are hypoxia activated and produce arousal and blood pressure increases when directly stimulated. Additionally, we assessed the contribution of the carotid bodies (CBs), the main peripheral chemoreceptors in mammals, to hypoxia and CO-induced arousal. In unanesthetized male rats, we tested whether ablation of the RTN, CBs, or C1 neurons affects arousal from sleep and respiratory responses to hypercapnia or hypoxia. The sleep-wake pattern was monitored by EEG and neck EMG recordings and breathing by whole-body plethysmography. The latency to arousal in response to hypoxia or hypercapnia was determined along with changes in ventilation coincident with the arousal. RTN lesions impaired CO-induced arousal but had no effect on hypoxia-induced arousal. CB ablation impaired arousal to hypoxia and, to a lesser extent, hypercapnia. C1 neuron ablation had no effect on arousal. Thus, the RTN contributes to CO-induced arousal, whereas the CBs contribute to both hypoxia and CO-induced arousal. Asphyxia-induced arousal likely requires the combined activation of RTN, CBs and other central chemoreceptors. Hypercapnia and hypoxia during sleep elicit arousal, which facilitates airway clearing in the case of obstruction and reinstates normal breathing in the case of hypoventilation or apnea. Arousal can also be detrimental to health by interrupting sleep. We sought to clarify how CO and hypoxia cause arousal. We show that the retrotrapezoid nucleus, a brainstem nucleus that mediates the effect of brain acidification on breathing, also contributes to arousal elicited by CO but not hypoxia. We also show that the carotid bodies contribute predominantly to hypoxia-induced arousal. Lesions of the retrotrapezoid nucleus or carotid bodies attenuate, but do not eliminate, arousal to CO or hypoxia; therefore, we conclude that these structures are not the sole trigger of CO or hypoxia-induced arousal.
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http://dx.doi.org/10.1523/JNEUROSCI.1268-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891059PMC
December 2019

The Retrotrapezoid Nucleus: Central Chemoreceptor and Regulator of Breathing Automaticity.

Trends Neurosci 2019 11 18;42(11):807-824. Epub 2019 Oct 18.

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.

The ventral surface of the rostral medulla oblongata has been suspected since the 1960s to harbor central respiratory chemoreceptors [i.e., acid-activated neurons that regulate breathing to maintain a constant arterial PCO (PaCO)]. The key neurons, a.k.a. the retrotrapezoid nucleus (RTN), have now been identified. In this review we describe their transcriptome, developmental lineage, and anatomical projections. We also review their contribution to CO homeostasis and to the regulation of breathing automaticity during sleep and wake. Finally, we discuss several mechanisms that contribute to the activation of RTN neurons by COin vivo: cell-autonomous effects of protons; paracrine effects of pH mediated by surrounding astrocytes and blood vessels; and excitatory inputs from other CO-responsive CNS neurons.
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http://dx.doi.org/10.1016/j.tins.2019.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825900PMC
November 2019

Rostral Ventrolateral Medulla and Hypertension.

Hypertension 2018 09;72(3):559-566

From the Department of Pharmacology, University of Virginia, Charlottesville.

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http://dx.doi.org/10.1161/HYPERTENSIONAHA.118.10921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324579PMC
September 2018

A Glutamatergic Hypothalamomedullary Circuit Mediates Thermogenesis, but Not Heat Conservation, during Stress-Induced Hyperthermia.

Curr Biol 2018 07 12;28(14):2291-2301.e5. Epub 2018 Jul 12.

Department of Neurology, Beth Israel-Deaconess Medical Center, Harvard Medical School, Blackfan Circle, Boston, MA 02215, USA. Electronic address:

Stress elicits a variety of autonomic responses, including hyperthermia (stress fever) in humans and animals. In this present study, we investigated the circuit basis for thermogenesis and heat conservation during this response. We first demonstrated the glutamatergic identity of the dorsal hypothalamic area (DHA) neurons that innervate the raphe pallidus nucleus (RPa) to regulate core temperature (Tc) and mediate stress-induced hyperthermia. Then, using chemogenetic and optogenetic methods to manipulate this hypothalamomedullary circuit, we found that activation of DHA neurons potently drove an increase in Tc, but surprisingly, stress-induced hyperthermia was only reduced by about one-third when they were inhibited. Further investigation showed that DHA neurons activate brown adipose tissue (BAT) but do not cause vasoconstriction, instead allowing reflex tail artery vasodilation as a response to BAT-induced hyperthermia. Retrograde rabies virus tracing revealed projections from DHA neurons to RPa, but not to RPa neurons, and identified a set of inputs to DHA → RPa neurons that are likely to mediate BAT activation. The dissociation of the DHA thermogenic pathway from the thermoregulatory vasoconstriction (heat-conserving) pathway may explain stress flushing (skin vasodilation but a feeling of being too hot) during stressful times.
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http://dx.doi.org/10.1016/j.cub.2018.05.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085892PMC
July 2018

Role of the median preoptic nucleus in the autonomic response to heat-exposure.

Temperature (Austin) 2018 6;5(1):4-6. Epub 2018 Feb 6.

Department of Neurology, Beth Israel-Deaconess Medical Center - Harvard Medical School, Boston, MA, USA.

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http://dx.doi.org/10.1080/23328940.2017.1413155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902207PMC
February 2018

Breathing regulation and blood gas homeostasis after near complete lesions of the retrotrapezoid nucleus in adult rats.

J Physiol 2018 07;596(13):2521-2545

Department of Pharmacology, University of Virginia, Charlottesville, VA, USA.

Key Points: The retrotrapezoid nucleus (RTN) drives breathing proportionally to brain PCO2 but its role during various states of vigilance needs clarification. Under normoxia, RTN lesions increased the arterial PCO2 set-point, lowered the PO2 set-point and reduced alveolar ventilation relative to CO production. Tidal volume was reduced and breathing frequency increased to a comparable degree during wake, slow-wave sleep and REM sleep. RTN lesions did not produce apnoeas or disordered breathing during sleep. RTN lesions in rats virtually eliminated the central respiratory chemoreflex (CRC) while preserving the cardiorespiratory responses to hypoxia; the relationship between CRC and number of surviving RTN Nmb neurons was an inverse exponential. The CRC does not function without the RTN. In the quasi-complete absence of the RTN and CRC, alveolar ventilation is reduced despite an increased drive to breathe from the carotid bodies.

Abstract: The retrotrapezoid nucleus (RTN) is one of several CNS nuclei that contribute, in various capacities (e.g. CO detection, neuronal modulation) to the central respiratory chemoreflex (CRC). Here we test how important the RTN is to PCO homeostasis and breathing during sleep or wake. RTN Nmb-positive neurons were killed with targeted microinjections of substance P-saporin conjugate in adult rats. Under normoxia, rats with large RTN lesions (92 ± 4% cell loss) had normal blood pressure and arterial pH but were hypoxic (-8 mmHg PaO ) and hypercapnic (+10 mmHg ). In resting conditions, minute volume (V ) was normal but breathing frequency (f ) was elevated and tidal volume (V ) reduced. Resting O consumption and CO production were normal. The hypercapnic ventilatory reflex in 65% FiO had an inverse exponential relationship with the number of surviving RTN neurons and was decreased by up to 92%. The hypoxic ventilatory reflex (HVR; FiO 21-10%) persisted after RTN lesions, hypoxia-induced sighing was normal and hypoxia-induced hypotension was reduced. In rats with RTN lesions, breathing was lowest during slow-wave sleep, especially under hyperoxia, but apnoeas and sleep-disordered breathing were not observed. In conclusion, near complete RTN destruction in rats virtually eliminates the CRC but the HVR persists and sighing and the state dependence of breathing are unchanged. Under normoxia, RTN lesions cause no change in V but alveolar ventilation is reduced by at least 21%, probably because of increased physiological dead volume. RTN lesions do not cause sleep apnoea during slow-wave sleep, even under hyperoxia.
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http://dx.doi.org/10.1113/JP275866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023841PMC
July 2018

Sympathoexcitation following intermittent hypoxia in rat is mediated by circulating angiotensin II acting at the carotid body and subfornical organ.

J Physiol 2018 08 19;596(15):3217-3232. Epub 2018 May 19.

Sydney Medical School, University of Sydney, Sydney, NSW, Australia.

Key Points: In anaesthetized rats, acute intermittent hypoxia increases sympathetic nerve activity, sympathetic peripheral chemoreflex sensitivity and central sympathetic-respiratory coupling. Renin-angiotensin system inhibition prevents the sympathetic effects of intermittent hypoxia, with intermittent injections of angiotensin II into the systemic circulation replicating these effects. Bilateral carotid body denervation reduces the sympathetic effects of acute intermittent hypoxia and eliminates the increases in chemoreflex sensitivity and sympathetic-respiratory coupling. Pharmacological inhibition of the subfornical organ also reduces the sympathetic effects of acute intermittent hypoxia, although it has no effect on the increases in chemoreflex sensitivity and central sympathetic-respiratory coupling. Combining both interventions eliminates the sympathetic effects of both intermittent hypoxia and angiotensin II.

Abstract: Circulating angiotensin II (Ang II) is vital for arterial pressure elevation following intermittent hypoxia in rats, although its importance in the induction of sympathetic changes is unclear. We tested the contribution of the renin-angiotensin system to the effects of acute intermittent hypoxia (AIH) in anaesthetized and ventilated rats. There was a 33.7 ± 2.9% increase in sympathetic nerve activity (SNA), while sympathetic chemoreflex sensitivity and central sympathetic-respiratory coupling increased by one-fold following AIH. The sympathetic effects of AIH were prevented by blocking angiotensin type 1 receptors with systemic losartan. Intermittent systemic injections of Ang II (Int.Ang II) elicited similar sympathetic responses to AIH. To identify the neural pathways responsible for the effects of AIH and Int.Ang II, we performed bilateral carotid body denervation, which reduced the increase in SNA by 56% and 45%, respectively. Conversely, pharmacological inhibition of the subfornical organ (SFO), an established target of circulating Ang II, reduced the increase in SNA following AIH and Int.Ang II by 65% and 59%, respectively, although it did not prevent the sensitization of the sympathetic peripheral chemoreflex, nor the increase in central sympathetic-respiratory coupling. Combined carotid body denervation and inhibition of the SFO eliminated the enhancement of SNA following AIH and Int.Ang II. Repeated systemic injections of phenylephrine caused an elevation in SNA similar to AIH, and this effect was prevented by a renin inhibitor, aliskiren. Our findings show that the sympathetic effects of AIH are the result of RAS-mediated activations of the carotid bodies and the SFO.
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http://dx.doi.org/10.1113/JP275804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068222PMC
August 2018

Interdependent feedback regulation of breathing by the carotid bodies and the retrotrapezoid nucleus.

J Physiol 2018 08 27;596(15):3029-3042. Epub 2017 Dec 27.

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.

The retrotrapezoid nucleus (RTN) regulates breathing in a CO - and state-dependent manner. RTN neurons are glutamatergic and innervate principally the respiratory pattern generator; they regulate multiple aspects of breathing, including active expiration, and maintain breathing automaticity during non-REM sleep. RTN neurons encode arterial /pH via cell-autonomous and paracrine mechanisms, and via input from other CO -responsive neurons. In short, RTN neurons are a pivotal structure for breathing automaticity and arterial homeostasis. The carotid bodies stimulate the respiratory pattern generator directly and indirectly by activating RTN via a neuronal projection originating within the solitary tract nucleus. The indirect pathway operates under normo- or hypercapnic conditions; under respiratory alkalosis (e.g. hypoxia) RTN neurons are silent and the excitatory input from the carotid bodies is suppressed. Also, silencing RTN neurons optogenetically quickly triggers a compensatory increase in carotid body activity. Thus, in conscious mammals, breathing is subject to a dual and interdependent feedback regulation by chemoreceptors. Depending on the circumstance, the activity of the carotid bodies and that of RTN vary in the same or the opposite directions, producing additive or countervailing effects on breathing. These interactions are mediated either via changes in blood gases or by brainstem neuronal connections, but their ultimate effect is invariably to minimize arterial fluctuations. We discuss the potential relevance of this dual chemoreceptor feedback to cardiorespiratory abnormalities present in diseases in which the carotid bodies are hyperactive at rest, e.g. essential hypertension, obstructive sleep apnoea and heart failure.
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http://dx.doi.org/10.1113/JP274357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068251PMC
August 2018

Supramammillary glutamate neurons are a key node of the arousal system.

Nat Commun 2017 11 10;8(1):1405. Epub 2017 Nov 10.

Department of Neurology, Beth Israel Deaconess Medical Center, Bostan, MA, 02215, USA.

Basic and clinical observations suggest that the caudal hypothalamus comprises a key node of the ascending arousal system, but the cell types underlying this are not fully understood. Here we report that glutamate-releasing neurons of the supramammillary region (SuM) produce sustained behavioral and EEG arousal when chemogenetically activated. This effect is nearly abolished following selective genetic disruption of glutamate release from SuM neurons. Inhibition of SuM neurons decreases and fragments wake, also suppressing theta and gamma frequency EEG activity. SuM neurons include a subpopulation containing both glutamate and GABA (SuM) and another also expressing nitric oxide synthase (SuM). Activation of SuM neurons produces minimal wake and optogenetic stimulation of SuM terminals elicits monosynaptic release of both glutamate and GABA onto dentate granule cells. Activation of SuM neurons potently drives wakefulness, whereas inhibition reduces REM sleep theta activity. These results identify SuM neurons as a key node of the wake-sleep regulatory system.
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http://dx.doi.org/10.1038/s41467-017-01004-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680228PMC
November 2017

Median preoptic glutamatergic neurons promote thermoregulatory heat loss and water consumption in mice.

J Physiol 2017 10 13;595(20):6569-6583. Epub 2017 Sep 13.

Department of Neurology, Beth Israel-Deaconess Medical Center - Harvard Medical School, Boston, MA, USA.

Key Points: Glutamatergic neurons in the median preoptic area were stimulated using genetically targeted Channelrhodopsin 2 in transgenic mice. Stimulation of glutamatergic median preoptic area neurons produced a profound hypothermia due to cutaneous vasodilatation. Stimulation also produced drinking behaviour that was inhibited as water was ingested, suggesting pre-systemic feedback gating of drinking. Anatomical mapping of the stimulation sites showed that sites associated with hypothermia were more anteroventral than those associated with drinking, although there was substantial overlap.

Abstract: The median preoptic nucleus (MnPO) serves an important role in the integration of water/electrolyte homeostasis and thermoregulation, but we have a limited understanding these functions at a cellular level. Using Cre-Lox genetic targeting of Channelrhodospin 2 in VGluT2 transgenic mice, we examined the effect of glutamatergic MnPO neuron stimulation in freely behaving mice while monitoring drinking behaviour and core temperature. Stimulation produced a strong hypothermic response in 62% (13/21) of mice (core temperature: -4.6 ± 0.5°C, P = 0.001 vs. controls) caused by cutaneous vasodilatation. Stimulating glutamatergic MnPO neurons also produced robust drinking behaviour in 82% (18/22) of mice. Mice that drank during stimulation consumed 912 ± 163 μl (n = 8) during a 20 min trial in the dark phase, but markedly less during the light phase (421 ± 83 μl, P = 0.0025). Also, drinking during stimulation was inhibited as water was ingested, suggesting pre-systemic feedback gating of drinking. Both hypothermia and drinking during stimulation occurred in 50% of mice tested. Anatomical mapping of the stimulation sites showed that sites associated with hypothermia were more anteroventral than those associated with drinking, although there was substantial overlap. Thus, activation of separate but overlapping populations of glutamatergic MnPO neurons produces effects on drinking and autonomic thermoregulatory mechanisms, providing a structural basis for their frequently being coordinated (e.g. during hyperthermia).
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http://dx.doi.org/10.1113/JP274667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638873PMC
October 2017

Reciprocal Control of Drinking Behavior by Median Preoptic Neurons in Mice.

J Neurosci 2016 08;36(31):8228-37

Department of Neurology, Beth Israel-Deaconess Medical Center-Harvard Medical School, Boston, Massachusetts 02215,

Unlabelled: Stimulation of glutamatergic neurons in the subfornical organ drives drinking behavior, but the brain targets that mediate this response are not known. The densest target of subfornical axons is the anterior tip of the third ventricle, containing the median preoptic nucleus (MnPO) and organum vasculosum of the lamina terminalis (OVLT), a region that has also been implicated in fluid and electrolyte management. The neurochemical composition of this region is complex, containing both GABAergic and glutamatergic neurons, but the possible roles of these neurons in drinking responses have not been addressed. In mice, we show that optogenetic stimulation of glutamatergic neurons in MnPO/OVLT drives voracious water consumption, and that optogenetic stimulation of GABAergic neurons in the same region selectively reduces water consumption. Both populations of neurons have extensive projections to overlapping regions of the thalamus, hypothalamus, and hindbrain that are much more extensive than those from the subfornical organ, suggesting that the MnPO/OVLT serves as a key link in regulating drinking responses.

Significance Statement: Neurons in the median preoptic nucleus (MnPO) and organum vasculosum of the lamina terminalis (OVLT) are known to regulate fluid/electrolyte homeostasis, but few studies have examined this issue with an appreciation for the neurochemical heterogeneity of these nuclei. Using Cre-Lox genetic targeting of Channelrhodospin-2 in transgenic mice, we demonstrate that glutamate and GABA neurons in the MnPO/OVLT reciprocally regulate water consumption. Stimulating glutamatergic MnPO/OVLT neurons induced water consumption, whereas stimulating GABAergic MnPO neurons caused a sustained and specific reduction in water consumption in dehydrated mice, the latter highlighting a heretofore unappreciated role of GABAergic MnPO neurons in thirst regulation. These observations represent an important advance in our understanding of the neural circuits involved in the regulation of fluid/electrolyte homeostasis.
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http://dx.doi.org/10.1523/JNEUROSCI.1244-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971367PMC
August 2016

Genetic identity of thermosensory relay neurons in the lateral parabrachial nucleus.

Am J Physiol Regul Integr Comp Physiol 2016 Jan 21;310(1):R41-54. Epub 2015 Oct 21.

Department of Neurology, Beth Israel Deaconess Medical Center; Harvard Medical School, Boston, Massachusetts;

The parabrachial nucleus is important for thermoregulation because it relays skin temperature information from the spinal cord to the hypothalamus. Prior work in rats localized thermosensory relay neurons to its lateral subdivision (LPB), but the genetic and neurochemical identity of these neurons remains unknown. To determine the identity of LPB thermosensory neurons, we exposed mice to a warm (36°C) or cool (4°C) ambient temperature. Each condition activated neurons in distinct LPB subregions that receive input from the spinal cord. Most c-Fos+ neurons in these LPB subregions expressed the transcription factor marker FoxP2. Consistent with prior evidence that LPB thermosensory relay neurons are glutamatergic, all FoxP2+ neurons in these subregions colocalized with green fluorescent protein (GFP) in reporter mice for Vglut2, but not for Vgat. Prodynorphin (Pdyn)-expressing neurons were identified using a GFP reporter mouse and formed a caudal subset of LPB FoxP2+ neurons, primarily in the dorsal lateral subnucleus (PBdL). Warm exposure activated many FoxP2+ neurons within PBdL. Half of the c-Fos+ neurons in PBdL were Pdyn+, and most of these project into the preoptic area. Cool exposure activated a separate FoxP2+ cluster of neurons in the far-rostral LPB, which we named the rostral-to-external lateral subnucleus (PBreL). These findings improve our understanding of LPB organization and reveal that Pdyn-IRES-Cre mice provide genetic access to warm-activated, FoxP2+ glutamatergic neurons in PBdL, many of which project to the hypothalamus.
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http://dx.doi.org/10.1152/ajpregu.00094.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747895PMC
January 2016

PHYSIOLOGY. Regulation of breathing by CO₂ requires the proton-activated receptor GPR4 in retrotrapezoid nucleus neurons.

Science 2015 Jun 11;348(6240):1255-60. Epub 2015 Jun 11.

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.

Blood gas and tissue pH regulation depend on the ability of the brain to sense CO2 and/or H(+) and alter breathing appropriately, a homeostatic process called central respiratory chemosensitivity. We show that selective expression of the proton-activated receptor GPR4 in chemosensory neurons of the mouse retrotrapezoid nucleus (RTN) is required for CO2-stimulated breathing. Genetic deletion of GPR4 disrupted acidosis-dependent activation of RTN neurons, increased apnea frequency, and blunted ventilatory responses to CO2. Reintroduction of GPR4 into RTN neurons restored CO2-dependent RTN neuronal activation and rescued the ventilatory phenotype. Additional elimination of TASK-2 (K(2P)5), a pH-sensitive K(+) channel expressed in RTN neurons, essentially abolished the ventilatory response to CO2. The data identify GPR4 and TASK-2 as distinct, parallel, and essential central mediators of respiratory chemosensitivity.
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http://dx.doi.org/10.1126/science.aaa0922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5171229PMC
June 2015

Optogenetic stimulation of adrenergic C1 neurons causes sleep state-dependent cardiorespiratory stimulation and arousal with sighs in rats.

Am J Respir Crit Care Med 2014 Dec;190(11):1301-10

Department of Pharmacology, University of Virginia, Charlottesville, Virginia.

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.
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http://dx.doi.org/10.1164/rccm.201407-1262OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315817PMC
December 2014

Vesicular glutamate transporter 2 is required for the respiratory and parasympathetic activation produced by optogenetic stimulation of catecholaminergic neurons in the rostral ventrolateral medulla of mice in vivo.

Eur J Neurosci 2014 Jan 18;39(1):98-106. Epub 2013 Nov 18.

Department of Pharmacology, University of Virginia, Charlottesville, VA, USA.

Catecholaminergic neurons of the rostral ventrolateral medulla (RVLM-CA neurons; C1 neurons) contribute to the sympathetic, parasympathetic and neuroendocrine responses elicited by physical stressors such as hypotension, hypoxia, hypoglycemia, and infection. Most RVLM-CA neurons express vesicular glutamate transporter (VGLUT)2, and may use glutamate as a ionotropic transmitter, but the importance of this mode of transmission in vivo is uncertain. To address this question, we genetically deleted VGLUT2 from dopamine-β-hydroxylase-expressing neurons in mice [DβH(Cre/0) ;VGLUT2(flox/flox) mice (cKO mice)]. We compared the in vivo effects of selectively stimulating RVLM-CA neurons in cKO vs. control mice (DβH(Cre/0) ), using channelrhodopsin-2 (ChR2-mCherry) optogenetics. ChR2-mCherry was expressed by similar numbers of rostral ventrolateral medulla (RVLM) neurons in each strain (~400 neurons), with identical selectivity for catecholaminergic neurons (90-99% colocalisation with tyrosine hydroxylase). RVLM-CA neurons had similar morphology and axonal projections in DβH(Cre/0) and cKO mice. Under urethane anesthesia, photostimulation produced a similar pattern of activation of presumptive ChR2-positive RVLM-CA neurons in DβH(Cre/0) and cKO mice. Photostimulation in conscious mice produced frequency-dependent respiratory activation in DβH(Cre/0) mice but no effect in cKO mice. Similarly, photostimulation under urethane anesthesia strongly activated efferent vagal nerve activity in DβH(Cre/0) mice only. Vagal responses were unaffected by α1 -adrenoreceptor blockade. In conclusion, two responses evoked by RVLM-CA neuron stimulation in vivo require the expression of VGLUT2 by these neurons, suggesting that the acute autonomic responses driven by RVLM-CA neurons are mediated by glutamate.
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http://dx.doi.org/10.1111/ejn.12421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4790426PMC
January 2014

C1 neurons: the body's EMTs.

Am J Physiol Regul Integr Comp Physiol 2013 Aug 22;305(3):R187-204. Epub 2013 May 22.

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908-0735, USA.

The C1 neurons reside in the rostral and intermediate portions of the ventrolateral medulla (RVLM, IVLM). They use glutamate as a fast transmitter and synthesize catecholamines plus various neuropeptides. These neurons regulate the hypothalamic pituitary axis via direct projections to the paraventricular nucleus and regulate the autonomic nervous system via projections to sympathetic and parasympathetic preganglionic neurons. The presympathetic C1 cells, located in the RVLM, are probably organized in a roughly viscerotopic manner and most of them regulate the circulation. C1 cells are variously activated by hypoglycemia, infection or inflammation, hypoxia, nociception, and hypotension and contribute to most glucoprivic responses. C1 cells also stimulate breathing and activate brain stem noradrenergic neurons including the locus coeruleus. Based on the various effects attributed to the C1 cells, their axonal projections and what is currently known of their synaptic inputs, subsets of C1 cells appear to be differentially recruited by pain, hypoxia, infection/inflammation, hemorrhage, and hypoglycemia to produce a repertoire of stereotyped autonomic, metabolic, and neuroendocrine responses that help the organism survive physical injury and its associated cohort of acute infection, hypoxia, hypotension, and blood loss. C1 cells may also contribute to glucose and cardiovascular homeostasis in the absence of such physical stresses, and C1 cell hyperactivity may contribute to the increase in sympathetic nerve activity associated with diseases such as hypertension.
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http://dx.doi.org/10.1152/ajpregu.00054.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743001PMC
August 2013

Chemoreception and asphyxia-induced arousal.

Respir Physiol Neurobiol 2013 Sep 19;188(3):333-43. Epub 2013 Apr 19.

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, United States. Electronic address:

Arousal protects against the adverse and potentially fatal effects of asphyxia during sleep. Asphyxia stimulates the carotid bodies and central chemoreceptors but the sequence of events leading to arousal is uncertain. In this review, the theoretical mechanisms leading to arousal from sleep are briefly summarized and the issue of whether central respiratory chemoreceptors (CRCs) or other types of CO2-responsive CNS neurons contribute to asphyxia-induced arousal is discussed. We focus on the role of the retrotrapezoid nucleus, the raphe and the locus coeruleus and emphasize the anatomical and neurophysiological evidence which suggests that these putative central chemoreceptors could contribute to arousal independently of their effects on breathing. Finally, we describe recent attempts to test the contribution of specific brainstem pathways to asphyxia-induced arousal using optogenetic and other tools and the possible contribution of a group of hypoxia-sensitive brainstem neurons (the C1 cells) to breathing and arousal.
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http://dx.doi.org/10.1016/j.resp.2013.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749262PMC
September 2013

Optogenetic stimulation of c1 and retrotrapezoid nucleus neurons causes sleep state-dependent cardiorespiratory stimulation and arousal in rats.

Hypertension 2013 Apr 25;61(4):835-41. Epub 2013 Feb 25.

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.

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 α1-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.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.111.00860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3666866PMC
April 2013

Selective optogenetic activation of rostral ventrolateral medullary catecholaminergic neurons produces cardiorespiratory stimulation in conscious mice.

J Neurosci 2013 Feb;33(7):3164-77

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.

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-hydroxylase(Cre/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% O(2)). In contrast, the effects of photostimulation were largely unaffected by hypercapnia (3 and 6% CO(2)). 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.
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http://dx.doi.org/10.1523/JNEUROSCI.1046-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596815PMC
February 2013

The respiratory chemoreception conundrum: light at the end of the tunnel?

Brain Res 2013 May 23;1511:126-37. Epub 2012 Oct 23.

Department of Pharmacology, University of Virginia, 1300 Jefferson Park Ave, Charlottesville, VA 22908, USA.

Arterial PCO₂ is tightly regulated via changes in breathing. A rise in PCO₂ activates the carotid bodies and exerts additional effects on neurons located within the CNS, causing an increase in lung ventilation. Central respiratory chemoreception refers to the component of this homeostatic reflex that is triggered by activation of receptors located within the brain (central chemoreceptors). Throughout the body, CO₂ generally operates via the proxy of pH. Since countless proteins, ion channels and neurons display some degree of pH-sensitivity, the notion that central respiratory chemoreception could rely on a few specialized neurons seems a priori counter-intuitive. Yet, two types of neurons currently stand out as critically important for breathing regulation by CO₂: the retrotrapezoid nucleus (RTN) and the raphe. RTN neurons are glutamatergic, strongly activated by hypercapnia in vivo and by CO₂ or protons in slices. These neurons target selectively the pontomedullary regions implicated in generating the respiratory rhythm and pattern. Their response to CO₂ seems to involve both cell-autonomous and paracrine effects of CO₂, the latter presumably mediated by the surrounding glia. The specific connections that these excitatory neurons establish with the rest of the breathing network are likely to be the main explanation of their importance to respiratory chemoreception. Serotonergic neurons have a powerful stimulatory effect on breathing, they facilitate the chemoreflexes and a subset of them likely function as CO₂ sensors. Opto- and pharmacogenetic methods have played an important role in assessing the contribution of RTN and serotonergic neurons as well as glial cells to respiration. These particular experiments are emphasized here for thematic reasons although the current perception of the importance of the RTN and serotonergic cells to respiratory chemoreception also relies on many other types of evidence. A small portion of this evidence is presented as background. This article is part of a Special Issue entitled Optogenetics (7th BRES).
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http://dx.doi.org/10.1016/j.brainres.2012.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570739PMC
May 2013

The retrotrapezoid nucleus and breathing.

Adv Exp Med Biol 2012 ;758:115-22

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908-0735, USA.

The retrotrapezoid nucleus (RTN) is located in the rostral medulla oblongata close to the ventral surface and consists of a bilateral cluster of glutamatergic neurons that are non-aminergic and express homeodomain transcription factor Phox2b throughout life. These neurons respond vigorously to increases in local pCO(2) via cell-autonomous and paracrine (glial) mechanisms and receive additional chemosensory information from the carotid bodies. RTN neurons exclusively innervate the regions of the brainstem that contain the respiratory pattern generator (RPG). Lesion or inhibition of RTN neurons largely attenuates the respiratory chemoreflex of adult rats whereas their activation increases respiratory rate, inspiratory amplitude and active expiration. Phox2b mutations that cause congenital central hypoventilation syndrome in humans prevent the development of RTN neurons in mice. Selective deletion of the RTN Phox2b-VGLUT2 neurons by genetic means in mice eliminates the respiratory chemoreflex in neonates.In short, RTN Phox2b-VGLUT2 neurons are a major nodal point of the CNS network that regulates pCO(2) via breathing and these cells are probable central chemoreceptors.
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http://dx.doi.org/10.1007/978-94-007-4584-1_16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111164PMC
April 2013

Wild-type microglia arrest pathology in a mouse model of Rett syndrome.

Nature 2012 Mar 18;484(7392):105-9. Epub 2012 Mar 18.

Department of Neuroscience, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.

Rett syndrome is an X-linked autism spectrum disorder. The disease is characterized in most cases by mutation of the MECP2 gene, which encodes a methyl-CpG-binding protein. Although MECP2 is expressed in many tissues, the disease is generally attributed to a primary neuronal dysfunction. However, as shown recently, glia, specifically astrocytes, also contribute to Rett pathophysiology. Here we examine the role of another form of glia, microglia, in a murine model of Rett syndrome. Transplantation of wild-type bone marrow into irradiation-conditioned Mecp2-null hosts resulted in engraftment of brain parenchyma by bone-marrow-derived myeloid cells of microglial phenotype, and arrest of disease development. However, when cranial irradiation was blocked by lead shield, and microglial engraftment was prevented, disease was not arrested. Similarly, targeted expression of MECP2 in myeloid cells, driven by Lysm(cre) on an Mecp2-null background, markedly attenuated disease symptoms. Thus, through multiple approaches, wild-type Mecp2-expressing microglia within the context of an Mecp2-null male mouse arrested numerous facets of disease pathology: lifespan was increased, breathing patterns were normalized, apnoeas were reduced, body weight was increased to near that of wild type, and locomotor activity was improved. Mecp2(+/-) females also showed significant improvements as a result of wild-type microglial engraftment. These benefits mediated by wild-type microglia, however, were diminished when phagocytic activity was inhibited pharmacologically by using annexin V to block phosphatydilserine residues on apoptotic targets, thus preventing recognition and engulfment by tissue-resident phagocytes. These results suggest the importance of microglial phagocytic activity in Rett syndrome. Our data implicate microglia as major players in the pathophysiology of this devastating disorder, and suggest that bone marrow transplantation might offer a feasible therapeutic approach for it.
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http://dx.doi.org/10.1038/nature10907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321067PMC
March 2012

Phox2b-expressing neurons of the parafacial region regulate breathing rate, inspiration, and expiration in conscious rats.

J Neurosci 2011 Nov;31(45):16410-22

Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.

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 (V(T)) and each inspiration was preceded by active expiration, denoting abdominal muscle contraction. During long-term pacing V(T) returned to prestimulation levels, suggesting that central chemoreceptors such as RTN-Phox2b neurons regulate V(T) 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.
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http://dx.doi.org/10.1523/JNEUROSCI.3280-11.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3236529PMC
November 2011

Central CO2 chemoreception and integrated neural mechanisms of cardiovascular and respiratory control.

J Appl Physiol (1985) 2010 Apr 14;108(4):995-1002. Epub 2010 Jan 14.

Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908-0735, USA.

In this review, we examine why blood pressure (BP) and sympathetic nerve activity (SNA) increase during a rise in central nervous system (CNS) P(CO(2)) (central chemoreceptor stimulation). CNS acidification modifies SNA by two classes of mechanisms. The first one depends on the activation of the central respiratory controller (CRG) and causes the much-emphasized respiratory modulation of the SNA. The CRG probably modulates SNA at several brain stem or spinal locations, but the most important site of interaction seems to be the caudal ventrolateral medulla (CVLM), where unidentified components of the CRG periodically gate the baroreflex. CNS P(CO(2)) also influences sympathetic tone in a CRG-independent manner, and we propose that this process operates differently according to the level of CNS P(CO(2)). In normocapnia and indeed even below the ventilatory recruitment threshold, CNS P(CO(2)) exerts a tonic concentration-dependent excitatory effect on SNA that is plausibly mediated by specialized brain stem chemoreceptors such as the retrotrapezoid nucleus. Abnormally high levels of P(CO(2)) cause an aversive interoceptive awareness in awake individuals and trigger arousal from sleep. These alerting responses presumably activate wake-promoting and/or stress-related pathways such as the orexinergic, noradrenergic, and serotonergic neurons. These neuronal groups, which may also be directly activated by brain acidification, have brainwide projections that contribute to the CO(2)-induced rise in breathing and SNA by facilitating neuronal activity at innumerable CNS locations. In the case of SNA, these sites include the nucleus of the solitary tract, the ventrolateral medulla, and the preganglionic neurons.
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http://dx.doi.org/10.1152/japplphysiol.00712.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853202PMC
April 2010

Photostimulation of channelrhodopsin-2 expressing ventrolateral medullary neurons increases sympathetic nerve activity and blood pressure in rats.

J Physiol 2009 Dec 12;587(Pt 23):5613-31. Epub 2009 Oct 12.

University of Virginia Health System, PO Box 800735, 1300 Jefferson Park Avenue, Charlottesville, VA 22908-0735, USA.

To explore the specific contribution of the C1 neurons to blood pressure (BP) control, we used an optogenetic approach to activate these cells in vivo. A lentivirus that expresses channelrhodopsin-2 (ChR2) under the control of the catecholaminergic neuron-preferring promoter PRSx8 was introduced into the rostral ventrolateral medulla (RVLM). After 2-3 weeks, ChR2 was largely confined to Phox2b-expressing neurons (89%). The ChR2-expressing neurons were non-GABAergic, non-glycinergic and predominantly catecholaminergic (54%). Photostimulation of ChR2-transfected RVLM neurons (473 nm, 20 Hz, 10 ms, 9 mW) increased BP (15 mmHg) and sympathetic nerve discharge (SND; 64%). Light pulses at 0.2-0.5 Hz evoked a large sympathetic nerve response (16 x baseline) followed by a silent period (1-2 s) during which another stimulus evoked a reduced response. Photostimulation activated most (75%) RVLM baroinhibited neurons sampled with 1/1 action potential entrainment to the light pulses and without accommodation during 20 Hz trains. RVLM neurons unaffected by either CO(2) or BP were light-insensitive. Bötzinger respiratory neurons were activated but their action potentials were not synchronized to the light pulses. Juxtacellular labelling of recorded neurons revealed that, of these three cell types, only the cardiovascular neurons expressed the transgene. In conclusion, ChR2 expression had no discernable effect on the putative vasomotor neurons at rest and was high enough to allow precise temporal control of their action potentials with light pulses. Photostimulation of RVLM neurons caused a sizable sympathoactivation and rise in blood pressure. These results provide the most direct evidence yet that the C1 neurons have a sympathoexcitatory function.
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http://dx.doi.org/10.1113/jphysiol.2009.177535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805374PMC
December 2009

Retrotrapezoid nucleus, respiratory chemosensitivity and breathing automaticity.

Respir Physiol Neurobiol 2009 Aug 13;168(1-2):59-68. Epub 2009 Feb 13.

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.

Breathing automaticity and CO(2) regulation are inseparable neural processes. The retrotrapezoid nucleus (RTN), a group of glutamatergic neurons that express the transcription factor Phox2b, may be a crucial nodal point through which breathing automaticity is regulated to maintain CO(2) constant. This review updates the analysis presented in prior publications. Additional evidence that RTN neurons have central respiratory chemoreceptor properties is presented, but this is only one of many factors that determine their activity. The RTN is also regulated by powerful inputs from the carotid bodies and, at least in the adult, by many other synaptic inputs. We also analyze how RTN neurons may control the activity of the downstream central respiratory pattern generator. Specifically, we review the evidence which suggests that RTN neurons (a) innervate the entire ventral respiratory column and (b) control both inspiration and expiration. Finally, we argue that the RTN neurons are the adult form of the parafacial respiratory group in neonate rats.
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http://dx.doi.org/10.1016/j.resp.2009.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2734912PMC
August 2009