Molecular cell types as functional units of the efferent vagus nerve.

The vagus nerve vitally connects the brain and body to coordinate digestive, cardiorespiratory, and immune functions. Its efferent neurons, which project their axons from the brainstem to the viscera, are thought to comprise "functional units" - neuron populations dedicated to the control of specific vagal reflexes or organ functions. Previous research indicates that these functional units differ from one another anatomically, neurochemically, and physiologically but have yet to define their identity in an experimentally tractable way. However, recent work with genetic technology and single-cell genomics suggests that genetically distinct subtypes of neurons may be the functional units of the efferent vagus. Here we review how these approaches are revealing the organizational principles of the efferent vagus in unprecedented detail.

Molecular organization of autonomic, respiratory, and spinally-projecting neurons in the mouse ventrolateral medulla.

The ventrolateral medulla (VLM) is a crucial region in the brain for visceral and somatic control, serving as a significant source of synaptic input to the spinal cord. Experimental studies have shown that gene expression in individual VLM neurons is predictive of their function. However, the molecular and cellular organization of the VLM has remained uncertain. This study aimed to create a comprehensive dataset of VLM cells using single-cell RNA sequencing in male and female mice. The dataset was enriched with targeted sequencing of spinally-projecting and adrenergic/noradrenergic VLM neurons. Based on differentially expressed genes, the resulting dataset of 114,805 VLM cells identifies 23 subtypes of neurons, excluding those in the inferior olive, and 5 subtypes of astrocytes. Spinally-projecting neurons were found to be abundant in 7 subtypes of neurons, which were validated through in-situ hybridization. These subtypes included adrenergic/noradrenergic neurons, serotonergic neurons, and neurons expressing gene markers associated with pre-motor neurons in the ventromedial medulla. Further analysis of adrenergic/noradrenergic neurons and serotonergic neurons identified 9 and 6 subtypes, respectively, within each class of monoaminergic neurons. Marker genes that identify the neural network responsible for breathing were concentrated in 2 subtypes of neurons, delineated from each other by markers for excitatory and inhibitory neurons. These datasets are available for public download and for analysis with a user-friendly interface. Collectively, this study provides a fine-scale molecular identification of cells in the VLM, forming the foundation for a better understanding of the VLM's role in vital functions and motor control.Significance statement The ventrolateral medulla (VLM) is an anatomically complex region of the brain that plays a crucial role in regulating vital functions, including autonomic and respiratory control, sleep-wake behaviors, cranial motor functions, and locomotion. This study comprehensively classifies VLM cell types and neuronal subtypes based on their molecular and anatomical features, by leveraging single-nuclei RNA sequencing, RNA fluorescence in situ hybridization, and axonal tract tracing. We present a dataset comprising 114,805 single-nuclei transcriptomes that identifies and validates the precise molecular characteristics of neurons involved in autonomic and motor systems functions. This publicly-available dataset offers new opportunities for comprehensive experimental studies to dissect the central organization of vital homeostatic functions and body movement.

Neuromedin B-Expressing Neurons in the Retrotrapezoid Nucleus Regulate Respiratory Homeostasis and Promote Stable Breathing in Adult Mice.

Respiratory chemoreceptor activity encoding arterial Pco2 and Po2 is a critical determinant of ventilation. Currently, the relative importance of several putative chemoreceptor mechanisms for maintaining eupneic breathing and respiratory homeostasis is debated. Transcriptomic and anatomic evidence suggests that bombesin-related peptide Neuromedin-B (Nmb) expression identifies chemoreceptor neurons in the retrotrapezoid nucleus (RTN) that mediate the hypercapnic ventilatory response, but functional support is missing. In this study, we generated a transgenic Nmb-Cre mouse and used Cre-dependent cell ablation and optogenetics to test the hypothesis that RTN Nmb neurons are necessary for the CO2-dependent drive to breathe in adult male and female mice. Selective ablation of ∼95% of RTN Nmb neurons causes compensated respiratory acidosis because of alveolar hypoventilation, as well as profound breathing instability and respiratory-related sleep disruption. Following RTN Nmb lesion, mice were hypoxemic at rest and were prone to severe apneas during hyperoxia, suggesting that oxygen-sensitive mechanisms, presumably the peripheral chemoreceptors, compensate for the loss of RTN Nmb neurons. Interestingly, ventilation following RTN Nmb -lesion was unresponsive to hypercapnia, but behavioral responses to CO2 (freezing and avoidance) and the hypoxia ventilatory response were preserved. Neuroanatomical mapping shows that RTN Nmb neurons are highly collateralized and innervate the respiratory-related centers in the pons and medulla with a strong ipsilateral preference. Together, this evidence suggests that RTN Nmb neurons are dedicated to the respiratory effects of arterial Pco2/pH and maintain respiratory homeostasis in intact conditions and suggest that malfunction of these neurons could underlie the etiology of certain forms of sleep-disordered breathing in humans.SIGNIFICANCE STATEMENT Respiratory chemoreceptors stimulate neural respiratory motor output to regulate arterial Pco2 and Po2, thereby maintaining optimal gas exchange. Neurons in the retrotrapezoid nucleus (RTN) that express the bombesin-related peptide Neuromedin-B are proposed to be important in this process, but functional evidence has not been established. Here, we developed a transgenic mouse model and demonstrated that RTN neurons are fundamental for respiratory homeostasis and mediate the stimulatory effects of CO2 on breathing. Our functional and anatomic data indicate that Nmb-expressing RTN neurons are an integral component of the neural mechanisms that mediate CO2-dependent drive to breathe and maintain alveolar ventilation. This work highlights the importance of the interdependent and dynamic integration of CO2- and O2-sensing mechanisms in respiratory homeostasis of mammals.

Vagus nerve stimulation activates two distinct neuroimmune circuits converging in the spleen to protect mice from kidney injury.

Acute kidney injury is highly prevalent and associated with high morbidity and mortality, and there are no approved drugs for its prevention and treatment. Vagus nerve stimulation (VNS) alleviates inflammatory diseases including kidney disease; however, neural circuits involved in VNS-induced tissue protection remain poorly understood. The vagus nerve, a heterogeneous group of neural fibers, innervates numerous organs. VNS broadly stimulates these fibers without specificity. We used optogenetics to selectively stimulate vagus efferent or afferent fibers. Anterograde efferent fiber stimulation or anterograde (centripetal) sensory afferent fiber stimulation both conferred kidney protection from ischemia-reperfusion injury. We identified the C1 neurons-sympathetic nervous system-splenic nerve-spleen-kidney axis as the downstream pathway of vagus afferent fiber stimulation. Our study provides a map of the neural circuits important for kidney protection induced by VNS, which is critical for the safe and effective clinical application of VNS for protection from acute kidney injury.

The astrocytic Na+ -HCO3 - cotransporter, NBCe1, is dispensable for respiratory chemosensitivity.

The interoceptive homeostatic mechanism that controls breathing, blood gases and acid-base balance in response to changes in CO2 /H+ is exquisitely sensitive, with convergent roles proposed for chemosensory brainstem neurons in the retrotrapezoid nucleus (RTN) and their supporting glial cells. For astrocytes, a central role for NBCe1, a Na+ -HCO3 - cotransporter encoded by Slc4a4, has been envisaged in multiple mechanistic models (i.e. underlying enhanced CO2 -induced local extracellular acidification or purinergic signalling). We tested these NBCe1-centric models by using conditional knockout mice in which Slc4a4 was deleted from astrocytes. In GFAP-Cre;Slc4a4fl/fl mice we found diminished expression of Slc4a4 in RTN astrocytes by comparison to control littermates, and a concomitant reduction in NBCe1-mediated current. Despite disrupted NBCe1 function in RTN-adjacent astrocytes from these conditional knockout mice, CO2 -induced activation of RTN neurons or astrocytes in vitro and in vivo, and CO2 -stimulated breathing, were indistinguishable from NBCe1-intact littermates; hypoxia-stimulated breathing and sighs were likewise unaffected. We obtained a more widespread deletion of NBCe1 in brainstem astrocytes by using tamoxifen-treated Aldh1l1-Cre/ERT2;Slc4a4fl/fl mice. Again, there was no difference in effects of CO2 or hypoxia on breathing or on neuron/astrocyte activation in NBCe1-deleted mice. These data indicate that astrocytic NBCe1 is not required for the respiratory responses to these chemoreceptor stimuli in mice, and that any physiologically relevant astrocytic contributions must involve NBCe1-independent mechanisms. KEY POINTS: The electrogenic NBCe1 transporter is proposed to mediate local astrocytic CO2 /H+ sensing that enables excitatory modulation of nearby retrotrapezoid nucleus (RTN) neurons to support chemosensory control of breathing. We used two different Cre mouse lines for cell-specific and/or temporally regulated deletion of the NBCe1 gene (Slc4a4) in astrocytes to test this hypothesis. In both mouse lines, Slc4a4 was depleted from RTN-associated astrocytes but CO2 -induced Fos expression (i.e. cell activation) in RTN neurons and local astrocytes was intact. Likewise, respiratory chemoreflexes evoked by changes in CO2 or O2 were unaffected by loss of astrocytic Slc4a4. These data do not support the previously proposed role for NBCe1 in respiratory chemosensitivity mediated by astrocytes.

Chemogenetic activation of noradrenergic A5 neurons increases blood pressure and visceral sympathetic activity in adult rats.

In mammals, the pontine noradrenergic system influences nearly every aspect of central nervous system function. A subpopulation of pontine noradrenergic neurons, called A5, are thought to be important in the cardiovascular response to physical stressors, yet their function is poorly defined. We hypothesized that activation of A5 neurons drives a sympathetically mediated increase in blood pressure (BP). To test this hypothesis, we conducted a comprehensive assessment of the cardiovascular effects of chemogenetic stimulation of A5 neurons in male and female adult rats using intersectional genetic and anatomical targeting approaches. Chemogenetic stimulation of A5 neurons in freely behaving rats elevated BP by 15 mmHg and increased cardiac baroreflex sensitivity with a negligible effect on resting HR. Importantly, A5 stimulation had no detectable effect on locomotor activity, metabolic rate, or respiration. Under anesthesia, stimulation of A5 neurons produced a marked elevation in visceral sympathetic nerve activity (SNA) and no change in skeletal muscle SNA, showing that A5 neurons preferentially stimulate visceral SNA. Interestingly, projection mapping indicates that A5 neurons target sympathetic preganglionic neurons throughout the spinal cord and parasympathetic preganglionic neurons throughout in the brainstem, as well as the nucleus of the solitary tract, and ventrolateral medulla. Moreover, in situ hybridization and immunohistochemistry indicate that a subpopulation of A5 neurons coreleases glutamate and monoamines. Collectively, this study suggests A5 neurons are a central modulator of autonomic function with a potentially important role in sympathetically driven redistribution of blood flow from the visceral circulation to critical organs and skeletal muscle.

EP3R-Expressing Glutamatergic Preoptic Neurons Mediate Inflammatory Fever.

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 (MnPOEP3R neurons). To investigate the identity of MnPOEP3R neurons, we first used in situ hybridization to show coexpression of EP3R and the VGluT2 transporter in MnPO neurons. Retrograde tracing showed extensive direct projections from MnPOVGluT2 but few from MnPOVgat neurons to a key site for fever production, the raphe pallidus. Ablation of MnPOVGluT2 but not MnPOVgat 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 MnPOEP3R neurons being GABAergic.SIGNIFICANCE STATEMENT 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.

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

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 Th-Cre rats to transduce selectively RTN (Phox2b+/Nmb+) or C1 neurons (Phox2b+/Th+) 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.SIGNIFICANCE STATEMENT 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 Pco2 stability.

Chemoreceptor mechanisms regulating CO2 -induced arousal from sleep.

Arousal from sleep in response to CO2 is a life-preserving reflex that enhances ventilatory drive and facilitates behavioural adaptations to restore eupnoeic breathing. Recurrent activation of the CO2 -arousal reflex is associated with sleep disruption in obstructive sleep apnoea. In this review we examine the role of chemoreceptors in the carotid bodies, the retrotrapezoid nucleus and serotonergic neurons in the dorsal raphe in the CO2 -arousal reflex. We also provide an overview of the supra-medullary structures that mediate CO2 -induced arousal. We propose a framework for the CO2 -arousal reflex in which the activity of the chemoreceptors converges in the parabrachial nucleus to trigger cortical arousal.

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

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 O2-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 CO2 and hypoxia-induced arousal: the retrotrapezoid nucleus (RTN), a CO2-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 CO2-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 CO2-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 CO2-induced arousal, whereas the CBs contribute to both hypoxia and CO2-induced arousal. Asphyxia-induced arousal likely requires the combined activation of RTN, CBs and other central chemoreceptors.SIGNIFICANCE STATEMENT 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 CO2 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 CO2 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 CO2 or hypoxia; therefore, we conclude that these structures are not the sole trigger of CO2 or hypoxia-induced arousal.

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

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.

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

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 CO2 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. CO2 detection, neuronal modulation) to the central respiratory chemoreflex (CRC). Here we test how important the RTN is to PCO2 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 PaO2 ) and hypercapnic (+10 mmHg ). In resting conditions, minute volume (VE ) was normal but breathing frequency (fR ) was elevated and tidal volume (VT ) reduced. Resting O2 consumption and CO2 production were normal. The hypercapnic ventilatory reflex in 65% FiO2 had an inverse exponential relationship with the number of surviving RTN neurons and was decreased by up to 92%. The hypoxic ventilatory reflex (HVR; FiO2 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 VE 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.

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

The retrotrapezoid nucleus (RTN) regulates breathing in a CO2 - 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 PCO2 /pH via cell-autonomous and paracrine mechanisms, and via input from other CO2 -responsive neurons. In short, RTN neurons are a pivotal structure for breathing automaticity and arterial PCO2 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 PCO2 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.

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

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.

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

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.

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

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).

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

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.

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.

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-ß-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.