Medullary Raphe Research Articles

Exploring Serotonergic mechanisms in Opioid Induced Respiratory Depression

Opioids profoundly depress breathing which can be fatal in the case of overdose. Despite growing knowledge of the pontine and medullary respiratory control circuits that are inhibited by opioids, the circuit and cellular mechanisms of opioid induced respiratory depression are not fully understood. Serotonin neurons located in the caudal medullary raphe modulate breathing and possess mu opioid receptors. Therefore, we hypothesized that serotonin-producing raphe neurons contribute to opioid induced respiratory depression. To test this, we employed to experimental approaches: 1) arterially perfused in situ working heart brainstem preparations and 2) ex vivo acute brain slice electrophysiology to identify and characterize serotonin neuron inputs onto brainstem respiratory neurons. To specifically activate serotonin neurons, we used ePet-Cre::Ai32 mice which Cre-dependently express channelrhodopsin-2, a light-activated cation channel, in serotonin neurons. At the circuit level, using the in situ preparation, we found that activation of caudal medullary raphe serotonin neurons increased respiratory rate by decreasing inspiratory and expiratory duration. Fentanyl application blocked the respiratory effects induced by serotonin neuron activation which were subsequently restored using naloxone. These results suggest serotonin neurons in the caudal medullary raphe are inhibited by opioids. To characterize the serotonin-mediated mechanisms at the cellular level, we made acute brain slices containing dorsal raphe neurons and lateral parabrachial neurons from ePet-Cre::Ai32 mice. We verified that optical stimulation resulted in action potentials and serotonin release from dorsal raphe serotonin neurons. Optically evoked serotonin release caused 5HT1A receptor-mediated outward currents in dorsal raphe serotonin neurons. To locate and characterize serotonin neuron inputs onto lateral parabrachial neurons, we performed whole-cell voltage clamp recordings from lateral parabrachial neurons and delivered paired light pulses (10 ms duration, 50 ms interval). We observed optically evoked inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA-A receptor antagonist gabazine (1 μM). Optically evoked IPSCs had long latency (> 5 ms) and high jitter (> 1 ms), indicating stimulation of serotonin terminals resulted in polysynaptic GABA release onto lateral parabrachial neurons. Future work will examine the influence of opioids on this raphe-parabrachial circuit. Uncovering the role of serotonin neurons in opioid induced respiratory depression may reveal targets for restoring breathing in the presence of opioids or other conditions where life-threatening apneas occur. This work was supported by the National Institutes of Health (NIDA) R01DA047978-05 grant to ESL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Central Mechanisms of Thermoregulation and Fever in Mammals.

Thermoregulation is a fundamental homeostatic function in mammals mediated by the central nervous system. The framework of the central circuitry for thermoregulation lies in the hypothalamus and brainstem. The preoptic area (POA) of the hypothalamus integrates cutaneous and central thermosensory information into efferent control signals that regulate excitatory descending pathways through the dorsomedial hypothalamus (DMH) and rostral medullary raphe region (rMR). The cutaneous thermosensory feedforward signals are delivered to the POA by afferent pathways through the lateral parabrachial nucleus, while the central monitoring of body core temperature is primarily mediated by warm-sensitive neurons in the POA for negative feedback regulation. Prostaglandin E2, a pyrogenic mediator produced in response to infection, acts on the POA to trigger fever. Recent studies have revealed that this circuitry also functions for physiological responses to psychological stress and starvation. Master psychological stress signaling from the medial prefrontal cortex to the DMH has been discovered to drive a variety of physiological responses for stress coping, including hyperthermia. During starvation, hunger signaling from the hypothalamus was found to activate medullary reticular neurons, which then suppress thermogenic sympathetic outflows from the rMR for energy saving. This thermoregulatory circuit represents a fundamental mechanism of the central regulation for homeostasis.

PHYSIOLOGY OF AFFERENT AND CENTRAL PARTS OF AUTONOMIC NERVOUS SYSTEM

The Autonomic Nervous System (ANS) and the endocrine system control the internal environment of the body. The organization of the ANS is on the basis of the reflex arc. Changes in chemical composition, blood pressure, osmotic pressure, stretch in viscera, and temperature are detected by autonomic receptors. The visceral afferent fibres are myelinated and they accompany the visceral motor fibres. The sensory autonomic neurons are pseudo-unipolar cells located in the dorsal root ganglia of somatic spinal nerves. Once the afferent fibres gain entrance to the spinal cord or the brainstem, they are thought to travel alongside the somatic afferent fibres to the autonomic centres in different parts of the brain. Information reaches the higher autonomic centres from viscera through an ascending system that involves the nucleus tractus solitarius, the parabrachial nucleus, the periaqueductal gray matter, and the hypothalamus. The hypothalamic paraventricular nucleus, pontine A5 cell group, rostral ventrolateral medulla, and medullary raphe nuclei send direct output to the preganglionic autonomic neurons. The amygdala, mesencephalic periaqueductal gray, caudal ventrolateral medulla, nucleus of the tractus solitarius, and medullary lateral tegmental field feed into these direct pathways. Pak J Physiol 2023;19(4):1-2

Protective effects of SSRI, Citalopram in mutant APP and mutant Tau expressed dorsal raphe neurons in Alzheimer's disease

Depression is among the most common neuropsychiatric comorbidities in Alzheimer's disease (AD) and other Tauopathies. Apart from its anti-depressive and anxiolytic effects, selective serotonin reuptake inhibitor (SSRI) treatment also offers intracellular modifications that may help to improve neurogenesis, reduce amyloid burden & Tau pathologies, and neuroinflammation in AD. Despite its multifaceted impact in the brain, the exact physiological and molecular mechanism by which SSRIs such as Citalopram improve neurogenesis and synaptogenesis in dementia is poorly understood. In the current study, we investigated the protective role of SSRI, Citalopram, in serotonergic, medullary raphe neurons (RN46A-B14). RN46A-B14 cells were transfected with wild-type and mutant APP and Tau cDNAs for 24 h and then treated with 20 μM Cit for 24 h. We then assessed mRNA and protein levels of pTau, total Tau, serotonin related proteins such as TPH2, SERT, and 5HTR1a, synaptic proteins and the cytoskeletal structure. We also assessed cell survival, mitochondrial respiration and mitochondrial morphology. The mutant APP and Tau transfected cells showed increased levels of serotonin related proteins and mRNA, while the mRNA and protein levels of synaptic proteins were downregulated. Citalopram treatment significantly reduced pathologically pTau level along with the serotonin related protein levels. On the other hand, there was a significant increase in the mRNA and protein levels of synaptic genes and cytoskeletal structure in the treated groups. Further, Citalopram also improved cell survival, mitochondrial respiration and mitochondrial morphology in the treated cells that express mAPP and mTau. Taken together these findings suggest Citalopram could not only be a promising therapeutic drug for treating patients with depression, but also for AD patients.

Essential roles of the hypothalamic A11 region and the medullary raphe nuclei in regulation of colorectal motility in rats.

The supraspinal brain regions controlling defecation reflex remain to be elucidated. The purpose of this study was to determine the roles of the hypothalamic A11 region and the medullary raphe nuclei in regulation of defecation. For chemogenetic manipulation of specific neurons, we used the double virus vector infection method in rats. hM3Dq or hM4Di was expressed in neurons of the A11 region and/or the raphe nuclei that send output to the lumbosacral defecation center. Immunohistological and functional experiments revealed that both the A11 region and the raphe nuclei directly connected with the lumbosacral spinal cord through descending pathways composed of stimulatory monoaminergic neurons. Stimulation of the hM3Dq-expressing neurons in the A11 region or the raphe nuclei enhanced colorectal motility only when GABAergic transmission in the lumbosacral spinal cord was blocked by bicuculline. Experiments using inhibitory hM4Di-expressing rats revealed that enhancement of colorectal motility caused by noxious stimuli in the colon is mediated by both the A11 region and the raphe nuclei. Furthermore, suppression of the A11 region and/or the raphe nuclei significantly inhibited water avoidance stress-induced defecation. These findings demonstrate that the A11 region and the raphe nuclei play an essential role in regulation of colorectal motility. This is important because brain regions that mediate both intracolonic noxious stimuli-induced defecation and stress-induced defecation have been clarified for the first time.

Analyzing the brainstem circuits for respiratory chemosensitivity in freely moving mice.

Regulation of systemic PCO2 is a life-preserving homeostatic mechanism. In the medulla oblongata, the retrotrapezoid nucleus (RTN) and rostral medullary Raphe are proposed as CO2 chemosensory nuclei mediating adaptive respiratory changes. Hypercapnia also induces active expiration, an adaptive change thought to be controlled by the lateral parafacial region (pFL). Here, we use GCaMP6 expression and head-mounted mini-microscopes to image Ca2+ activity in these nuclei in awake adult mice during hypercapnia. Activity in the pFL supports its role as a homogenous neuronal population that drives active expiration. Our data show that chemosensory responses in the RTN and Raphe differ in their temporal characteristics and sensitivity to CO2, raising the possibility these nuclei act in a coordinated way to generate adaptive ventilatory responses to hypercapnia. Our analysis revises the understanding of chemosensory control in awake adult mouse and paves the way to understanding how breathing is coordinated with complex non-ventilatory behaviours.

Sleep architecture and sleep-disordered breathing in fatal insomnia

Study objectivesFatal insomnia (FI) is a rare prion disease severely affecting sleep architecture. Breathing during sleep has not been systematically assessed. Our aim was to characterize the sleep architecture, respiratory patterns, and neuropathologic findings in FI. MethodsEleven consecutive FI patients (ten familial, one sporadic) were examined with video-polysomnography (vPSG) between 2002 and 2017. Wake/sleep stages and respiration were evaluated using a modified scoring system. Postmortem neuropathology was assessed in seven patients. ResultsMedian age at onset was 48 years and survival after vPSG was 1 year. All patients had different combinations of breathing disturbances including increased respiratory rate variability (RRV; n = 7), stridor (n = 9), central sleep apnea (CSA) (n = 5), hiccup (n = 6), catathrenia (n = 7), and other expiratory sounds (n = 10). RRV in NREM sleep correlated with ambiguous and solitary nuclei degeneration (r = 0.9, p = 0.008) and reduced survival (r = −0.7, p = 0.037). Two new stages, Subwake1 and Subwake2, present in all patients, were characterized. NREM sleep (conventional or undifferentiated) was identifiable in ten patients but reduced in duration in eight. REM sleep occurred in short segments in nine patients, and their reduced duration correlated with medullary raphe nuclei degeneration (r = −0.9, p = 0.005). Seven patients had REM without atonia. Three vPSG patterns were identified: agitated, with aperiodic, manipulative, and finalistic movements (n = 4); quiet-apneic, with CSA (n = 4); and quiet-non-apneic (n = 3). ConclusionsFI patients show frequent breathing alterations, associated with respiratory nuclei damage, and, in addition to NREM sleep distortion, have severe impairment of REM sleep, related with raphe nuclei degeneration. Brainstem impairment is crucial in FI.

An oxytocinergic neural pathway that stimulates thermogenic and cardiac sympathetic outflow.

Oxytocin alters autonomic functions besides social behaviors. However, the central neuronal links between hypothalamic oxytocinergic neurons and the autonomic nervous system remain unclear. Here we show that oxytocinergic neurons in the rat paraventricular hypothalamic nucleus (PVH), a pivotal site for energy homeostasis, innervate sympathetic premotor neurons in the rostral medullary raphe region (rMR) to stimulate brown adipose tissue (BAT) thermogenesis and cardiovascular functions. Oxytocin receptor stimulation in the rMR evokes BAT thermogenesis and tachycardia. Invivo optogenetic stimulation of the PVH→rMR long-range oxytocinergic pathway, using a virus-mediated system for amplified gene expression in oxytocinergic neurons, not only elicits BAT thermogenic and cardiac responses but also potentiates sympathetic responses evoked by glutamatergic transmission in the rMR. The PVH→rMR oxytocinergic pathway connects the hypothalamic circuit for energy homeostasis to thermogenic and cardiac sympathetic outflow, and, therefore, its defects may cause obesity and impaired thermoregulation, as seen in Prader-Willi syndrome.

Raphe and ventrolateral medulla proteomics in epilepsy and sudden unexpected death in epilepsy.

Brainstem nuclei dysfunction is implicated in sudden unexpected death in epilepsy. In animal models, deficient serotonergic activity is associated with seizure-induced respiratory arrest. In humans, glia are decreased in the ventrolateral medullary pre-Botzinger complex that modulate respiratory rhythm, as well as in the medial medullary raphe that modulate respiration and arousal. Finally, sudden unexpected death in epilepsy cases have decreased midbrain volume. To understand the potential role of brainstem nuclei in sudden unexpected death in epilepsy, we evaluated molecular signalling pathways using localized proteomics in microdissected midbrain dorsal raphe and medial medullary raphe serotonergic nuclei, as well as the ventrolateral medulla in brain tissue from epilepsy patients who died of sudden unexpected death in epilepsy and other causes in diverse epilepsy syndromes and non-epilepsy control cases (n = 15–16 cases per group/region). Compared with the dorsal raphe of non-epilepsy controls, we identified 89 proteins in non-sudden unexpected death in epilepsy and 219 proteins in sudden unexpected death in epilepsy that were differentially expressed. These proteins were associated with inhibition of EIF2 signalling (P-value of overlap = 1.29 × 10−8, z = −2.00) in non-sudden unexpected death in epilepsy. In sudden unexpected death in epilepsy, there were 10 activated pathways (top pathway: gluconeogenesis I, P-value of overlap = 3.02 × 10−6, z = 2.24) and 1 inhibited pathway (fatty acid beta-oxidation, P-value of overlap = 2.69 × 10−4, z = −2.00). Comparing sudden unexpected death in epilepsy and non-sudden unexpected death in epilepsy, 10 proteins were differentially expressed, but there were no associated signalling pathways. In both medullary regions, few proteins showed significant differences in pairwise comparisons. We identified altered proteins in the raphe and ventrolateral medulla of epilepsy patients, including some differentially expressed in sudden unexpected death in epilepsy cases. Altered signalling pathways in the dorsal raphe of sudden unexpected death in epilepsy indicate a shift in cellular energy production and activation of G-protein signalling, inflammatory response, stress response and neuronal migration/outgrowth. Future studies should assess the brain proteome in relation to additional clinical variables (e.g. recent tonic–clonic seizures) and in more of the reciprocally connected cortical and subcortical regions to better understand the pathophysiology of epilepsy and sudden unexpected death in epilepsy.

Patch-to-Seq and Transcriptomic Analyses Yield Molecular Markers of Functionally Distinct Brainstem Serotonin Neurons.

Acute regulation of CO2 and pH homeostasis requires sensory feedback from peripheral (carotid body) and central (central) CO2/pH sensitive cells – so called respiratory chemoreceptors. Subsets of brainstem serotonin (5-HT) neurons in the medullary raphe are CO2 sensitive or insensitive based on differences in embryonic origin, suggesting these functionally distinct subpopulations may have unique transcriptional profiles. Here, we used Patch-to-Seq to determine if the CO2 responses in brainstem 5-HT neurons could be correlated to unique transcriptional profiles and/or unique molecular markers and pathways. First, firing rate changes with hypercapnic acidosis were measured in fluorescently labeled 5-HT neurons in acute brainstem slices from transgenic, Dahl SS (SSMcwi) rats expressing T2/ePet-eGFP transgene in Pet-1 expressing (serotonin) neurons (SSePet1–eGFP rats). Subsequently, the transcriptomic and pathway profiles of CO2 sensitive and insensitive 5-HT neurons were determined and compared by single cell RNA (scRNAseq) and bioinformatic analyses. Low baseline firing rates were a distinguishing feature of CO2 sensitive 5-HT neurons. scRNAseq of these recorded neurons revealed 166 differentially expressed genes among CO2 sensitive and insensitive 5-HT neurons. Pathway analyses yielded novel predicted upstream regulators, including the transcription factor Egr2 and Leptin. Additional bioinformatic analyses identified 6 candidate gene markers of CO2 sensitive 5-HT neurons, and 2 selected candidate genes (CD46 and Iba57) were both expressed in 5-HT neurons determined via in situ mRNA hybridization. Together, these data provide novel insights into the transcriptional control of cellular chemoreception and provide unbiased candidate gene markers of CO2 sensitive 5-HT neurons. Methodologically, these data highlight the utility of the patch-to-seq technique in enabling the linkage of gene expression to specific functions, like CO2 chemoreception, in a single cell to identify potential mechanisms underlying functional differences in otherwise similar cell types.

5‐HT7 receptors expressed in the mouse parafacial region are not required for respiratory chemosensitivity

A brainstem homeostatic system senses CO2/H+ to regulate ventilation, blood gases and acid‐base balance. Neurons of the retrotrapezoid nucleus (RTN) and medullary raphe are both implicated in this mechanism as respiratory chemosensors, but recent pharmacological work suggested that the CO2/H+‐sensitivity of RTN neurons is mediated indirectly – by raphe‐derived serotonin acting on 5‐HT7 receptors. To investigate this further, we characterized Htr7 transcript expression in phenotypically identified RTN neurons using multiplex single cell qRT‐PCR and RNAscope. Although present in multiple neurons in the parafacial region of the ventrolateral medulla, Htr7 expression was undetectable in most RTN neurons (Nmb+/Phox2b+) concentrated in the densely packed cell group ventrolateral to the facial nucleus. Where detected, Htr7 expression was modest and often associated with RTN neurons that extend dorsolaterally to partially encircle the facial nucleus. These more dorsolateral Nmb+/Htr7+ neurons tended to express high levels of Nmb and low levels of the intrinsic RTN proton detectors, Gpr4 and Kcnk5. In mouse brainstem slices, CO2‐stimulated firing in RTN neurons was mostly unaffected by a 5‐HT7 receptor antagonist, SB269970 (n=11/12). At the whole animal level, microinjection of SB269970 into the RTN of conscious mice blocked respiratory stimulation by co‐injected LP‐44, a 5‐HT7 receptor agonist, but had no effect on CO2‐stimulated breathing in those same mice. We conclude that Htr7 is expressed by a minor subset of RTN neurons with a molecular profile distinct from the established chemoreceptors and that 5‐HT7 receptors have negligible effects on CO2‐evoked firing activity in RTN neurons or on CO2‐stimulated breathing in mice.

Distribution of c‐Fos Labeling Elicited by Intragastric Copper Sulfate: Comparison with Labeling Induced by Galvanic Vestibular Stimulation

The neural pathways that produce nausea and emesis are poorly defined. Although it is widely postulated that a “final common pathway” is activated during emetic responses elicited by any stimulus, this hypothesis has not been adequately tested. In a prior study (Balaban et al., PLoS ONE 9(1): e86730, 2014) we mapped the distribution of Fos labeling in the feline brainstem elicited by galvanic vestibular stimulation (GVS) that evoked retching and other indicators of nausea. As a comparison, in the present study we mapped Fos labeling patterns induced by intragastric administration of copper sulfate (CuSO4), which produces emesis by activating vagal afferent fibers. Emetic responses slowly build‐up during GVS, but occur rapidly following CuSO4 administration. In 10 felines (6 male, 4 female), doses of CuSO4 ranging from 83 to 322 mg were administered through an intragastric catheter or gavage, and ~2 hours later animals were perfused with paraformaldehyde. The brain was sectioned and processed for Fos labeling as in our prior study (Balaban et al., PLoS ONE 9(1): e86730, 2014). Three additional animals served as controls and did not receive intragastric CuSO4. Some brainstem areas exhibited Fos labeling following either GVS or CuSO4 treatments, as shown in Fig. 1: the commissural, medial and lateral regions of nucleus tractus solitarii, lateral reticular nucleus, medullary lateral tegmental field, subtrigeminal nucleus, medullary raphe nuclei, parabrachial nucleus, and locus coeruleus. However, the labeling patterns also diverged: CuSO4 administration produced virtually no labeling in the vestibular nuclei (which was heavy following GVS), more labeling in nucleus tractus solitarii and the medullary raphe nuclei, and less labeling in the pontine raphe nuclei. These findings support the hypothesis that some brainstem areas mediate nausea and emesis induced by any stimulus, independent of how quickly the responses develop, but also suggest that unique pathways may be activated by particular emetic stimuli.

The 5‐HT1A Agonist 8‐OH‐DPAT Enhances Submental Laryngeal Elevator EMG Amplitude During Swallow

Swallow is a critical behavior for sustaining life, important for both for the ingestion of nutrients and for protecting the airways. To produce an effective swallow, neural circuits must coordinate over 20 muscles across multiple cranial/spinal nerves to produce a rostral‐caudal pressure gradient to move the bolus from the mouth to the stomach. The medullary raphe nuclei are the main source of serotonin in the brainstem, and this region has been implicated in modulation of swallow and other airway protective behaviors. In order to explore the role of serotonin in swallow, we conducted experiments in adult cats (n = 6). Animals were anesthetized with intravenous sodium pentobarbital and tracheostomized. Fine wire intramuscular electromyograms (EMGs) were recorded from swallow and respiratory related muscles. The 5‐HT1A receptor agonist 8‐OH‐DPAT was delivered either intravenously through the femoral artery (n = 3) or intra‐arterially through the vertebral artery (n = 3) as a series of six cumulative doses (0.1‐30 ug/kg i.v.; 1‐300 ug/kg i.a.). Swallow was elicited by oral infusion water (3 ml) via syringe and was confirmed by the presence coordinated activity on swallow‐related EMGs. EMG amplitude was normalized prior to analysis. Administration of 8‐OH‐DPAT demonstrated a significant effect on the two primary submental muscles responsible for hyolaryngeal elevation necessary for full occlusion of the airway by the epiglottis. Mylohyoid amplitude increased by 45% (p = 0.006) and geniohyoid amplitude increased by 64% (p = 0.04) at the highest dose when compared to control. This is evidence of the raphe and its serotonergic network coordinating distributed aspects of the swallow control network as these two muscles are innervated via separate cranial nerves: the trigeminal for the mylohyoid and hypoglossal for the geniohyoid. Disordered hyolaryngeal function is a leading cause of aspiration across a multitude of neuro‐ degenerative and ‐traumatic diseases, and pharmacologic intervention with serotonergic agonists represents a promising agent for therapeutic interventions.

5-HT7 receptors expressed in the mouse parafacial region are not required for respiratory chemosensitivity.

A brainstem homeostatic system senses CO2/H+ to regulate ventilation, blood gases and acid–base balance. Neurons of the retrotrapezoid nucleus (RTN) and medullary raphe are both implicated in this mechanism as respiratory chemosensors, but recent pharmacological work suggested that the CO2/H+ sensitivity of RTN neurons is mediated indirectly, by raphe‐derived serotonin acting on 5‐HT7 receptors. To investigate this further, we characterized Htr7 transcript expression in phenotypically identified RTN neurons using multiplex single cell qRT‐PCR and RNAscope. Although present in multiple neurons in the parafacial region of the ventrolateral medulla, Htr7 expression was undetectable in most RTN neurons (Nmb +/Phox2b+) concentrated in the densely packed cell group ventrolateral to the facial nucleus. Where detected, Htr7 expression was modest and often associated with RTN neurons that extend dorsolaterally to partially encircle the facial nucleus. These dorsolateral Nmb +/Htr7 + neurons tended to express Nmb at high levels and the intrinsic RTN proton detectors Gpr4 and Kcnk5 at low levels. In mouse brainstem slices, CO2‐stimulated firing in RTN neurons was mostly unaffected by a 5‐HT7 receptor antagonist, SB269970 (n = 11/13). At the whole animal level, microinjection of SB269970 into the RTN of conscious mice blocked respiratory stimulation by co‐injected LP‐44, a 5‐HT7 receptor agonist, but had no effect on CO2‐stimulated breathing in those same mice. We conclude that Htr7 is expressed by a minor subset of RTN neurons with a molecular profile distinct from the established chemoreceptors and that 5‐HT7 receptors have negligible effects on CO2‐evoked firing activity in RTN neurons or on CO2‐stimulated breathing in mice.Key points Neurons of the retrotrapezoid nucleus (RTN) are intrinsic CO2/H+ chemosensors and serve as an integrative excitatory hub for control of breathing.Serotonin can activate RTN neurons, in part via 5‐HT7 receptors, and those effects have been implicated in conferring an indirect CO2 sensitivity.Multiple single cell molecular approaches revealed low levels of 5‐HT7 receptor transcript expression restricted to a limited population of RTN neurons.Pharmacological experiments showed that 5‐HT7 receptors in RTN are not required for CO2/H+‐stimulation of RTN neuronal activity or CO2‐stimulated breathing.These data do not support a role for 5‐HT7 receptors in respiratory chemosensitivity mediated by RTN neurons.

In Transgenic Erythropoietin Deficient Mice, an Increase in Respiratory Response to Hypercapnia Parallels Abnormal Distribution of CO2/H+-Activated Cells in the Medulla Oblongata.

Erythropoietin (Epo) and its receptor are expressed in central respiratory areas. We hypothesized that chronic Epo deficiency alters functioning of central respiratory areas and thus the respiratory adaptation to hypercapnia. The hypercapnic ventilatory response (HcVR) was evaluated by whole body plethysmography in wild type (WT) and Epo deficient (Epo-TAgh) adult male mice under 4%CO2. Epo-TAgh mice showed a larger HcVR than WT mice because of an increase in both respiratory frequency and tidal volume, whereas WT mice only increased their tidal volume. A functional histological approach revealed changes in CO2/H+-activated cells between Epo-TAgh and WT mice. First, Epo-TAgh mice showed a smaller increase under hypercapnia in c-FOS-positive number of cells in the retrotrapezoid nucleus/parafacial respiratory group than WT, and this, independently of changes in the number of PHOX2B-expressing cells. Second, we did not observe in Epo-TAgh mice the hypercapnic increase in c-FOS-positive number of cells in the nucleus of the solitary tract present in WT mice. Finally, whereas hypercapnia did not induce an increase in the c-FOS-positive number of cells in medullary raphe nuclei in WT mice, chronic Epo deficiency leads to raphe pallidus and magnus nuclei activation by hyperacpnia, with a significant part of c-FOS positive cells displaying an immunoreactivity for serotonin in the raphe pallidus nucleus. All of these results suggest that chronic Epo-deficiency affects both the pattern of ventilatory response to hypercapnia and associated medullary respiratory network at adult stage with an increase in the sensitivity of 5-HT and non-5-HT neurons of the raphe medullary nuclei leading to stimulation of f R for moderate level of CO2.

Contribution of the caudal medullary raphe to opioid induced respiratory depression

BackgroundOpioid-induced respiratory depression can be partially antagonized in the preBötzinger Complex and Parabrachial Nucleus/Kölliker-Fuse Complex. We hypothesized that additional opioid antagonism in the caudal medullary raphe completely reverses the opioid effect. MethodsIn adult ventilated, vagotomized, decerebrate rabbits, we administrated remifentanil intravenously at “analgesic”, “apneic”, and “very high” doses and determined the reversal with sequential naloxone microinjections into the bilateral Parabrachial Nucleus/Kölliker-Fuse Complex, preBötzinger Complex, and caudal medullary raphe. In separate animals, we injected opioid antagonists into the raphe without intravenous remifentanil. ResultsSequential naloxone microinjections completely reversed respiratory rate depression from “analgesic” and “apneic” remifentanil, but not “very high” remifentanil concentrations. Antagonist injection into the caudal medullary raphe without remifentanil independently increased respiratory rate. ConclusionsOpioid-induced respiratory depression results from a combined effect on the respiratory rhythm generator and respiratory drive. The effect in the caudal medullary raphe is complex as we also observed local antagonism of endogenous opioid receptor activation, which has not been described before.

Central sympathetic network for thermoregulatory responses to psychological stress

In mammals, many types of psychological stressors elicit a variety of sympathoexcitatory responses paralleling the classic fight-or-flight response to a threat to survival, including increased body temperature via brown adipose tissue thermogenesis and cutaneous vasoconstriction, and increased skeletal muscle blood flow via tachycardia and visceral vasoconstriction. Although these responses are usually supportive for stress coping, aberrant sympathetic responses to stress can lead to clinical issues in psychosomatic medicine. Sympathetic stress responses are mediated mostly by sympathetic premotor drives from the rostral medullary raphe region (rMR) and partly by those from the rostral ventrolateral medulla (RVLM). Hypothalamomedullary descending pathways from the dorsomedial hypothalamus (DMH) to the rMR and RVLM mediate important, stress-driven sympathoexcitatory transmission to the premotor neurons to drive the thermal and cardiovascular responses. The DMH also likely sends an excitatory input to the paraventricular hypothalamic nucleus to stimulate stress hormone release. Neurons in the DMH receive a stress-related excitation from the dorsal peduncular cortex and dorsal tenia tecta (DP/DTT) in the ventromedial prefrontal cortex. By connecting the corticolimbic emotion circuit to the central sympathetic and somatic motor systems, the DP/DTT → DMH pathway plays as the primary mediator of the psychosomatic signaling that drives a variety of sympathetic and behavioral stress responses. These brain regions together with other stress-related regions constitute a central neural network for physiological stress responses. This network model is relevant to understanding the central mechanisms by which stress and emotions affect autonomic regulations of homeostasis and to developing new therapeutic strategies for various stress-related disorders.

Melanin-concentrating hormone neurons affect adipose tissues and modulate weight gain.

Melanin-concentrating hormone neurons affect adipose tissues and modulate weight gain.

Role of hydrogen sulfide in ventilatory responses to hypercapnia in the medullary raphe of adult rats.

What is the central question of this study? There is evidence that H2 S plays a role in the control of breathing: what are its actions on the ventilatory and thermoregulatory responses to hypercapnia via effects in the medullary raphe, a brainstem region that participates in the ventilatory adjustments to hypercapnia? What is the main finding and its importance? Hypercapnia increased the endogenous production of H2 S in the medullary raphe. Inhibition of endogenous H2 S attenuated the ventilatory response to hypercapnia in unanaesthetized rats, suggesting its excitatory action via the cystathionine β-synthase-H2 S pathway in the medullary raphe. Hydrogen sulfide (H2 S) has been recently recognized as a gasotransmitter alongside carbon monoxide (CO) and nitric oxide (NO). H2 S seems to modulate the ventilatory and thermoregulatory responses to hypoxia and hypercapnia. However, the action of the H2 S in the medullary raphe (MR) on the ventilatory responses to hypercapnia remains to be elucidated. The present study aimed to assess the role of H2 S in the MR (a brainstem region that contains CO2 -sensitive cells and participates in the ventilatory adjustments to hypercapnia) in the ventilatory responses to hypercapnia in adult unanaesthetized Wistar rats. To do so, aminooxyacetic acid (AOA; a cystathionine β-synthase (CBS) enzyme inhibitor), propargylglycine (PAG; a cystathionine γ-lyase enzyme inhibitor) and sodium sulfide (Na2 S; an H2 S donor) were microinjected into the MR. Respiratory frequency (fR ), tidal volume (VT ), ventilation ( ), oxygen consumption ( ) and body temperature (Tb ) were measured under normocapnic (room air) and hypercapnic (7% CO2 ) conditions. H2 S concentration within the MR was determined. Microinjection of the drugs did not affect fR , VT and during normocapnia when compared to the control group. However, the microinjection of AOA, but not PAG, attenuated fR and during hypercapnia in comparison to the vehicle group, but had no effects on Tb . In addition, we observed an increase in the endogenous production of H2 S in the MR during hypercapnia. Our findings indicate that endogenously produced H2 S in the MR plays an excitatory role in the ventilatory response to hypercapnia, acting through the CBS-H2 S pathway.

5-HT neurons of the medullary raphe contribute to respiratory control in toads

Air-breathing vertebrates undergo respiratory adjustments when faced with disturbances in the gas composition of the environment. In mammals, the medullary raphe nuclei are involved in the neuronal pathway that mediates the ventilatory responses to hypoxia and hypercarbia. We investigate whether the serotoninergic neurons of the medullary raphe nuclei of toads (Rhinella diptycha) play a functional role in respiratory control during resting conditions (room air), hypercarbia (5% CO2), and hypoxia (5% O2). The raphe nuclei were located and identified based on the location of the serotoninergic neurons in the brainstem. We then lesioned the medullary raphe (raphe pallidus, obscurus and magnus) with anti-SERT-SAP and measured ventilation in both control and lesioned groups and we observed that serotonin (5-HT) specific chemical lesions of the medullary raphe caused reduced respiratory responses to both hypercarbia and hypoxia. In summary, we report that the serotoninergic neurons of the medullary raphe of the cururu toad Rhinella diptycha participate in the chemoreflex responses during hypercarbia and hypoxia, but not during resting conditions. This current evidence in anurans, together with the available data in mammals, brings insights to the evolution of brain sites, such as the medullary raphe, involved in the ventilatory chemoreflex in vertebrates.