Medullary Raphe Nuclei Research Articles

RNA sequencing to profile transcriptional changes within the medullary raphe: potential mechanisms of central neuroplasticity driving the recovery of eupneic ventilation after bilateral carotid body denervation (713.7)

Bilateral carotid body denervation (CBD) in rats elicits eupneic (resting) hypoventilation 1‐2 d post‐CBD, which relatively rapidly returns to baseline values (14‐15 d). We have previously shown increases in brainstem serotonin (5‐HT) turnover and the 5‐HT transporter (SERT) acutely after CBD, leading to the hypothesis that the recovery of eupneic ventilation following CBD may be associated and/or initiated by activation of the medullary raphe nuclei (MRN). To test this hypothesis, we collected medullary raphe tissue punches (~1mm3) from rats euthanized acutely (1‐2 days) after sham (n=11) or CBD (n=10), or 15 days after sham (n=10) or CBD (n=10) surgery. Eupneic PaCO2 was increased acutely (p<0.001) and chronically (p<0.001) after CBD compared to pre‐CBD control, but PaCO2 15 days post‐CBD was not different than time‐control sham rats demonstrating recovery following CBD. Total RNA was collected using a phenol‐chloroform extraction protocol and quantified/qualified using an Agilent Bioanalyzer. 100 – 600 ng of total RNA was collected with an integrity number of 7 or greater for all samples, indicating high RNA quality. 12 cDNA libraries were created (3 or 4 per group x 4 groups) using the Illumina TruSeq RNA Sample Prep LS protocol for mRNA sequencing, from which all 12 libraries yielded pure cDNA. Ongoing mRNA sequencing will allow for the eventual coupling of our comprehensive physiologic measurements with raphe gene expression to provide unique insights into potential mechanisms of recovery or plasticity ventilatory control after CBD.Grant Funding Source: Supported by NIHHL097033, T32HL007852.

Purinergic signaling contributes to CO2‐sensitivity of neurons in the retrotrapezoid nucleus but not nucleus of the solitary tract or medullary raphe (872.6)

Central chemoreception is the mechanism by which the brain controls breathing in response to changes in tissue CO2/H+. Several brainstem regions are thought to contribute to chemoreception including the caudal nucleus of the solitary tract (cNTS), medullary raphe and retrotrapezoid nucleus (RTN). Although the molecular basis of chemoreception remains unclear, evidence suggests that RTN chemoreception involves intrinsic H+ sensing by an unidentified K+ channel and purinergic signaling possibly from CO2‐sensitive astrocytes. However, it is not clear whether purinergic signaling influences CO2‐responsiveness of other putative chemoreceptors. The goal of this study is to determine whether purinergic signaling modulates basal activity or CO2‐responsivness of neurons in the cNTS or medullary raphe. We use cell‐attached current clamp techniques to characterize firing rate responses to 15% CO2 and focal application of ATP (1mM) under control conditions and in the presence of a P2‐receptor blocker (PPADS, 100 µM). Chemosensitive neurons in the cNTS or medullary raphe were identified in brain slices (300 µm thick) from neonatal rat pups (P7‐12) bases on a 蠅 0.5 Hz firing rate response to 15% CO2. We found that CO2‐sensitive neurons in the cNTS, but not the medullary raphe, respond to focal ATP application with ~3 fold increase in firing rate. However, bath application of PPADS had no effect on baseline activity or CO2‐responsivness of neurons in either region. Immunohistochemical analysis using an astrocyte‐specific marker (aldehyde dehydrogenase 1L1) suggests that the RTN has a higher density of astrocytes compared to the cNTS or medullary raphe. Together, these results suggest that purinergic signaling is a unique feature of RTN chemoreception.Grant Funding Source: Supported by NLBI (HL 104101) and AHA (11PRE7580037)

Purinergic signalling contributes to chemoreception in the retrotrapezoid nucleus but not the nucleus of the solitary tract or medullary raphe

Several brain regions are thought to function as important sites of chemoreception including the nucleus of the solitary tract (NTS), medullary raphe and retrotrapezoid nucleus (RTN). In the RTN, mechanisms of chemoreception involve direct H(+)-mediated activation of chemosensitive neurons and indirect modulation of chemosensitive neurons by purinergic signalling. Evidence suggests that RTN astrocytes are the source of CO2-evoked ATP release. However, it is not clear whether purinergic signalling also influences CO2/H(+) responsiveness of other putative chemoreceptors. The goals of this study are to determine if CO2/H(+)-sensitive neurons in the NTS and medullary raphe respond to ATP, and whether purinergic signalling in these regions influences CO2 responsiveness in vitro and in vivo. In brain slices, cell-attached recordings of membrane potential show that CO2/H(+)-sensitive NTS neurons are activated by focal ATP application; however, purinergic P2-receptor blockade did not affect their CO2/H(+) responsiveness. CO2/H(+)-sensitive raphe neurons were unaffected by ATP or P2-receptor blockade. In vivo, ATP injection into the NTS increased cardiorespiratory activity; however, injection of a P2-receptor blocker into this region had no effect on baseline breathing or CO2/H(+) responsiveness. Injections of ATP or a P2-receptor blocker into the medullary raphe had no effect on cardiorespiratory activity or the chemoreflex. As a positive control we confirmed that ATP injection into the RTN increased breathing and blood pressure by a P2-receptor-dependent mechanism. These results suggest that purinergic signalling is a unique feature of RTN chemoreception.

Central and Peripheral Expression and Distribution of NUCB2/nesfatin-1

Nesfatin-1 is the N-terminal fragment of nucleobindin-2 (NUCB2). The antibody against nesfatin-1 recognizes both full length of NUCB2 and nesfatin-1, thus the immunolabeling represents NUCB2/nesfatin-1. It has been found that NUCB2/nesfatin-1 is widely distributed in the rodent central nervous system. The immunoreactivity is more intensive in the brain autonomic centers that regulate feeding, neuroendocrine and cardiovascular functions, such as the hypothalamic paraventricular nucleus, supraoptic nucleus, lateral hypothalamic area, Edinger-Westphal nucleus, locus coeruleus, dorsal vagal complex and medullary raphe nuclei. In neurons, NUCB2/nesfatin-1 is located in the soma and primary dendrites, not in nerve fibers. NUCB2/nesfatin-1 is co-localized with several neurotransmitters involved in regulation of food intake, autonomic and neuroendocrine functions, including oxytocin, vasopressin, neuropeptide Y, cocaine- and amphetamine-regulated transcript, proopiomelanocortin, α-melanocyte-stimulating hormone, melanin-concentrating hormone, leptin, mammalian target of rapamycin, urocortin-1, corticotropin-releasing factor and serotonin. In the periphery, NUCB2/nesfatin-1 is located mainly in the pituitary, gastric mucosa where it coexists with ghrelin, and pancreatic endocrine cells containing insulin. Nesfatin-1 is detectable in the cerebrospinal fluid of rats. NUCB2/nesfatin-1 is measurable in the plasma, and altered under different conditions in rodents and humans, such as immune challenge, high fat diet and exercise, anorexia nervosa, anxiety and depression. Anatomical data suggest that nesfatin-1 is a unique neuroendocrine peptide that may be involved in regulation of homeostasis.

Orexin neurons are indispensable for prostaglandin E2‐induced fever and defence against environmental cooling in mice

We recently showed using prepro-orexin knockout (ORX-KO) mice and orexin neuron-ablated (ORX-AB) mice that orexin neurons in the hypothalamus, but not orexin peptides per se, are indispensable for stress-induced thermogenesis. To examine whether orexin neurons are more generally involved in central thermoregulatory mechanisms, we applied other forms of thermogenic perturbations, including brain prostaglandin E2 (PGE2) injections which mimic inflammatory fever and environmental cold exposure, to ORX-KO mice, ORX-AB mice and their wild-type (WT) litter mates. ORX-AB mice, but not ORX-KO mice, exhibited a blunted PGE2-induced fever and intolerance to cold (5°C) exposure, and these findings were similar to the results previously obtained with stress-induced thermogenesis. PGE2-induced shivering was also attenuated in ORX-AB mice. Both mutants responded similarly to environmental heating (39°C). In WT and ORX-KO mice, the administration of PGE2 and cold exposure activated orexin neurons, as revealed by increased levels of expression of c-fos. Injection of retrograde tracer into the medullary raphe nucleus revealed direct and indirect projection from the orexin neurons, of which the latter seemed to be preserved in the ORX-AB mice. In addition, we found that glutamate receptor antagonists (D-(-)-2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione) but not orexin receptor antagonists (SB334867 and OX2 29) successfully inhibited PGE2-induced fever in WT mice. These results suggest that orexin neurons are important in general thermogenic processes, and their importance is not restricted to stress-induced thermogenesis. In addition, these results indicate the possible involvement of glutamate in orexin neurons implicated in PGE2-induced fever.

P1-20. Respiratory changes after lesions of the medullary raphe nuclei

It is known that respiratory movements change after chemical or electrical stimulation of the midline regions of the brain stem, including raphe obscurus, raphe pallidus and raphe magnus. We previously reported that neurons located in these regions had respiratory-related activity and a part of the neurons projected to the phrenic nuclei, suggesting the midline regions are involved in the control of respiration. However, it is unclear whether or not these neurons play a crucial role in respiration. To elucidate the role, we tested effects of chemical lesions of the medullary raphe nuclei on respiration using spontaneously breathing Sprague–Dawley rats which were anesthetized with pentobarbital sodium. Local injection of kainic acid into the medullary midline induced a transient increase in amplitude and frequency of diaphragm activities, and the frequency subsequently decreased. Then, apnea was elicited 20 min after the injection. Injection sites were located 1.5–3.0 mm rostral to the obex. The results suggest that the caudal medullary raphe nuclei may participate in the control of respiratory movements in rats.

P.3.a.001 Sex differences in early adolescent functioning among persons later hospitalised for schizophrenia

In order to study the regulation of co-localized monoamine and peptide neurotransmitters in the medullary raphe nuclei (MRN), we determined whether inhibition of serotonin (5-HT) synthesis affected levels of preprotachykinin (PPT; the prohormone precursor of substance P) mRNA in the MRN. Adult rats received p-chlorophenylalanine (pCPA), an irreversible inhibitor of tryptophan hydroxylase (TPH), via Alzet minipumps. TPH activity was inhibited by 70–80% for 3 weeks following pump implantation. During this period Northern mRNA analysis revealed that PPT mRNA levels in the MRN were increased 1.5–2-fold. The pCPA-induced increase was specific for PPT mRNA since no change was detected in mRNA coding for neuron-specific enolase (NSE; a constitutive neuronal protein) or 28 S ribosomal RNA. To determine whether fetal inhibition of 5-HT synthesis affected development of PPT mRNA in the MRN, pregnant rats were administered pCPA via Alzet minipump implanted on embryonic day 8. In pCPA-treated litters TPH activity was decreased by 60–70% from E16 to postnatal day 3 (P3), returning to control levels by P8. Northern mRNA analysis revealed that PPT mRNA levels increased 2.4-fold of control levels at P1. Infusion of pCPA for one week resulted in an earlier increase in PPT mRNA levels, suggesting that birth was not required to elicit the surge in PPT message. These results support the hypothesis that alterations in 5-HT metabolism have regulatory consequences for co-localized substance P formation in the MRN.

Distinct regional and subcellular localization of the actin‐binding protein filamin a in the mature rat brain

Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include regulation of protein trafficking and surface expression. Although the functions of FLNa during brain development are well studied, little is known on its expression, distribution, and function in the adult brain. Here we characterize in detail the neuroanatomical distribution and subcellular localization of FLNa in the mature rat brain, by using two antisera directed against epitopes at either the N' or the C' terminus of the protein, further validated by mRNA expression. FLNa was widely and selectively expressed throughout the brain, and the intensity of immunoreactivity was region dependent. The most intensely FLNa-labeled neurons were found in discrete neuronal systems, including basal forebrain structures, anterior nuclear group of thalamus, and hypothalamic parvocellular neurons. Pyramidal neurons in neocortex and hippocampus and magnocellular cells in basolateral amygdaloid nucleus were also intensely FLNa immunoreactive, and strong FLNa labeling was evident in the pontine and medullary raphe nuclei and in sensory and spinal trigeminal nuclei. The subcellular localization of FLNa was evaluated in situ as well as in primary hippocampal neurons. Punctate expression was found in somata and along the dendritic shaft, but FLNa was not detected in dendritic spines. These subcellular distribution patterns were recapitulated in hippocampal and neocortical pyramidal neurons in vivo. The characterization of the expression and subcellular localization of FLNa may provide new clues to the functional roles of this cytoskeletal protein in the adult brain.

Selective innervation of upper and lower thoracic spinal segments by medullary raphe neurons in felines

The medullary raphe nuclei mediate a variety of physiological responses, including the regulation of blood pressure. We have demonstrated that vestibular stimulation results in distinct changes in blood flow in the upper and lower body, indicating that the nervous system has the capacity to elicit regionally‐specific changes in blood flow. Prior physiological studies have shown that neurons in the rostral ventrolateral medulla (RVLM) are not responsible for this response patterning. In the present study, we ascertained whether neurons in the medullary raphe nuclei have the capacity to independently regulate sympathetic outflow to different body regions. For this purpose, multiple injections of the fluorescent dye Fast Blue were made into the vicinity of the intermediolateral cell column (IML) in T4, whereas multiple injections of Fluoro‐Ruby were made near the IML in T10. Retrogradely labeled neurons were mapped in the medullary raphe nuclei. Neurons that were single‐labeled for each of the dyes and those that were double‐labeled were noted in approximately equal numbers. These findings show that although some medullary raphe neurons regulate sympathetic outflow from multiple spinal segments, others have the capacity to control activity in specific segments.

Exercise-Induced Pain Requires NMDA Receptor Activation in the Medullary Raphe Nuclei

Pain in response to physical activity is common in people with chronic musculoskeletal pain and is likely a barrier to regular exercise, which would lead to a sedentary lifestyle. We recently developed a model of exercise-induced pain that is associated with increased activation of neurons in the medullary raphe nuclei, i.e., the nucleus raphe obscurus (NRO) and nucleus raphe pallidus (NRP). Because the NRO and NRP not only modulate motor output but also respond to noxious stimuli, we hypothesized that the NRO and NRP were key nuclei in the interaction between pain and exercise. We tested whether exercise enhances hyperalgesia through activation of N-methyl D-aspartate (NMDA) receptors in the NRO/NRP. Muscle insult was induced by two injections of pH 5.0 saline 5 d apart into one gastrocnemius muscle. We initially tested whether hyperalgesia developed in mice injected with acidic saline (pH 5.0) into the gastrocnemius muscle immediately after a 30-min or 2-h exercise task or 2 h after a 2-h exercise task. Next, we tested whether blockade of NMDA receptors in the NRO/NRP during the exercise task prevented the development of exercise-induced hyperalgesia. Finally, we evaluated changes in phosphorylation of the NR1 subunit of the NMDA receptor (pNR1) after the exercise task at times in which muscle insult was given in behavioral experiments, i.e., immediately after a 30-min or 2-h exercise task or 2 h after the 2-h exercise task. All exercise conditions enhanced nociception (hyperalgesia) after combining with two injections of pH 5.0 saline. Microinjection of AP5 (1.0-0.1 nmol; 2-amino-5-phophonopenanoate) dose-dependently prevented the development of exercise-induced hyperalgesia. All exercise conditions increased pNR1 in the NRO and NRP. Thus, exercise-induced pain in sedentary mice is associated with increased phosphorylation and activation of NMDA receptors in the NRO/NRP, suggesting that changes in central excitability mediate an interaction between unaccustomed exercise and pain.

Postnatal loss of brainstem serotonin neurones compromises the ability of neonatal rats to survive episodic severe hypoxia

Pet-1(-/-) mice with a prenatal, genetically induced loss of 5-hydroxytryptamine (5-HT, serotonin) neurones are compromised in their ability to withstand episodic environmental anoxia via autoresuscitation. Given the prenatal role of 5-HT neurones in the development of neural networks, here we ask if a postnatal loss of 5-HT neurones also compromises autoresuscitation. We treated neonatal rat pups at postnatal day (P)2-3 with an intra-cisternal injection of 5,7-dihydroxytryptamine (5,7-DHT; ~40 μg; n = 8) to pharmacologically lesion the 5-HT system, or vehicle (control; n = 14). At P7-10 we exposed unanaesthetized treated and control pups to 15 episodes of environmental anoxia (97% N(2), 3% CO(2)). Medullary 5-HT content was reduced 80% by 5,7-DHT treatment (P < 0.001). Baseline ventilation (V(E)), metabolic rate (V(O(2))), ventilatory equivalent (V(E)/V(O(2))), heart rate (HR), heart rate variability (HRV) and arterial haemoglobin saturation (S(aO(2))) were no different in 5-HT-deficient pups compared to controls. However, only 25% of 5-HT-deficient pups survived all 15 episodes of environmental anoxia, compared to 79% of control littermates (P = 0.007). High mortality of 5,7-DHT-treated pups was associated with delayed onset of gasping (P < 0.001), delayed recovery of HR from hypoxic-induced bradycardia (P < 0.001), and delayed recovery of eupnoea from hypoxic-induced apnoea (P < 0.001). Treatment with 5,7-DHT affected neither the gasping pattern once initiated, nor HR, V(E)/V(O(2)) or S(aO(2)) during the intervening episodes of room air. A significant increase in HRV occurred in all animals with repeated exposure, and in 5-HT-deficient pups this increase occurred immediately prior to death. We conclude that a postnatal loss of brainstem 5-HT content compromises autoresuscitation in response to environmental anoxia. This report provides new evidence in rat pups that 5-HT neurones serve a physiological role in autoresuscitation. Our data may be relevant to understanding the aetiology of the sudden infant death syndrome (SIDS), in which there is medullary 5-HT deficiency and in some cases evidence of severe hypoxia and failed autoresuscitation.

Properties of respiratory neurons in medullary raphe nuclei of rats

Properties of respiratory neurons in medullary raphe nuclei of rats

Respiratory Effects of Opioids in Perioperative Medicine

Opioids are widely used to treat acute and chronic pain as well as respiratory distress. There is great variability in opioid-induced side effects due to individual biological factors, patient co-morbidities and drug interactions. Normal respiratory rhythm generation is decreased primarily via inhibititory effects within the pre-Bötzinger complex. Central chemosensitivity to hypercapnia and hypoxia are blunted by opioids at the levels of the retrotrapezoid nucleus, medullary raphe nucles and nucleus tractus solitarius. Opioids also decrease central drive to both respiratory pump muscles and the upper airway dilator muscles. Opioid-induced respiratory depression can be reversed by naloxone, and recent data suggest that 5-HT4(a) agonists and ampakines are effective to reverse some of the opioid-induced respiratory depressant effects. The potentially fatal side effects of respiratory depression within the acute peri-operative setting necessitates effective monitoring of respiratory function in all patients receiving opioid therapy. Each institution needs to develop an optimal organization structure locally to define appropriate methods for avoiding medication errors, titrating opioids to target effect, and monitoring for respiratory side effects.

Active projections to medullary raphe during conditioned fear in the rat

Psychological stress leads to sympathetic and behavioural responses that are mediated by the medullary raphe nuclei (MR). These nuclei receive direct projections from the hypothalamus, which drive the tachycardia, and the periaqueductal gray (PAG), which drives behaviour. To confirm that these projections are active during conditioned fear, we combined fear-induced Fos expression with cholera toxin B retrograde tracing from the MR. Rats were either placed for 30 min in a context where they had previously experienced footshocks (conditioned fear, N=5) or left undisturbed in their homecages (control, N=5) prior to sacrifice. Relative to the total number of double labelled neurons, most of these were found in the hypothalamus (24.5±6.8%) and PAG (34.2±8.8%). Within the PAG, double labelling was found in the dorsomedial (14.9±5.0%), lateral (9.9±2.4%) and ventrolateral columns (9.4±1.9%). The anterior hypothalamus had about the same number of double labelled neurons (5.6±2.1%) as the dorsomedial (5.8±1.9%), paraventricular (5.8±1.3%) and perifornical nuclei (4.4±2.0%). The prefrontal cortex was the third most labelled region (15.0±3.8%). Double labelling was also found in the midbrain tegmentum (11.3±4.6%), nucleus cuneiformis (5.3±2.4%) and A5 (2.0±0.7%) area. These results confirm that the most important active afferents to MR during conditioned fear are in the hypothalamus and PAG.

Projection of respiratory neurons in rat medullary raphe nuclei to the retrotrapezoid nucleus

Projection of respiratory neurons in rat medullary raphe nuclei to the retrotrapezoid nucleus

Fos-Tau-LacZ mice reveal sex differences in brainstem c- fos activation in response to mild carbon dioxide exposure

There are sex differences in the neurochemistry of brainstem nuclei that participate in the control of breathing as well as sex differences in respiratory responses to hypoxia. Central chemoreception refers to the detection within the brain of minute changes in carbon dioxide (CO 2) levels and the subsequent modulation of breathing. Putative central chemoreceptor sites are widespread and include cells located near the ventral surface of the brainstem in the retrotrapezoid nucleus (RTN), in the medullary midline raphe nuclei, and, more dorsally in the medulla, in the nucleus of the solitary tract and in the locus caeruleus at the pontomedullary junction as well as in the fastigial nucleus of the cerebellum. In this study, we ask if the cells that respond to CO 2 differ between the sexes. We used a transgenic mouse with a c- fos promoter driven tau-lacZ reporter construct (FTL) to map the locations of cells in the mouse brainstem and cerebellum that responded to exposure of mice of both sexes to 5% CO 2 or room air (control). X-gal (5-bromo-4-chloro-3-indolyl-beta- d-galactopyranoside) histochemical staining to detect the beta-galactosidase enzyme produced staining in the brains of mice of both sexes in all of the previously identified putative chemoreceptor sites, with the exception of the fastigial nucleus. Notably, the male RTN region contained significantly more x-gal-labeled cells than the female RTN region. In addition to new observations regarding potential sex differences in the retrotrapezoid region, we found the FTL mouse to be a useful tool for identifying cells that respond to the exposure of the whole animal to relatively low concentrations of CO 2.

Effect of Treadmill Exercise on Serotonin Immunoreactivity in Medullary Raphe Nuclei and Spinal Cord Following Sciatic Nerve Transection in Rats

The serotoninergic system modulates nociceptive and locomotor spinal cord circuits. Exercise improves motor function and changes dopaminergic, noradrenergic, and serotonergic central systems. However, the direct relationship between serotonin, peripheral nerve lesion and aerobic treadmill exercise has not been studied. Using immunohistochemistry and optic densitometry, this study showed that the sciatic nerve transection increased the serotoninergic immunoreactivity in neuronal cytoplasm of the magnus raphe nuclei of trained and sedentary rats. In the dorsal raphe nucleus the increase only occurred in sedentary-sham-operated rats. In the spinal cord of trained, transected rats, the ventral horn showed significant changes, while the change in dorsal horn was insignificant. Von Frey's test indicated analgesia in all exercise-trained rats. The sciatic nerve functional index indicated recovery in the trained group. Thus, both the aerobic treadmill exercise training and the nervous lesion appear to contribute to changes in serotonin immunoreactivity.

Localization of serotoninergic neurons that participate in regulating diaphragm activity in the cat

Although a considerable body of literature indicates that serotoninergic neurons affect diaphragm activity both through direct inputs to phrenic motoneurons and multisynaptic connections involving the brainstem respiratory groups, the locations of the serotoninergic neurons that modulate breathing have not been well defined. The present study identified these neurons in cats by combining the transneuronal retrograde transport of rabies virus from the diaphragm with the immunohistochemical detection of the N-terminal region of tryptophan hydroxylase-2 (TPH2), the brain-specific isoform of the enzyme responsible for the initial and rate-limiting step in serotonin synthesis. TPH2-immunopositive neurons were present in the midline raphe nuclei, formed a column in the ventrolateral medulla near the lateral reticular nucleus, and were spread across the dorsal portion of the pons just below the fourth ventricle. In most animals, only a small fraction of neurons (typically <20%) labeled for TPH2 in each of the medullary raphe nuclei and the medullary ventrolateral column were infected with rabies virus. However, the percentage of medullary neurons dual-labeled for both rabies and TPH2 was much higher in animals with very advanced infections where virus had spread transneuronally through many synapses. Furthermore, in all cases, TPH2-immunopositive neurons that were infected by rabies virus were significantly less prevalent in the pons than the medulla. These findings suggest that although serotoninergic neurons with direct influences on diaphragm activity are widely scattered in the brainstem, the majority of these neurons are located in the medulla. Many non-serotoninergic neurons in the raphe nuclei were also infected with rabies virus, indicating that midline cells utilizing multiple neurotransmitters participate in the control of breathing.

Differences between two inbred rat strains in number of neurons expressing K+ ion channels in the medullary raphe nucleus (MRN)

Male (M) and female (F) Dahl salt sensitive (SS) and Brown Norway (BN) inbred rat strains increase pulmonary ventilation by 183 and 37% respectively when exposed to 7% inspired CO2. Since K+ ion channels are hypothesized determinants of ventilatory CO2 sensitivity, we presently determined whether there is a difference between these two rat strains in number of neurons expressing K+ channels in the chemosensitive MRN. Rats were sacrificed, and the brains were perfused, extracted, cyrosectioned at 25μm into 4 series, and alternate sections were stained for Nissl and Task‐1, Kv1.4, or Kir2.3 K+ channel antibodies. From 0‐2mm rostral to obex, the stained neurons in the MRN were microscopically visualized and counted. We found more Nissl stained MRN neurons in BN than in SS rats in both M (6%, P&lt;0.001) and F (8.2%, P&lt;0.001) rats. However, for Task‐1, Kv1.4, and Kir2.3, there were 12, 13.8, and 13.9% respectively more (P&lt;0.001) immunostained MRN neurons in SS than in BN rats. Additionally, F BN rats had 5.3 (P=0.014), 7.3 (P=0.040), and 7.6% (P=0.005) more Task‐1, Kv1.4, and Kir2.3 respectively stained MRN neurons than in F SS rats. We conclude that the number of neurons expressing K+ channels in the MRN is genetically determined. Supported by NIH 25739, HL066579, and the Department of Veterans Affairs.

Stimulation of the rat medullary raphe nuclei induces differential responses in respiratory muscle activity

Neural control circuits that coordinate the motor activity of the diaphragm (DIA) and the geniohyoid muscle (GH) are potentially involved in pathological conditions such as various forms of sleep apnea. Here we investigated a differential role of the raphe magnus (RMg), pallidus (RPa) and the obscurus (ROb) nuclei in the neural control of DIA and GH muscle activity in rats under volatile anesthesia. In order to characterize a topographical organization of the raphe nuclei we analyzed changes in DIA and GH during high-frequency stimulation (HFS, 10-130 Hz, 60 micros pulse width, 40-160 microA, 30s). HFS of the RMg and the ROb induced apnea, in the latter case apnea was associated with massive tonic discharge in the GH. By contrast, HFS of the RPa induced tachypnea. At caudal stimulation sites the tachypnea was accompanied by tonic DIA activity and cessation of GH. These data suggest a differential distribution of inhibitory and excitatory drives of DIA and GH muscles within distinct raphe nuclei.