Central Respiratory Neurons Research Articles

Adaptation of Respiratory-Related Brain Regions to Long-Term Hypercapnia: Focus on Neuropeptides in the RTN.

Long-term hypercapnia is associated with respiratory conditions including obstructive sleep apnea, chronic obstructive pulmonary disease and obesity hypoventilation syndrome. Animal studies have demonstrated an initial (within hours) increase in ventilatory drive followed by a decrease in this response over the long-term (days–weeks) in response hypercapnia. Little is known about whether changes in the central respiratory chemoreflex are involved. Here we investigated whether central respiratory chemoreceptor neurons of the retrotrapezoid nucleus (RTN), which project to the respiratory pattern generator within the ventral respiratory column (VRC) have a role in the mechanism of neuroplasticity associated with long-term hypercapnia. Adult male C57BL/6 mice (n = 5/group) were used. Our aims were (1) to determine if galanin, neuromedin B and gastrin-releasing peptide gene expression is altered in the RTN after long-term hypercapnia. This was achieved using qPCR to measure mRNA expression changes of neuropeptides in the RTN after short-term hypercapnia (6 or 8 h, 5 or 8% CO2) or long-term hypercapnia exposure (10 day, 5 or 8% CO2), (2) in the mouse brainstem, to determine the distribution of preprogalanin in chemoreceptors, and the co-occurrence of the galanin receptor 1 (GalR1:Gi-coupled receptor) with inhibitory GlyT2 ventral respiratory column neurons using in situ hybridization (ISH) to better characterize galaninergic RTN-VRC circuitry, (3) to investigate whether long-term hypercapnia causes changes to recruitment (detected by cFos immunohistochemistry) of respiratory related neural populations including the RTN neurons and their galaninergic subset, in vivo. Collectively, we found that hypercapnia decreases neuropeptide expression in the RTN in the short-term and has the opposite effect over the long-term. Following long term hypercapnia, the number of RTN galanin neurons remains unchanged, and their responsiveness to acute chemoreflex is sustained; in contrast, we identified multiple respiratory related sites that exhibit blunted chemoreflex activation. GalR1 was distributed in 11% of preBötC and 30% of BötC glycinergic neurons. Our working hypothesis is that during long-term hypercapnia, galanin co-release from RTN neurons may counterbalance glutamatergic inputs to respiratory centers to downscale energetically wasteful hyperventilation, thereby having a role in neuroplasticity by contributing to a decrease in ventilation, through the inhibitory effects of galanin.

The General Anesthetic Isoflurane Bilaterally Modulates Neuronal Excitability

SummaryVolatile anesthetics induce hyperactivity during induction while producing anesthesia at higher concentrations. They also bidirectionally modulate many neuronal functions. However, the neuronal mechanism is unclear. The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices, including sodium leak (NALCN) currents and voltage-gated sodium channels (Nav) currents. Isoflurane at sub-anesthetic concentrations increased the spontaneous firing rate of CA3 pyramidal neurons, whereas anesthetic concentrations of isoflurane decreased the firing rate. Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance but minimally inhibited Nav currents. Isoflurane at anesthetic concentrations depressed Nav currents and action potential amplitudes. Isoflurane at sub-anesthetic concentrations depolarized resting membrane potential (RMP) of neurons, whereas hyperpolarized the RMP at anesthetic concentrations. Isoflurane at low concentrations induced hyperactivity in vivo, which was diminished in NALCN knockdown mice. In conclusion, enhancement of NALCN by isoflurane contributes to its bidirectional modulation of neuronal excitability and the hyperactivity during induction.

Effects of eugenol on respiratory burst generation in newborn rat brainstem-spinal cord preparations

Eugenol is contained in several plants including clove and is used as an analgesic drug. In the peripheral and central nervous systems, this compound modulates neuronal activity through action on voltage-gated ionic channels and/or transient receptor potential channels. However, it is unknown whether eugenol exerts any effects on the respiratory center neurons in the medulla. We examined the effects of eugenol on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn rat (P0-P3). The preparations were superfused by artificial cerebrospinal fluid at 25-26°C, and inspiratory C4 ventral root activity was monitored. Membrane potentials of respiratory neurons were recorded in the parafacial region of the rostral ventrolateral medulla. Bath application of eugenol (0.5-1mM) decreased respiratory rhythm accompanied by strong inhibition of the burst activity of pre-inspiratory neurons. After washout, respiratory rhythm partly recovered, but the inspiratory burst duration was extremely shortened, and this continued for more than 60min after washout. The shortening of C4 inspiratory burst by eugenol was not reversed by capsazepine (TRPV1 antagonist) or HC-030031 (TRPA1 antagonist), whereas the depression was partially blocked by GABAA antagonist bicuculline and glycine antagonist strychnine or GABAB antagonist phaclofen. A spike train of action potentials in respiratory neurons induced by depolarizing current pulse was depressed by application of eugenol. Eugenol decreased the negative slope conductance of pre-inspiratory neurons, suggesting blockade of persistent Na+ current. These results suggest that changes in both membrane excitability and synaptic connections are involved in the shortening of respiratory neuron bursts by eugenol.

Neural Control of the Upper Airway: Respiratory and State-Dependent Mechanisms.

Upper airway muscles subserve many essential for survival orofacial behaviors, including their important role as accessory respiratory muscles. In the face of certain predisposition of craniofacial anatomy, both tonic and phasic inspiratory activation of upper airway muscles is necessary to protect the upper airway against collapse. This protective action is adequate during wakefulness, but fails during sleep which results in recurrent episodes of hypopneas and apneas, a condition known as the obstructive sleep apnea syndrome (OSA). Although OSA is almost exclusively a human disorder, animal models help unveil the basic principles governing the impact of sleep on breathing and upper airway muscle activity. This article discusses the neuroanatomy, neurochemistry, and neurophysiology of the different neuronal systems whose activity changes with sleep-wake states, such as the noradrenergic, serotonergic, cholinergic, orexinergic, histaminergic, GABAergic and glycinergic, and their impact on central respiratory neurons and upper airway motoneurons. Observations of the interactions between sleep-wake states and upper airway muscles in healthy humans and OSA patients are related to findings from animal models with normal upper airway, and various animal models of OSA, including the chronic-intermittent hypoxia model. Using a framework of upper airway motoneurons being under concurrent influence of central respiratory, reflex and state-dependent inputs, different neurotransmitters, and neuropeptides are considered as either causing a sleep-dependent withdrawal of excitation from motoneurons or mediating an active, sleep-related inhibition of motoneurons. Information about the neurochemistry of state-dependent control of upper airway muscles accumulated to date reveals fundamental principles and may help understand and treat OSA. © 2016 American Physiological Society. Compr Physiol 6:1801-1850, 2016.

Effects of the Olfactory Bulbs on the Activity of Reticular Neurons of the Bulbar Respiratory Center in Hypoxia

The study reported here seeks to explain the roles of different limbic system formations in controlling respiration in conditions of oxygen insufficiency. The effects of stimulation of the olfactory bulbs (OB) on medulla oblongata respiratory center (RC) neurons during exposure to hypoxia were studied in a laboratory barochamber. Stimulation of the OB in normoxia had a predominantly activatory influence on the baseline activity of neurons. During hypoxia, the responses of medulla oblongata respiratory center neurons to stimulation of the OB were phasic in nature. At the first stage of hypoxia (at an altitude of 4000–5000 m), on the background of hypoxic activation of neurons, the activatory influence of this formation on the spike activity of neurons persisted, albeit weakly. At an altitude of 7500–8000 m, (severe hypoxia phase), on the background of hypoxic suppression of medulla oblongata neuron activity and respiration overall, the activatory effect of stimulation of the OB on the spiked activity of neurons in the bulbar respiratory center was more marked than in the first stage. The results of these experiments provide grounds for the suggestion that the OB is among the limbic system formations with predominantly activatory influences on RC neurons. After lowering the animals to normal atmospheric pressure, there was gradual restoration of initial values for both baseline neuron activity and their responses to stimulation.

Blockade of Na+/H+ exchanger type 3 causes intracellular acidification and hyperexcitability via inhibition of pH-sensitive K+ channels in chemosensitive respiratory neurons of the dorsal vagal nucleus in rats

Extracellular pH (pHe) and intracellular pH (pHi) are important factors for the excitability of chemosensitive central respiratory neurons that play an important role in respiration and obstructive sleep apnea. It has been proposed that inhibition of central Na(+)/H(+) exchanger 3 (NHE-3), a key pHi regulator in the brainstem, decreases the pHi, leading to membrane depolarization for the maintenance of respiration. However, how intracellular pH affects the neuronal excitability of respiratory neurons remains largely unknown. In this study, we showed that NHE-3 mRNA is widely distributed in respiration-related neurons of the rat brainstem, including the dorsal vagal nucleus (DVN). Whole-cell patch clamp recordings from DVN neurons in brain slices revealed that the standing outward current (Iso) through pH-sensitive K(+) channels was inhibited in the presence of the specific NHE-3 inhibitor AVE0657 that decreased the pHi. Exposure of DVN neurons to an acidified pHe and AVE0657 (5 μmol/L) resulted in a stronger effect on firing rate and Iso than acidified pHe alone. Taken together, our results showed that intracellular acidification by blocking NHE-3 results in inhibition of a pH-sensitive K(+) current, leading to synergistic excitation of chemosensitive DVN neurons for the regulation of respiration.

Amyloid β (Aβ) Peptide Directly Activates Amylin-3 Receptor Subtype by Triggering Multiple Intracellular Signaling Pathways

The two age-prevalent diseases Alzheimer disease and type 2 diabetes mellitus share many common features including the deposition of amyloidogenic proteins, amyloid β protein (Aβ) and amylin (islet amyloid polypeptide), respectively. Recent evidence suggests that both Aβ and amylin may express their effects through the amylin receptor, although the precise mechanisms for this interaction at a cellular level are unknown. Here, we studied this by generating HEK293 cells with stable expression of an isoform of the amylin receptor family, amylin receptor-3 (AMY3). Aβ1-42 and human amylin (hAmylin) increase cytosolic cAMP and Ca(2+), trigger multiple pathways involving the signal transduction mediators protein kinase A, MAPK, Akt, and cFos. Aβ1-42 and hAmylin also induce cell death during exposure for 24-48 h at low micromolar concentrations. In the presence of hAmylin, Aβ1-42 effects on HEK293-AMY3-expressing cells are occluded, suggesting a shared mechanism of action between the two peptides. Amylin receptor antagonist AC253 blocks increases in intracellular Ca(2+), activation of protein kinase A, MAPK, Akt, cFos, and cell death, which occur upon AMY3 activation with hAmylin, Aβ1-42, or their co-application. Our data suggest that AMY3 plays an important role by serving as a receptor target for actions Aβ and thus may represent a novel therapeutic target for development of compounds to treat neurodegenerative conditions such as Alzheimer disease.

Muscles bring breathing alive

Increases in ventilation, heart rate and systolic blood pressure accompany muscle exercise. Two neural mechanisms contribute to these effects. One of these is centrally mediated motor command, in which there is parallel activation of locomotor and autonomic brain circuits, while the other is feedback from muscle afferent receptors. The relative influence of the latter has been the focus of much attention in recent years.The population of afferent fibres in skeletal muscle is dominated by the small myelinated group III and unmyelinated group IV nerve fibres. Many of these thin fibre muscle afferents are sensitive to metabolites of muscle contraction, and it is well accepted that they play a significant role in the cardiovascular and respiratory response to exercise. Amongst the group III afferents are those that respond to mechanical deformation, and in the last 10 years good evidence has been provided to show that they also play a role in the cardiovascular changes associated with exercise (Hayes & Kaufman, 2001; Gladwell & Coote, 2002). Surprisingly, the role of these thin fibre muscle mechanoreceptors in respiratory control during exercise has been somewhat neglected, although it would be expected that cardiovascular and ventilatory changes are tightly integrated. The paper by Bruce & White (2011) in this issue of Experimental Physiology addresses this issue. In the early 1960s, Kao and colleagues published a series of papers describing cross-perfusion experiments in anaesthetized dogs in order to isolate potential sources of respiratory stimuli. They concluded that ‘It seems necessary to postulate a receptor in skeletal muscle which is responsible for the great intensity of ventilation during muscle exercise’ (see Kao, 1963). In human studies it is less clear, there being evidence for and against an effect of muscle contractions on ventilation. In particular, the close linear relationship between the work of muscles and oxygen consumption argues against a significant role for skeletal muscle mechanoreceptors. This would not be the case for muscle metaboreceptors. In humans, the metaboreceptors can be studied without the presence of command signals, by trapping metabolites produced during contraction with circulatory occlusion of the limb. In the last 10 years, a method has been developed that enables the muscle mechanoreceptor reflex to be studied, also in isolation of central command. This is achieved by passive stretch of a muscle group by dorsiflexion of the foot (Gladwell & Coote, 2002). These techniques are cleverly exploited in the paper by Bruce & White (2011), in which the influence on ventilation, the heart and blood pressure of either metaboreceptors or mechanoreceptors and both together are tested. The ingenious part of the study was to increase the subliminal fringe of the central neuronal circuits by increasing the excitability of central respiratory and other autonomic neurones by raising the inspired carbon dioxide concentration. This enabled the central synaptic effects to summate sufficiently to activate the respiratory and cardiovascular effectors so they could be observed and measured. The experiments controlled for the effects of metabolite sensitization of muscle afferents, hypercapnia-induced elevation of central respiratory drive and central command. The results convincingly show that facilitation of central neurones by hypercapnia results in a significant increase in ventilation when either muscle mechanoreceptors alone or muscle metaboreceptors alone are activated. The cardiovascular responses were also enhanced. This is the first clear indication that, during natural exercise, it is likely that thin fibre mechanoreceptors in skeletal muscles provide a significant signal to central autonomic pools of neurones that act together with the metaboreceptors and central command signals to ensure that the oxygen demands of exercise are precisely met. These results are not only important to the understanding of normal cardiorespiratory control during exercise but may also help to explain the reduced exercise capacity in patients with heart failure, hypertension and chronic obstructive pulmonary disease. In these patients, abnormal cardiorespiratory responses and intolerance of exercise may be linked to inappropriate reflex information generated by the muscle, as recently highlighted by Koba et al. (2009). The paper by Bruce & White (2011) adds weight to the conclusion of the study by Koba et al. (2009) that in these patients, accentuation of muscle mechanoreceptor signals is responsible for the enhanced cardiorespiratory responses in these disease states.

Anesthetic Activation of Central Respiratory Chemoreceptor Neurons Involves Inhibition of a THIK-1-Like Background K+ Current

At surgical depths of anesthesia, inhalational anesthetics cause a loss of motor response to painful stimuli (i.e., immobilization) that is characterized by profound inhibition of spinal motor circuits. Yet, although clearly depressed, the respiratory motor system continues to provide adequate ventilation under these same conditions. Here, we show that isoflurane causes robust activation of CO(2)/pH-sensitive, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) of the rodent brainstem, in vitro and in vivo. In brainstem slices from Phox2b-eGFP mice, the firing of pH-sensitive RTN neurons was strongly increased by isoflurane, independent of prevailing pH conditions. At least two ionic mechanisms contributed to anesthetic activation of RTN neurons: activation of an Na(+)-dependent cationic current and inhibition of a background K(+) current. Single-cell reverse transcription-PCR analysis of dissociated green fluorescent protein-labeled RTN neurons revealed expression of THIK-1 (TWIK-related halothane-inhibited K(+) channel, K(2P)13.1), a channel that shares key properties with the native RTN current (i.e., suppression by inhalational anesthetics, weak rectification, inhibition by extracellular Na(+), and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO(2) levels. In these animals, isoflurane transiently enhanced activity of the respiratory system, an effect that was most prominent at low levels of respiratory drive and mediated primarily by an increase in respiratory frequency. These data indicate that inhalational anesthetics cause activation of RTN neurons, which serve an important integrative role in respiratory control; the increased drive provided by enhanced RTN neuronal activity may contribute, in part, to maintaining respiratory motor activity under immobilizing anesthetic conditions.

Cough reflex is additively potentiated by inputs from the laryngeal and tracheobonchial receptors and enhanced by stimulation of the central respiratory neurons

The cough is an essential airway defense reflex. In this study we investigated the coordination of inputs from the laryngeal and tracheobronchial receptors in the cough reflex. In 15 beagle dogs (7-9 kg) lightly anesthetized with intravenous profobol (20-30 mg/kg/h), the cough response was elicited with mechanical stimulation of either the vocal chord or tracheal bifurcation. Simultaneous stimulation of both sites increased all the parameters of cough strength, that is, mean pleural pressure (P (pl)), mean expiratory flow, number of cough bouts, and cough duration, in comparison with stimulation of the sites individually. The increases in mean P (pl) and cough duration reached statistical significance (13.3 vs. 18.4 cmH(2)O and 13.3 vs. 18.2 s, respectively). When the anesthetic level became deeper, the prolongation of cough duration almost disappeared, but the augmentation of mean P (pl) was much less affected. During stimulation of the central respiratory neurons by intravenous dimorphoramine or acute hyperoxic hypercapnia, the cough strength increased significantly. We concluded that inputs from the laryngeal and tracheobonchial cough receptors acted in concert and potentiated the cough reflex. Furthermore, stimulation of the central respiratory neurons may increase the intensity of a cough response.

Hypoxia-induced modulation of the respiratory CPG

Despite recent advances in our understanding of the neural control of breathing, the precise cellular, synaptic, and molecular mechanisms underlying the generation and modulation of respiratory rhythm remain largely unknown. This lack of fundamental knowledge in the field of neural control of respiration is likely due to the complexity of the mammalian brain where synaptic connectivity between central respiratory neurons, motor neurons and their peripheral counterparts cannot be mapped reliably. We have therefore developed an invertebrate model system wherein the essential elements of the central pattern generator (CPG), the motor neurons and the peripheral chemosensory cells involved in respiratory control have been worked out both in vivo and in vitro. We discuss our recent identification of peripheral, hypoxia sensitive chemoreceptor elements in a sensory organ of the pulmonate freshwater pond snail Lymnaea stagnalis, which provide an excitatory drive to the respiratory CPG neuron RPeD1 via direct chemical synaptic connections. Further studies using this unique invertebrate model system may reveal highly conserved principles of CPG neuromodulation that will remain relevant to more complex mammalian systems.

Effects of leucine-enkephalin on potassium currents in neurons in the rat respiratory center in vitro

Experiments to identify the neuronal mechanisms underlying the respiratory activity of the opioid peptide leucine-enkephalin were performed on transverse slices of the rat brainstem in voltage-clamped conditions; studies addressed the effects of this peptide (10 nM-1 microM) on the potassium A current and the inward potassium current of neurons in two areas of the respiratory center: the ventrolateral area of the solitary tract nucleus and the pre-Bötzinger complex. The parameters of the A current assessed in all respiratory center neurons studied showed no change in the presence of leucine-enkephalin. At the same time, leucine-enkephalin produced reversible increases in the amplitude of the inward potassium current. These results provide evidence that the inhibitory effect of leucine-enkephalin at the level of respiratory center neurons is at least in part explained by its stimulatory action on the inward potassium current but is not associated with modulation of the potassium A current.

High Sensitivity to Neuromodulator-Activated Signaling Pathways at Physiological [K+] of Confocally Imaged Respiratory Center Neurons in On-Line-Calibrated Newborn Rat Brainstem Slices

The pre-Bötzinger complex (PBC) inspiratory center remains active in a transverse brainstem slice. Such slices are studied at high (8-10 mM) superfusate [K+], which could attenuate the sensitivity of the PBC to neuromodulators such as opiates. Findings may also be confounded because slice boundaries, drug injection sites, or location of rhythmogenic interneurons are rarely verified histologically. Thus, we first generated PBC slices with defined boundaries using novel "on-line histology" based on our finding that rostrocaudal extensions of brainstem respiratory marker nuclei are constant in newborn rats between postnatal days 0-4. At physiological superfusate [K+] (3 mM), 500- and 600-microm-thick slices with the PBC in the center and the caudal boundary 0.70 and 0.76 mm caudal to the facial motonucleus generated rhythm for >2 and approximately 4 h, respectively. Rhythm was abolished by low nanomolar concentrations of the mu-opiate receptor agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin). After spontaneous arrest of bursting, rhythm was reactivated at clinically relevant or physiological concentrations by 3,5-dihydroxyphenylglycine, thyrotropin-releasing hormone, or rolipram, each affecting distinct second-messenger pathways. Two-photon/confocal Ca2+ imaging revealed that these agents reactivated the same PBC neurons initially active in 3 mM [K+]. The data show that "calibrated" PBC slices at physiological [K+] generate rhythm with a high sensitivity to neuromodulators for extended time periods, whereas spontaneous "in vitro apnea" is an important tool to study the interaction of signaling pathways that modulate rhythm. Our approaches and findings provide the basis for a pharmacological and structure-function analysis of the isolated respiratory center in a histologically well defined substrate at physiological [K+].

The effects of leucine-enkephalin on the membrane potential and activity of rat respiratory center neurons in vitro

Studies of transverse slices of Wistar rat brainstem using a patch clamp technique addressed the effects of the opioid peptide leucine-enkephalin (10 nM-1 microM) on the membrane potential and pattern of spontaneous activity of neurons in two parts of the respiratory center: the ventrolateral area of the solitary tract nucleus and the pre-Bötzinger complex. Leucine-enkephalin induced membrane hyperpolarization of respiratory center neurons and decreased the level of spike activity in spontaneously active cells. In pre-Bötzinger complex neurons showing a burst pattern of activity, leucine-enkephalin decreased the burst frequency, and two cells showed a transition from burst activity to tonic activity. These results provide evidence that the mechanism of the central respiratory activity of leucine-enkephalin results from its direct action on the membranes of respiratory center neurons.

Central respiratory modulation of barosensitive neurones in rat caudal ventrolateral medulla

The sympathetic nerves that maintain blood pressure are modulated by the central respiratory generator. Neurones in the rostral ventrolateral medulla (RVLM) that drive this sympathetic nerve activity (SNA) also display central respiratory drive (CRD)-related activity, suggesting integration of respiratory and cardiovascular regulatory systems within the brainstem. Whether CRD-related activity in the RVLM is due to direct inputs from central respiratory neurones or modulation of cardiovascular-related neurones that influence the RVLM is not known. The caudal ventrolateral medulla (CVLM) contains GABAergic neurones that tonically inhibit presympathetic RVLM neurones and are essential for the production of numerous cardiovascular reflexes. The present study sought to determine whether cardiovascular-related GABAergic neurones in the CVLM display CRD-related activity. The firing patterns of individual barosensitive CVLM neurones were examined in relation to phrenic nerve activity in chloralose-anaesthetized, ventilated, neuromuscularly blocked, vagotomized rats. Histograms of phrenic-triggered CVLM neuronal activity showed that all baro-activated CVLM neurones displayed one of four patterns of CRD-related activity: (i) inspiratory peak (n = 15), (ii) inspiratory depression (n = 15), (iii) inspiratory peak with postinspiratory depression (n = 10), and (iv) postinspiratory peak (n = 9). A subset of each type of CVLM neurone was identified as GABAergic by individually filling the recorded neurone with biotinamide and observing expression of GAD67 mRNA by in situ hybridization (n = 10). These data suggest that the activity of GABAergic neurones in the CVLM is regulated by cardiovascular and respiratory inputs, and baro-activated GABAergic CVLM neurones may contribute to CRD-related modulation of presympathetic RVLM neurones and SNA.

Thyroliberin blocks the potassium A-current in neurons in the respiratory center of adult rats in vitro

Thyroliberin is a neuropeptide with marked respiratory activity. The neuronal mechanisms underlying this activity were addressed in experiments on transverse slices of brainstem from adult rats in conditions of membrane potential clamping to study effect effects of thyroliberin (10 nM) on the potassium A-current in neurons of two areas of the respiratory center--the ventrolateral areas of the solitary tract nucleus and the pre-Betzinger complex. The A-current, seen in all study neurons in the respiratory center, was partially and reversibly blocked by thyroliberin. A significant reduction in the amplitude of the current was accompanied by an increase in the inactivation constant. The effect of thyroliberin on the amplitude of the A-current was analogous to that of 5 mM 4-aminopyridine. These results show that the stimulatory effects of thyroliberin at the level of respiratory center neurons is at least partly explained by its ability to block the potassium A-current.

Malfunction of Respiratory-Related Neuronal Activity in Na+, K+-ATPase α2 Subunit-Deficient Mice Is Attributable to Abnormal Cl-Homeostasis in Brainstem Neurons

Na+, K+-ATPase 2 subunit gene (Atp1a2) knock-out homozygous mice (Atp1a2-/-) died immediately after birth resulting from lack of breathing. The respiratory-related neuron activity in Atp1a2-/- was investigated using a brainstem-spinal cord en bloc preparation. The respiratory motoneuron activity recorded from the fourth cervical ventral root (C4) was defective in Atp1a2-/- fetuses of embryonic day 18.5. The C4 response to electrical stimulation of the ventrolateral medulla (VLM) recovered more slowly in Atp1a2-/- than in wild type during superfusion with Krebs' solution, consistent with the high extracellular GABA in brain of Atp1a2-/-. Lack of inhibitory neural activities in VLM of Atp1a2-/- was observed by optical recordings. High intracellular Cl- concentrations in neurons of the VLM of Atp1a2-/- were detected in gramicidin-perforated patch-clamp recordings. The alpha2 subunit and a neuron-specific K-Cl cotransporter KCC2 were coimmunoprecipitated in a purified synaptic membrane fraction of wild-type fetuses. Based on these results, we propose a model for functional coupling between the Na+, K+-ATPase alpha2 subunit and KCC2, which excludes Cl- from the cytosol in respiratory center neurons.

Pontine cholinergic mechanisms and their impact on respiratory regulation

Activation of pontomedullary cholinergic neurons may directly and indirectly cause depression of respiratory motoneuronal activity, activation of respiratory premotor neurons and acceleration of the respiratory rate during REM sleep, as well as activation of breathing during active wakefulness. These effects may be mediated by distinct subpopulations of cholinergic neurons. The relative inactivity of cholinergic neurons during slow-wave sleep also may contribute to the depressant effects of this state on breathing. Cholinergic muscarinic and nicotinic receptors are expressed in central respiratory neurons and motoneurons, thus allowing cholinergic neurons to act on the respiratory system directly. Additional effects of cholinergic activation are mediated indirectly by noradrenergic, serotonergic and other neurons of the reticular formation. Excitatory and suppressant respiratory effects with features of natural states of REM sleep or active wakefulness can be elicited in urethane-anesthetized rats by pontine microinjections of the cholinergic agonist, carbachol. Carbachol models help elucidate the neural basis of respiratory disorders associated with central cholinergic activation.

Effects of thyroliberin on membrane potential and the pattern of spontaneous activity of neurons in the respiratory center in in vitro studies in rats.

Patch-clamp experiments on transverse brainstem slices from rats were performed to study the effects of thyroliberin (10(-8) M) on the membrane potential and spontaneous activity of neurons in two areas of the respiratory center: the ventrolateral area of the solitary tract nucleus and the pre-Botzinger complex. Thyroliberin induced membrane depolarization of neurons in the respiratory center and increased their spike activity. The pattern of activity of neurons in the pre-Botzinger complex showed decreases in the time intervals between the beginnings of bursts in response to thyroliberin. In some cases, thyroliberin led to the appearance of spike activity in initially "silent" neurons; "silent" neurons in the solitary tract nucleus became tonically active, while those in the pre-Botzinger complex showed burst activity. These results provide evidence for the existence of an indirect regulatory influence for thyroliberin on respiratory center neurons, operating at the membrane level.

Effects of chronic dermal exposure to nonlethal doses of methyl parathion on brain regional acetylcholinesterase and muscarinic cholinergic receptors in female rats

The in vivo and in vitro effects of methyl parathion, a phosphorothionate insecticide, on cholinergic neurotransmitter systems in the brain of rats were investigated. Three groups of adult female rats received 0, 0.1, or 1.0 mg/kg methyl parathion via dermal exposure for 95 days. Exposure to 0.1 mg/kg methyl parathion produced inhibition of AChE in the caudate-putamen and thalamic nuclei, whereas 1.0 mg/kg resulted in inhibition of AChE in most brain regions. The same doses of methyl parathion had no effect on [(3)H]QNB binding to muscarinic receptors in the brain regions examined. The in vitro study demonstrated that methyl parathion causes preferential inhibition of AChE and [(3)H]QNB binding in specific brain regions. As an inhibitor of AChE, methyl paraoxon was 1,000-fold more potent than was methyl parathion. Similarly, methyl paraoxon showed brain region-specific inhibition of the enzyme. Generally, the brain stem was highly sensitive to organophosphate-induced inhibition of AChE activity and [(3)H]QNB binding. Because central respiratory neurons gather in the brain stem, preferential effects there and in other brain regions may underlie lethal toxicity of methyl parathion and other organophosphates.