Astrocytic contribution to glutamate-related central respiratory chemoreception in vertebrates.

Central respiratory chemoreceptors play a key role in the respiratory homeostasis by sensing CO2 and H+ in brain and activating the respiratory neural network. This ability of specific brain regions to respond to acidosis and hypercapnia is based on neuronal and glial mechanisms. Several decades ago, glutamatergic transmission was proposed to be involved as a main mechanism in central chemoreception. However, a complete identification of mechanism has been elusive. At the rostral medulla, chemosensitive neurons of the retrotrapezoid nucleus (RTN) are glutamatergic and they are stimulated by ATP released by RTN astrocytes in response to hypercapnia. In addition, recent findings show that caudal medullary astrocytes in brainstem can also contribute as CO2 and H+ sensors that release D-serine and glutamate, both gliotransmitters able to activate the respiratory neural network. In this review, we describe the mammalian astrocytic glutamatergic contribution to the central respiratory chemoreception trying to trace in vertebrates the emergence of several components involved in this process.

d-serine regulation of the timing and architecture of the inspiratory burst in neonatal mice.

d-serine, released from mouse medullary astrocytes in response to increased CO2 levels, boosts the respiratory frequency to adapt breathing to physiological demands. We analyzed in mouse neonates, the influence of d-serine upon inspiratory/expiratory durations and the architecture of the inspiratory burst, assessed by pwelch's power spectrum density (PSD) and continuous wavelet transform (CWT) analyses. Suction electrode recordings were performed in slices from the ventral respiratory column (VRC), site of generation of the respiratory rhythm, and in brainstem-spinal cord (en bloc) preparations, from the C5 ventral roots, containing phrenic fibers that in vivo innervate and drive the diaphragm, the main inspiratory muscle. In en bloc and slice preparations, d-serine (100 µM) reduced the expiratory, but not the inspiratory duration, and increased the frequency and the regularity of the respiratory rhythm. In en bloc preparations, d-serine (100 µM) also increased slightly the amplitude of the integrated inspiratory burst and the area under the curve of the integrated inspiratory burst, suggesting a change in the recruitment or the firing pattern of neurons within the burst. Time-frequency analyses revealed that d-serine changed the burst architecture of phrenic roots, widening their frequency spectrum and shifting the position of the core of firing frequencies towards the onset of the inspiratory burst. At the VRC, no clear d-serine induced changes in the frequency-time domain could be established. Our results show that d-serine not only regulates the timing of the respiratory cycle, but also the recruitment strategy of phrenic motoneurons within the inspiratory burst.

D-serine released by astrocytes in brainstem regulates breathing response to CO2 levels.

Central chemoreception is essential for adjusting breathing to physiological demands, and for maintaining CO2 and pH homeostasis in the brain. CO2-induced ATP release from brainstem astrocytes stimulates breathing. NMDA receptor (NMDAR) antagonism reduces the CO2-induced hyperventilation by unknown mechanisms. Here we show that astrocytes in the mouse caudal medullary brainstem can synthesize, store, and release D-serine, an agonist for the glycine-binding site of the NMDAR, in response to elevated CO2 levels. We show that systemic and raphe nucleus D-serine administration to awake, unrestrained mice increases the respiratory frequency. Application of D-serine to brainstem slices also increases respiratory frequency, which was prevented by NMDAR blockade. Inhibition of D-serine synthesis, enzymatic degradation of D-serine, or the sodium fluoroacetate-induced impairment of astrocyte functions decrease the basal respiratory frequency and the CO2-induced respiratory response in vivo and in vitro. Our findings suggest that astrocytic release of D-serine may account for the glutamatergic contribution to central chemoreception.Astrocytes are involved in chemoreception in brainstem areas that regulate breathing rhythm, and astrocytes are known to release D-serine. Here the authors show that astrocyte release of D-serine contributes to CO2 sensing and breathing in brainstem slices, and in vivo in awake unrestrained mice.

Development and pH sensitivity of the respiratory rhythm of fetal mice in vitro.

In newborn and adult mammals, chemosensory drive exerted by CO(2) and H(+) provides an essential tonic input: without it the rhythm of respiration is abolished. It is not known, however, whether this chemosensory drive and the respiratory rhythm appear simultaneously during development. In isolated brainstem-spinal cord preparations from fetal mice, we determined at what stage of fetal life the respiratory rhythm appeared in third to fifth cervical ventral roots (phrenic motoneurons) and whether this fetal rhythm was sensitive to chemosensory inputs. A respiratory-like rhythm consisting of short duration bursts of discharges recurring at 2-16 min(-1) was detected in two of nine embryonic day 13 fetuses; it was abolished by transection of the spinal cord between the first to second cervical segments and was phase-related to rhythmic activity from medullary units of the ventral respiratory group. At embryonic day 13, it coexisted with a slow rhythm (0.1-2.0 min(-1)) of long duration bursts of action potentials which was generated by the spinal cord. At later fetal stages, the respiratory-like rhythm became more robust and of higher frequency, while the spinal cord rhythm became less obvious. At all fetal stages, acidification of the superfusion medium from pH 7.5-7.2 or 7.4-7.3 or 7.4 to 7.2 increased the frequency of both the respiratory-like and the spinal cord rhythms. In addition, acidification reduced the amplitude of the integrated burst activity of the spinal cord rhythm of embryonic day 13-embryonic day 16 fetuses and the respiratory-like rhythm of embryonic day 17 and older fetuses. Our results indicate that the rhythms transmitted by phrenic motoneurons during fetal development are chemosensitive from early fetal stages. Through its effects on induction and patterning of the rhythm, chemosensory drive may play a role in activity-dependent formation of respiratory neural networks.

Optical recording from respiratory pattern generator of fetal mouse brainstem reveals a distributed network.

Unfailing respiration depends on neural mechanisms already present in mammals before birth. Experiments were made to determine how inspiratory and expiratory neurons are grouped in the brainstem of fetal mice. A further aim was to assess whether rhythmicity arises from a single pacemaker or is generated by multiple sites in the brainstem. To measure neuronal firing, a fluorescent calcium indicator dye was applied to embryonic central nervous systems isolated from mice. While respiratory commands were monitored electrically from third to fifth cervical ventral roots, activity was measured optically over areas containing groups of respiratory neurones, or single neurones, along the medulla from the facial nucleus to the pre-Bötzinger complex. Large optical signals allowed recordings to be made during individual respiratory cycles. Inspiratory and expiratory neurones were intermingled. A novel finding was that bursts of activity arose in a discrete area intermittently, occurring during some breaths, but failing in others. Raised CO2 partial pressure or lowered pH increased the frequency of respiration; neurons then fired reliably with every cycle. Movies of activity revealed patterns of activation of inspiratory and expiratory neurones during successive respiratory cycles; there was no evidence for waves spreading systematically from region to region. Our results suggest that firing of neurons in immature respiratory circuits is a stochastic process, and that the rhythm does not depend on a single pacemaker. Respiratory circuits in fetal mouse brainstem appear to possess a high safety factor for generating rhythmicity, which may or may not persist as development proceeds.

In vitro approach to the chemical drive of breathing.

Since its introduction two decades ago, the isolated brain stem-spinal cord preparation of neonatal rodents has been the preferred method used to reveal the mystery underlying the genesis of the respiratory rhythm. Little research using this in vitro approach has focused on the study of the central respiratory chemosensitivity. Some unexpected findings obtained with the brain stem-spinal cord preparation have added new questions that challenge our previous theoretic framework. Some of these findings are addressed here.

Control of respiration in the isolated central nervous system of the neonatal opossum, Monodelphis domestica.

Respiration represents an unusual motor activity with respect to its development. As newly born mammals enter the world, their limb movements are not coordinated; time and experience are required for effective performance to be achieved. Yet the rhythm of respiration is of necessity functionally perfected and unfailing at birth. Inspiratory and expiratory motor neurons are already able to fire at appropriate rates, under the command of rhythmically active neurons in the medulla. In this review, we discuss refinements of control present in the newborn opossum, particularly with respect to mechanisms that allow adaptation of respiration to changes in the level of activity or in the outside environment. Our own studies have been aimed at analyzing respiration at the earliest stages, and at establishing the way in which important variables influence inspiration and expiration. To this end, we have used the central nervous system (CNS) of a neonatal opossum, isolated in its entirety and maintained in culture. Although the opossum is unable to walk and highly immature at birth, its respiration is regular and unfailing. The isolated CNS survives, undergoes development, and maintains its neural activity and fine structure in vitro. Moreover, fictive respiration persists for over a day or longer at rates similar to those of the intact pup. The effects of altered pH, of increased temperature, and of drugs known to alter respiratory rhythm in intact animals can be measured directly, by electrical recordings made from medullary neurons or ventral roots. As in a slice, fluids of different composition can be applied focally, through micropipettes to the surface of the ventral medulla, or diffusely to the brainstem, With highly localized application of procaine hydrochloride (2%) to selected areas of the ventral medulla, the respiratory rhythm is reduced or abolished. As in adult mammals, both the rate and the amplitude of respiration simultaneously increase in response to lowered pH (6.5-.7.1) or to topical application of 1.0 microM carbachol. Conversely, as expected, the rate and amplitude decrease in response to increased pH (pH 7.5-7.7), or 100 microM scopolamine. Two characteristic features of the control of respiration in the neonatal opossum are evident from such tests. First, changes in rate are achieved by changes in the duration of the expiratory phase of respiration. This result suggests that the timing of the respiratory cycle in the neonatal opossum is controlled by an expiratory instead of an inspiratory "off-switch". Second, the rate and the amplitude of the respiratory excursions can be controlled independently, depending on the stimulus. For example, an increase in temperature increases the rate of fictive respiration without changing its amplitude, whereas noradrenaline decreases the rate while increasing the amplitude. Thus, changes of timing and amplitude need not go hand in hand. The opossum CNS offers a favorable preparation for the analysis of neural mechanisms that generate and modulate a motor rhythm, as the animal develops from embryonic to adult stages.

Why does the central nervous system not regenerate after injury?

Spinal cord injuries in humans and in other mammals are never followed by regrowth. In recent years, considerable progress has been made in analyzing mechanisms that promote and inhibit regeneration. The focus of this review is changes that occur in the transition period in development when the central nervous system (CNS) changes from being able to regenerate to the adult state of failure. In our experiments we have used the neonatal opossum (Monodelphis domestica), which corresponds to a 14-day embryonic rat or mouse. The CNS isolated from an opossum pup and maintained in culture shows dramatic regeneration. Fibers grow through and beyond lesions and reform synaptic connections with their targets. Similarly, anesthetized neonatal pups attached to the mother recover the ability to walk after complete spinal cord transection. Although the CNS isolated from a 9-day-old animal will regenerate in vitro, CNS from a 12-day-old will not. This is the stage at which glial cells in the CNS develop. Present research is devoted toward molecular screening to determine which growth-promoting molecules decrease during development, which inhibitory molecules increase, and which receptors on growing axons become altered. Despite progress in many laboratories, major hurdles must be overcome before patients can hope to be treated. Nevertheless, the picture today is not as discouraging as it was: one can think of strategies for research on spinal cord injury so as to promote regeneration and restore function.

Chemosensory and cholinergic stimulation of fictive respiration in isolated CNS of neonatal opossum.

1. The aim of the present experiments was to characterize the central chemical drive of fictive respiration in the isolated CNS of the newborn opossum, Monodelphis domestica. This opossum preparation, in contrast to those of neonatal rats and mice, produces respiratory rhythm of high frequency in vitro. 2. Fictive respiration was recorded from C3-C5 ventral roots of the isolated CNS of 4- to 14-day-old opossums using suction electrodes. At room temperature (21-23 degrees C) the frequency of respiration was 43 +/- 5.3 min-1 (mean +/- S.E.M., n = 50) in basal medium Eagle's medium (BMEM) equilibrated with 5% CO2-95% O2, pH 7.37-7.40. Respiratory discharges remained regular throughout 8 h experiments and continued for more than 20 h in culture. 3. Superfusion of the brainstem confirmed that solutions of pH 6.3-7.2 increased both the amplitude and frequency of respiration. High pH solutions (7.5-7.7) had the opposite effect and abolished the rhythm at pH 7.7. Addition of ACh (50-100 microM) or carbachol (0.01-10 microM) to the brainstem superfusion also increased the amplitude and frequency of respiratory activity, as did physostigmine (50-100 microM) or neostigmine (20-50 microM). Conversely, scopolamine (50-100 microM) reduced the amplitude and frequency of the basal respiratory rhythm by about 30%. 4. H(+)- and cholinergic-sensitive areas on the surface of the isolated CNS were explored with a small micropipette (outer tip diameter, 100 microns) filled with BMEM (pH 6.5) or 1 microM carbachol. Carbachol applied to H(+)- and cholinergic-sensitive areas in the ventral medulla mimicked the changes of respiratory pattern produced by low pH application. Responses to altered pH and carbachol were abolished by scopolamine (50 microM). Histochemistry demonstrated several medullary groups of neurons stained for acetylcholinesterase. The superficial location of one of these groups coincided with a functional and anatomically well-defined pH- and carbachol-sensitive area placed medial to the hypoglossal roots. 5. Exploration of chemosensitive areas revealed that application of drugs or solutions of different pH to a single well-defined spot could have selective and distinctive effects upon amplitude and frequency of respiratory activity. 6. These results show that fictive respiration in the isolated CNS of the newborn opossum is tonically driven by chemical- and cholinergic-sensitive areas located on the ventral medulla, the activity of which regulates frequency and amplitude of respiration. They suggest that a cholinergic relay, although not essential for rhythm generation, is involved in the central pH chemosensory mechanism, or that cholinergic and chemical inputs converge upon the same input pathway to the respiratory pattern generator.

Incorporation of amino acids into the axoplasm is enhanced by electrical stimulation of the fiber.

The effect of sustained electrical stimulation upon the incorporation of amino acids into the axoplasm was studied in the goldfish Mauthner (M) axon with light autoradiography. An extracellular pulse of tracers applied between M-axons in the medulla resulted in a local and substantial labeling of the M-axoplasm and a faint labeling of the M-perikaryon 4-5 mm away from the site of injection. After 18 h of direct electrical stimulation of the M-axon at 0.3-0.8 Hz, the local incorporation of amino acids into the M-axoplasm doubled. This enhancement declined to reach the baseline within 24 h. A 4 h electrical stimulation did not enhance the incorporation. Transynaptic activation of the M-neuron through the auditory input at 0.1-0.2 Hz for 18 h did not raise the amino acid incorporation in the M-axoplasm. We conclude that electrical discharge of the axon modulates the local incorporation of amino acids into the axoplasm.

Generation of the respiratory rhythm: modelling the inspiratory off switch as a neural integrator.

Neural integration is involved in the respiratory central processing of vagal afferent activity originating from lung mechanoreceptors. In the present work it is assumed that not only vagal activity, but also the incoming activities of the other inputs which converge into the "inspiratory off-switch", are processed there by integration. Moreover, integration is considered to be essential for the inspiratory off-switch function. This assumption leads to a novel mathematical model, in which constancy of the inspiratory off-switch threshold and absence of a central inspiratory activity input to the inspiratory off-switch are postulated. The model accurately predicts the relationship between timing and depth of breathing measured experimentally under several steady-state conditions, including peripheral and central chemoreceptor stimulation, increased body temperature, increased elastic and flow resistive loads, and bilateral vagotomy. The model also provides adequate simulation of the integrative vagal processing and questions the idea that "accommodative" vagal processing originates from a separate process different to integration at the inspiratory off-switch. The assumption that integration is essential for the inspiratory off-switching suggests a simple basis for interpreting, electrophysiological properties of bulbo-pontine inspiratory neurons in terms of a neuronal circuit that acts like an integrator. The prediction of a new type of inspiratory neuron is discussed.

The chick chorioallantoic membrane promotes survival of co-transplanted rat carotid bodies and nodose ganglia.

Carotid bodies and nodose ganglia, removed from adult rats, were co-implanted onto the chorioallantois of 6- to 12-day chick embryos. Implants were rapidly vascularized and incorporated into the chorioallantoic membrane, where they survived and grew for up to 12 days. The morphological characteristics of grafted tissues were largely preserved. Regenerating axons from nodose neurons invaded the carotid body and contacted some glomus cells through morphologically immature synapses. Thus, the chick chorioallantoic membrane may be a useful substrate to study carotid chemoreceptor-sensory neuron interactions.

Functional recovery of the ventilatory chemoreflexes after partial chronic denervation of the nucleus tractus solitarius.

In pentobarbitone-anesthetized cats breathing spontaneously, we studied whether excision of one petrosal ganglion would modify the reflex efficacy of the remaining carotid and aortic chemoafferences in ventilatory control. Resting ventilation was not affected shortly after the ganglionectomy, but decreased sensitivities and reactivities for changes in tidal volume and respiratory frequency were revealed by dose-response curves for ventilatory chemoreflexes evoked by NaCN i.v. After 2 weeks of ganglionectomy, basal tidal volume was increased, being slightly reduced by contralateral carotid neurotomy, but persisting above control after section of all buffer nerves. The ventilatory chemosensory drive--tested by breathing 100% O2--was unmodified with respect to the acute condition, but the tonic ventilatory influence exerted by the right carotid nerve was diminished. Dose-response curves for reflex changes in tidal volume exhibited increased sensitivity, while those for changes in respiratory frequency showed increased reactivity. Thus, partial chemosensory denervation of the nucleus tractus solitarius triggers a slowly developing increase in the reflex efficacy of the remaining chemosensory inputs. The recovery of sensitivity for reflex changes in tidal volume required the presence of contralateral carotid afferents, while the increased reactivity in respiratory frequency needed the integrity of aortic afferents. The results also suggest an enhanced contribution of central structures other than chemosensory inputs in respiratory control after partial deafferentation.

Correlative contribution of carotid and aortic afferences to the ventilatory chemosensory drive in steady-state normoxia and to the ventilatory chemoreflexes induced by transient hypoxia.

The contributions of the peripheral arterial chemoreceptors to the tonic and phasic reflex ventilatory regulation were studied in spontaneously breathing pentobarbitone anesthetized adult cats. The chemosensory drive during eucapnic normoxia was inferred from the transient ventilatory effects induced by anesthetic blockade of the buffer nerves. Aortic nerves block did not modify ventilation. Carotid nerves block provoked transient ventilatory depression, decreasing VT by 46% and fR by 26%, followed by recovery to steady-state values in VT, fR and PETCO2. Changes in PETCO2 were correlated with those in VT, but not with those in fR. The ventilatory effects of blocking a given carotid nerve were more intense when the contralateral carotid nerve was already blocked. This effect may be an expression of hypoadditive interactions between carotid nerves inputs with respect to chemosensory drive of ventilation. Analysis of the dose-response curves for the ventilatory reflexes evoked by NaCN i.v., before and after blockade of the buffer nerves, revealed major contributions of the carotid nerves, with small contributions of the aortic nerves to the those responses to high doses of NaCN. The contributions of each carotid nerve to the tonic chemosensory drive and to the phasic ventilatory chemoreflexes were highly correlated (rs = 0.90; p less than 0.01). We propose that a family of modulatory functions may describe the effects exerted by the peripheral arterial chemoreceptors upon the tonic ventilatory drive in normoxia and the phasic reflex responses evoked by hypoxia. While the carotid nerves mediated modulation is evident in normoxia, that provided by both aortic nerves is only expressed during pronounced hypoxia.