Activation of microglia and astrocytes in the nucleus tractus solitarius during ventilatory acclimatization to 10% hypoxia in unanesthetized mice.

Nucleus tractus solitarius (NTS) is the integrative sensory relay of autonomic functions in the brainstem. To explore the nonneuronal cellular basis of central chemosensitivity during the first 24 hr of ventilatory acclimatization to hypoxia (VHA), we have investigated glial activation markers in the NTS. Conscious mice (C57/BL6) were placed in a hermetic hypoxia chamber containing a plethysmograph to record ventilation. After 4 days of habituation to the normoxic environment, mice were subjected to physiological hypoxia (10% O2 ) for 1, 6, or 24 hr. To dissociate interactions between microglia and astrocytes, another group received daily minocycline, a microglia activation blocker. By immunochemical localization of astrocytes (GFAP), activated microglia (Cd11b), and total microglia (Iba-1), we identified an oxygen-sensing glial layer in the NTS, in which astrocytes are first activated after 1-6 hr of hypoxia, followed by microglia after 6-24 hr of hypoxia. Minocycline administration suppressed microglial activation and decreased astrocyte activation at 6 hr and VHA at 24 hr of hypoxia. These results suggest that astrocytes contribute to the neuronal response during the first hour of hypoxia, whereas microglial cells, via cross-talk with astrocytes, are involved in the VHA during the first 24 hr of acclimatization.

Extracellular calcium induces quiescence of the low-frequency embryonic motor rhythm in the mouse isolated brainstem.

Although extracellular calcium ionic concentration ([Ca](o) ) is known to increase during late gestation and to drop after parturition, little is known about the influence of [Ca](o) on fetal brain function. We have investigated the influence of [Ca](o) , calcium-sensing receptors/nonselective cation currents (CaSR/NSCC), and GABAergic inhibitions on maturation of brainstem-spinal motor activities: the primary low-frequency embryonic rhythm [LF; silent since embryonic day (E)16] and the fetal respiratory rhythm (RR; emerging at E14-E15). Using in vitro isolated brainstem-spinal cord preparations of mice at different fetal and postnatal (P) stages (E16-P1), we demonstrate that reducing fetal [Ca](o) from 1.2 mM to 0.7 mM at E16-E18 or blocking GABA(A) receptors at E16-P0 reactivates LF and reveals LF-related disturbance of RR at E16-E18. This LF is stopped by adding gadolinium or spermidine (CaSR/NSCC agonists) at E18-P0 or GABA(A) receptor agonists at E16-E18. In contrast, [Ca](o) -induced slowing of RR at E16-E18 is not reproduced by gadolinium and spermidine. We conclude that perinatal CaSR/NSCC and GABA(A) inhibition allow quiescence of the LF, thereby improving functional maturation of the RR.

Developmental basis of the rostro-caudal organization of the brainstem respiratory rhythm generator.

The Hox genetic network plays a key role in the anteroposterior patterning of the rhombencephalon at pre- and early-segmental stages of development of the neural tube. In the mouse, it controls development of the entire brainstem respiratory neuronal network, including the pons, the parafacial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC). Inactivation of Krox20/Egr2 eliminates the pFRG activity, thereby causing life-threatening neonatal apnoeas alternating with respiration at low frequency. Another respiratory abnormality, the complete absence of breathing, is induced when neuronal synchronization fails to develop in the preBötC. The present paper summarizes data on a third type of respiratory deficits induced by altering Hox function at pontine levels. Inactivation of Hoxa2, the most rostrally expressed Hox gene in the hindbrain, disturbs embryonic development of the pons and alters neonatal inspiratory shaping without affecting respiratory frequency and apnoeas. The same result is obtained by the Phox2a(+/-) mutation modifying the number of petrosal chemoafferent neurons, by eliminating acetylcholinesterase and by altering Hox-dependent development of the pons with retinoic acid administration at embryonic day 7.5. In addition, embryos treated with retinoic acid provide a mouse model for hyperpnoeic episodic breathing, widely reported in pre-term neonates, young girls with Rett's syndrome, patients with Joubert syndrome and adults with Cheyne-Stokes respiration. We conclude that specific respiratory deficits in vivo are assignable to anteroposterior segments of the brainstem, suggesting that the adult respiratory neuronal network is functionally organized according to the rhombomeric, Hox-dependent segmentation of the brainstem in embryos.

Pregnenolone sulfate enhances long-term potentiation in CA1 in rat hippocampus slices through the modulation of N-methyl-D-aspartate receptors.

Among the different steroids found in the brain, pregnenolone sulfate (3beta-hydroxy-5-pregnen-20-one-3-sulfate; PREGS) is known to enhance hippocampal-associated memory. The present study employs rat hippocampal slices to investigate the ability of PREGS to modulate long-term potentiation (LTP), a phenomenon considered as a model of synaptic plasticity related to memory processes. LTP (3 x 100 Hz/1 sec within 2 min), implicated essentially glutamatergic transmission, for which the different synaptic events could be pharmacologically dissociated. We show that PREGS enhances LTP in CA1 pyramidal neurons at nanomolar concentrations and exhibits a bell-shaped concentration-response curve. The maximal effect of PREGS on both induction and maintenance phases of LTP is observed at 300 nM and requires 10 min of superfusion. Although PREGS does not change the N-methyl-D-aspartate (NMDA) component of the field potentials (fEPSPs) isolated in the presence of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in Mg2+-free artificial cerebrospinal fluid, PREGS does enhance the response induced by NMDA application (50 microM, 20 sec). PREGS does not modify the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component of the fEPSPs isolated in the presence of 100 microM DL-2-amino-7-phosphopentanoic acid (DL-AP5) or its potentiation induced by a single tetanic stimulation and the response induced by AMPA application (10 microM, 10 sec). Furthermore, PREGS does not affect the recurrent inhibition of the fEPSPs mediated by gamma-aminobutyric acid type A (GABA(A)) receptor. In conclusion, this study shows the ability of PREGS to enhance LTP in CA1 by accentuating the activity of NMDA receptors. This modulation of LTP might mediate the steroid-induced enhancement of memory.

Protein kinase C-dependent potentiation of intracellular calcium influx by sigma1 receptor agonists in rat hippocampal neurons.

Intracellular calcium concentration ([Ca2+]i) plays a major role in neuronal excitability, especially that triggered by the N-methyl-d-aspartate (NMDA)-sensitive glutamatergic receptor. We have previously shown that sigma1 receptor agonists potentiate NMDA receptor-mediated neuronal activity in the hippocampus and recruit Ca2+-dependent second messenger cascades (e.g., protein kinase C; PKC) in brainstem motor structures. The present study therefore assessed whether the potentiating action of sigma1 agonists on the NMDA response observed in the hippocampus involves the regulation of [Ca2+]i and PKC. For this purpose, [Ca2+]i changes after NMDA receptor activation were monitored in primary cultures of embryonic rat hippocampal pyramidal neurons using microspectrofluorometry of the Ca2+-sensitive indicator Fura-2/acetoxymethyl ester in the presence of sigma1 agonists and PKC inhibitors. We show that successive activations of the sigma1 receptor by 1-min pulses of (+)-benzomorphans or (+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (JO-1784) concomitantly with glutamate time dependently potentiated before inconstantly inhibiting the NMDA receptor-mediated increase of [Ca2+]i, whereas 1,3-di-o-tolyl-guanidine, a mixed sigma1/sigma2 agonist, did not significantly modify the glutamate response. Both potentiation and inhibition were prevented by the selective sigma1 antagonist N,N-dipropyl-2-[4-methoxy-3-(211phenylethoxy) phenyl]-ethylamine monohydrochloride (NE-100). Furthermore, only (+)-benzomorphans could induce [Ca2+]i influx by themselves after a brief pulse of glutamate. A pretreatment with the conventional PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2,3-a] pyrrolo [3,4-c] carbazole (Gö-6976) prevented the potentiating effect of (+)-benzomorphans on the glutamate response. Our results provide further support for a general mechanism for the intracellular sigma1 receptor to regulate Ca2+-dependent signal transduction and protein phosphorylation.

Respiratory function in adult mice lacking the mu-opioid receptor: role of delta-receptors.

Mice lacking the mu-opioid receptor (MOR) provide a unique model to determine whether opioid receptors are functionally interactive. Recent results have shown that respiratory depression produced by delta-opioid receptor agonists is suppressed in mice lacking the mu-opioid receptor. Here we investigated the involvement of mu- and delta-opioid receptors in the control of ventilation and mu/delta receptor interactions in brainstem rhythm-generating structures. Unrestrained MOR-/- and wild-type mice showed similar ventilatory patterns at rest and similar chemosensory responses to hyperoxia (100% O2), hypoxia (10% O2) or hypercapnia (5%CO2-95%O2). Blockade of delta-opioid receptors with naltrindole affected neither the ventilatory patterns nor the ventilatory responses to hypoxia in MOR-/- and wild-type mice. In-vitro, respiratory neurons were recorded in the pre-Bötzinger complex of thick brainstem slices of MOR-/- and wild-type young adult mice. Respiratory frequency was not significantly different between these two groups. The delta2 receptor agonist deltorphin II (0.1-1.0 microM) decreased respiratory frequency in both groups whereas doses of the delta1 receptor agonist enkephalin[D-Pen2,5] (0.1-1.0 microM) which were ineffective in wild-type mice significantly decreased respiratory frequency in MOR-/- mice. We conclude that deletion of the mu-opioid receptor gene has no significant effect on ensuing respiratory rhythm generation, ventilatory pattern, or chemosensory control. In MOR-/- mice, the loss of respiratory-depressant effects of delta2-opioid receptor agonists previously observed in vivo does not result from a blunted response of delta receptors in brainstem rhythm-generating structures. These structures show an unaltered response to delta2-receptor agonists and an augmented response to delta1-receptor agonists.

Intracellular sigma1 receptor modulates phospholipase C and protein kinase C activities in the brainstem.

Most physiological effects of sigma1 receptor ligands are sensitive to pertussis toxin, suggesting a coupling with cell membrane-bound G proteins. However, the cloning of the sigma1 receptor has allowed the identification of an intracellular protein anchored on the endoplasmic reticulum. Here, we show, using the isolated adult guinea pig brainstem preparation, that activation of the sigma1 receptor results in its translocation from the cytosol to the vicinity of the cell membrane and induces a robust and rapid decrease in hypoglossal activity, which is mediated by phospholipase C. The subsequent activation of protein kinase C beta1 and beta2 isoforms and the phosphorylation of a protein of the same molecular weight as the cloned sigma1 receptor lead to a desensitization of the sigma1 motor response. Our results indicate that the intracellular sigma1 receptor regulates several components implicated in plasma membrane-bound signal transduction. This might be an example of a mechanism by which an intracellular receptor modulates metabotropic responses.

Coherent inspiratory oscillation of cranial nerve discharges in perfused neonatal cat brainstem in vitro.

1. To understand the neural organization of respiratory movement control and its developmental transformation, we studied the temporal characteristics of inspiratory activities, especially nerve-to-nerve short-term synchronization, in an in vitro preparation of the isolated, perfused brainstem of kittens aged 0-14 days (postnatal day (P) 0-14). 2. In the inspiratory discharges of facial, vagus, glossopharyngeal and hypoglossal nerves, a stable oscillation with a period of 30-40 ms (i.e. approximately 30 Hz) was observed in all preparations examined. In addition, we demonstrated that this oscillation presents a strong short-term synchrony between distinct inspiratory nerves. This nerve-to-nerve synchronization was already apparent at approximately 12 h after birth. The degree of synchronization as evaluated by coherence spectral analysis was larger than 0.85 in all cases at any age examined. 3. This nerve-to-nerve coherence was not affected by changes in temperature (28-36 degrees C), whereas respiratory rate, oscillation frequency and oscillation amplitude as estimated by power spectral analysis were highly temperature sensitive. 4. The nerve-to-nerve synchronization, as well as the approximately 30 Hz oscillation, remained unchanged after a pontomedullary transection, indicating that the medullary network, completely isolated from other structures and afferents, is sufficient to produce both fast oscillation and nerve-to-nerve synchronization. 5. Based on these observations in vitro, we conclude that nerve-to-nerve coherent inspiratory oscillation generated in the brainstem is already functional early in life.

Involvement of NMDA receptors in the respiratory phase transition is different in the adult guinea pig in vivo and in the isolated brain stem preparation.

1. We investigated the involvement of N-methyl-D-aspartate (NMDA) receptors in the respiratory pattern in an in vitro preparation of adult brain stem compared with in vivo conditions in the guinea pig. 2. In vivo, combining administration of the NMDA channel blocker dizocilpine (MK-801) (3 mg/kg) with a surgical section of the vagus nerves induced an apneustic type of respiration characterized by long inspiratory "holds," as has been shown in other species. The same effect was observed in hypothermic animals (30 degrees C). 3. The isolated in vitro brain stems from these apneustic animals did not present a prolonged inspiratory phase. A second dose of dizocilpine (100 microM perfused vascularly did not induce apneusis, even after increasing brain stem temperature to 35.5 degrees C. 4. In another group of isolated brain stems of adult guinea pigs anesthetized with pentobarbital sodium before decapitation, we perfused dizocilpine and NMDA through the basilar artery. The duration of periodic inspiratory motor activity recorded from the hypoglossal nerve was unaffected by dizocilpine (1-100 microM) or the competitive NMDA antagonist D- or DL-2-amino-5-phosphonopentanoic acid (100 microM and 1 mM), although respiratory frequency decreased. The increase in respiratory activity produced by vascularly perfused NMDA (25-100 microM) was blocked by dizocilpine (100 microM). 5. We conclude that the central mechanism of inspiratory termination in the vagotomized adult guinea pig requires the activation of NMDA receptors in vivo but not in vitro. This difference is not due to the hypothermic environment in vitro. Possible mechanisms for phase switching in vitro are discussed.

Localization of chemosensitive structures in the isolated brainstem of adult guinea-pig.

1. Central respiratory chemosensitivity has been intensively examined but some questions remain unsolved; namely, what is the nature of the stimulus (fixed acid and/or CO2) and where is the site of brainstem chemosensitivity (near the ventral medullary surface or structures deeper within the brainstem)? To examine these questions, we used the in vitro isolated brainstem of adult guinea-pig perfused independently through the basilar artery and the bath. 2. Respiratory motor output was recorded with a suction electrode from cranial hypoglossal (XII) roots. Changes in pH and CO2 in the Krebs perfusate were made by changing either the bicarbonate concentration or the PCO2 saturating the Krebs solution. 3. Changes in basilar artery perfusate consisting of (i) an acidifying increase in PCO2 (hypercapnic acidic Krebs solution), (ii) an increase in PCO2 with no change in pH (hypercapnic Krebs solution), or (iii) a decrease in pH with no change in PCO2 (acidic Krebs solution) evoked increases in respiratory frequency and a concomitant decrease in inspiratory burst amplitude. 4. Bath superfusion with hypercapnic acidic Krebs solution increased the inspiratory burst amplitude with no effect on respiratory burst frequency. 5. Bath superfusion with hypercapnic non-acidic Krebs solution increased the inspiratory burst amplitude and decreased the respiratory frequency, while normocapnic acidic Krebs solution increased the respiratory frequency with no change in burst amplitude. 6. These results show that respiratory responses to changes in CO2 and pH depend upon the sites of action. While a CO2 increase or a pH decrease affected the respiratory frequency in the deep brainstem structures (perfused through the basilar artery), CO2 respiratory chemosensitivity at the ventral surface could be differentiated from the hydrogen ion chemosensitivity. This suggests that different mechanisms mediated respiratory responses when deep versus superficial brainstem structures were stimulated.

Brain stem chemosensitivity: its implication in central respiratory regulation.

Central brain stem chemosensitivity plays an essential role in acid-base regulation. The site of the chemosensitive neuronal elements has not yet been clearly established. To address this question, we used an in vitro adult guinea pig isolated brain stem preparation, maintained in survival conditions by intrabasilar and bath perfusions of Krebs solution saturated with 95% O2 and 5% CO2. Hypercapnic stimulation, produced by modifying the CO2 concentration of the medium perfusing brain stem structures via the basilar perfusion system, increased the respiratory burst frequency recorded from the hypoglossal nerve. Hypercapnia evoked by ventral surface superfusion increased the respiratory burst amplitude. These data suggest that chemosensitive neuronal elements could be located at the ventral surface as well as in the deeper brainstem structures. However, their characteristics may be different.

Oxygen supply and respiratory-like activity in the isolated perfused brainstem of the adult guinea pig.

In isolated brainstem preparations of mature guinea pigs the respiratory network remains functional only if perfused internally via the basilar artery with Krebs solution equilibrated with 95% O2/5% CO2. In order to determine the oxygen availability in this preparation, we measured tissue partial oxygen pressures at the level of respiratory-related neurons using oxygen-sensitive microelectrodes. To estimate oxygen consumption we studied the effects of ischemia and cyanide-induced blockade of oxidative metabolism in relation to the respiratory-like rhythmic activity recorded from the hypoglossal nerve. The pO2 profiles obtained from 9 stepwise measurements from the ventral to the dorsal surface decreased from 423 +/- 32 (SE) mmHg on the ventral surface to 219 +/- 64 mmHg at 1900 microns and stabilized near this value up to a depth of 5000 microns. In the superfused preparation without internal perfusion pO2 was 0 mmHg within the first 500 microns. An interruption of perfusion resulted in a rapid (less than 2 min) decrease of tissue pO2 to 0 mmHg. During the ischemic period, respiratory-like neural activity exhibited first an increase in frequency and tonic discharge, followed by a marked decrease in both parameters. Cyanide added to the perfusate caused an immediate increase of tissue pO2 and the drop of tissue pO2 associated with ischemia was abolished. We conclude that there is a considerable oxygen consumption but no hypoxic or anoxic core in the isolated perfused brainstem at the level of the respiratory-related neurons.

CholecystokininA and cholecystokininB receptors in neurons of the brainstem solitary complex of the rat: pharmacological identification.

The brainstem solitary complex, which receives projections from primary sensory afferents of the vagus nerve, appears to be a crucial site for the action of the cholecystokinin octapeptide (CCK8), because both peripheral-type (CCKA) and central-type (CCKB) binding sites are present in this structure. In the present study, we investigated the effects of recently developed receptor-specific pharmacological tools on neurons recorded in rat coronal brainstem slices, to ascertain whether CCK acts differently on each type of receptor. CCK8, which interacts with both binding sites, had three effects on neuronal discharge, brief excitation, prolonged excitation and delayed inhibition. BC 264, a novel CCK analog endowed with high affinity and selectivity for CCKB receptors, produced exclusively prolonged excitation. L-365,260, a novel nonpeptide antagonist of CCKB receptors, blocked the prolonged excitation induced by BC 264 or CCK8. L-364,718, a potent antagonist of CCKA receptors, blocked delayed inhibition and replaced brief CCK8-induced excitation by prolonged excitation. Altogether, these results show that CCK8 exerts, on neurons of the solitary complex, mixed effects due to simultaneous activation of CCKA inhibitory and CCKB excitatory binding sites. The hypothesis that exogenous CCK8 acts, in the solitary complex, through CCKA sites to slow down the motility of the digestive tract and through CCKB sites to modulate anxiety will be developed.

Control of breathing by endogenous opioid peptides: possible involvement in sudden infant death syndrome.

Experiments have been performed in order to evaluate the respiratory consequences of a suppression or accumulation of endogenous opioid peptides, in the neuronal network which generates the motor respiratory activity. Iontophoretic application of naloxone onto respiratory neurons increases their firing activity and increases their respiratory modulation. On the other hand the local injection of kelatorphan (an enkephalinase inhibitor) decreases the firing of respiratory neurons and thus reduces the respiratory modulation. This effect of kelatorphan mimics the effect on respiratory neuron of an iontophoretic application of met-enkephalin. Furthermore the local injection of kelatorphan reduces the frequency of the respiratory output recorded from the phrenic nerve. This effect is reversed by systemic administration of naloxone. The results demonstrate the involvement of endogenous opioid peptides in the control of breathing suggesting that in Sudden Infant Death Syndrome a possible dysregulation in opioidergic system could occur.

Respiratory network remains functional in a mature guinea pig brainstem isolated in vitro.

We previously developed a perfused isolated brainstem preparation in the adult guinea pig (Morin-Surun and Denavit-Saubie 1989a) which permitted us to describe several types of rhythmic neuronal discharge. In the present study, we demonstrate that nearly all the periodic neuronal activity we recorded in the ventral respiratory areas were directly related to the respiratory-like periodic output of the hypoglossal nerve. This respiratory-like activity lasted several hours only when the brainstem was perfused by the basilar artery. This shows the necessity of the intraarterial perfusion to preserve a functional respiratory network. Analysis of the characteristics of hypoglossal respiratory-like activity shows that (1) two types of respiratory rhythms can be recorded; one with long respiratory phases (inspiratory and expiratory) and one with short respiratory phases. Depending on the preparation, either type occurs alone or intermingled with the other. (2) The shape of the inspiratory-like activity can change throughout the recording period while the periodicity remains stable. This preparation generates a respiratory rhythm and enables us to dissociate the different mechanisms involved in respiratory neurogenesis.

Rhythmic discharges in the perfused isolated brainstem preparation of adult guinea pig.

An isolated brainstem preparation of adult guinea pig was used for an in vitro electrophysiological study of rhythmic neuronal discharge patterns. More than half of the spontaneously active neurons (40/71) exhibited a rhythmic discharge. Rhythmic discharges were recorded dorsally in the nucleus tractus solitarius and ventrally in the ambiguus and paragigantocellular reticular nuclei. Three types of rhythmic patterns of discharge were identified in these areas: repetitive single discharge, repetitive bursting discharge and spontaneous periodic discharge. No rhythmic patterns were recorded in other explored parts of the brainstem. Comparison of these data with those from brainstem slices shows that spontaneous periodic discharges may require extended networks within the brainstem.

Respiratory actions induced by cholecystokinin at the brainstem level.

A functional differentiation of the action of cholecystokinin octapeptide (CCK-8) on the respiratory centers was accomplished by the topical application to the ventral surface of the medulla and to the dorso-rostral pontine surface in cats. In the medulla, CCK-8S at doses ranging from 0.09 nmol to 0.88 nmol, stimulated tidal volume in a dose-dependent fashion, with minimal or no changes in frequency. The antagonist proglumide (30 nmol) inhibited specifically the action on the respiratory amplitude. In the pons, CCK-8S did not modify the respiratory activity even at the dose of 8.8 nmol. The results suggest a specific involvement of CCK-8S in the mechanisms controlling respiratory amplitude, which appear mostly restricted to the medullary level. The lack of effect of the peptide in the pons is in agreement with the absence of CCK receptors in the respiration related nuclei located at that level, as evidenced by autoradiographic studies.

Effects of pentobarbital and flurazepam on respiratory neurons in undrugged cats.

Two hypnotic drugs known to enhance GABAergic transmission, a barbiturate (pentobarbital) and a benzodiazepine (flurazepam), were applied locally to respiratory-related neurons (RN) located in the ventral respiratory area in the medulla of non-anaesthetized cats which were either decerebrated or chronically implanted. Pentobarbital applied iontophoretically depressed the spontaneous discharge rate of most RN tested as well as the increase in firing of RN discharge induced by iontophoretic application of glutamate; pentobarbital also potentiated inhibition induced by iontophoresed GABA. Flurazepam applied by iontophoresis or pressure pulses depressed a minority of RN tested and did not enhance GABA-induced inhibition. These results suggest: that inhibition of RN activity through GABAergic mechanisms can be affected by drugs which act at the chloride ionophore but not those acting indirectly through the benzodiazepine binding site, and the effects of the agents on medullary neuronal activity are independent of an effect on the states of consciousness or on structures rostral to the medulla.

Excitability of 'silent' respiratory neurons during sleep-waking states: an iontophoretic study in undrugged chronic cats.

An iontophoretic study of respiratory-related neurons (RN) was conducted in the medullary ventral respiratory area of chronically implanted, undrugged cats during states of sleep and wakefulness. Most RN recorded were unaffected by sleep-wake states but a few RN decreased their firing rate during sleep (sleep sensitive cells). The excitability of RN was assessed in the different states by local application of L-glutamate. Glutamate iontophoresis revealed the presence of 5 cells which were silent during sleep and completely or mostly silent during undisturbed wakefulness but always discharged with a respiratory-modulated pattern of the expiratory type in response to glutamate application. Arousing stimuli induced spontaneous firing of these cells and REM sleep reduced glutamate effectiveness. It was concluded that silent RN and RN which become inactive during sleep permanently receive subthreshold respiratory-modulated inputs which are amplified or depressed by state-dependent tonic inputs.

The excitation by neurotensin of nucleus tractus solitarius neurons induces apneustic breathing.

The possible involvement of neurotensin in the regulation of respiratory drive has been tested on single brainstem respiratory related neurons and on the global respiratory output. The neuropeptide was locally applied either by microiontophoresis or by pressure injection in the dorsal and ventral respiratory areas of the anesthetized bivagotomized cat. Effects of neurotensin applications were studied, on the one hand on the firing discharge of respiratory related neurons and on the other hand on the phrenic nerve activity and on arterial blood pressure. An increase of the firing frequency of respiratory related neurons was induced by neurotensin applied by iontophoresis or by pressure injection (0.005-33.5 fmol/s) on single neurons. In the latter case, neurotensin was active at concentration 10(3) times lower than glutamate. A bilateral apneustic pattern was induced on the phrenic nerve activities by microinjection of neurotensin (0.23-0.54 pmol/s) in one ventrolateral nucleus tractus solitarius without alteration of arterial blood pressure. These results suggest that the release of neurotensin in the nucleus tractus solitarius regulates respiratory rhythmogenesis by increasing the inspiratory duration.