Marie-Pierre Morin-Surun

Different effects of μ and δ opiate agonists on respiration

Abstract The involvement of different opiate receptor subtypes in opiate-induced respiratory depression was studied in the unanaesthetized rat. Synthetic opioid agonists, specific for μ or δ receptors, were administered intraperitoneally in freely moving rats while respiratory parameters were recorded by means of the whole body plethysmographic method. TRIMU-4 (Tyr-D-Ala-Gly-NH-CH(CH 3 )-CH 2 -CH(CH 3 ) 2 ), a specific agonist of the μ receptor, reduced the tidal volume and did not change the respiratory frequency. DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr), a relatively specific agonist of the δ receptor subtype, reduced respiratory frequency and was significantly less effective on tidal volume than was TRIMU-4. It is concluded that the respiratory depression occurring after the administration of opiates in clinical practice is a dual complementary effect involving μ and δ receptors.

Excitatory effects of iontophoretically applied substance P on neurons in the nucleus tractus solitarius of the cat: lack of interaction with opiates and opioids

The effects of iontophoretically applied substance P (SP), (D-Pro2, D-Trp7,9)-SP and (D-Pro4, D-Trp7,9,10)-SP were studied on neurons identified by their histological location in the nucleus tractus solitarius (NTS), their response to vagal or carotid sinus nerve stimulation and eventually their functional correlation with the central respiratory drive. Potent and consistent excitatory effects of SP were found supporting its role as a putative excitatory transmitter in the NTS. The effects of SP and L-glutamate (Glu) were differentiated by the relative insensitivity of SP-induced excitations to levorphanol and Met-enkephalin.

Pharmacological identification of δ and μ opiate receptors on bulbar respiratory neurons

Abstract The opiate receptors of central neurons related to the generation of the respiratory rhythm were identified using microiontophoresis of the synthetic opioid peptides DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and TRIMU-4 (Tyr-D-Ala-Gly-NH-CH (CH 3 )-CH 2 -CH(CH 3 ) 2 ), exhibiting a high selectivity for δ and μ receptors respectively. Both agonists induced depressions of spontaneous and L-glutamate-induced discharges; the effects were antagonized by naloxone and not mimicked by a related almost inactive peptide DSLLET (Tyr-D-Ser-Gly-Leu-Leu-Thr). The effect of DSLET had a faster time course than that of TRIMU-4 and persisted after prolonged applications of TRIMU-4. It is concluded that δ and μ receptor subtypes are distinct and are both present on central respiratory-related neurons.

Respiratory function in adult mice lacking the µ-opioid receptor: role of δ-receptors

Mice lacking the µ‐opioid receptor (MOR) provide a unique model to determine whether opioid receptors are functionally interactive. Recent results have shown that respiratory depression produced by δ‐opioid receptor agonists is suppressed in mice lacking the µ‐opioid receptor. Here we investigated the involvement of µ‐ and δ‐opioid receptors in the control of ventilation and µ/δ 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 δ‐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 δ2 receptor agonist deltorphin II (0.1–1.0 µm) decreased respiratory frequency in both groups whereas doses of the δ1 receptor agonist enkephalin[D‐Pen2,5] (0.1–1.0 µm) which were ineffective in wild‐type mice significantly decreased respiratory frequency in MOR–/– mice. We conclude that deletion of the µ‐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 δ2‐opioid receptor agonists previously observed in vivo does not result from a blunted response of δ receptors in brainstem rhythm‐generating structures. These structures show an unaltered response to δ2‐receptor agonists and an augmented response to δ1‐receptor agonists.

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.

Differentiation of two respiratory areas in the cat medulla using kainic acid

Kainic acid (KA) was used to destroy neuronal perikarya in different areas of the brainstem. Single KA microinjections were performed in 30 anaesthetized, vagotomized, artificially ventilated cats. Consequences were studied on the phrenic nerve activity (PNA) and blood pressure. We observed changes of the PNA unrelated to blood pressure alteration. Destruction of the dorsal respiratory area (DRA) including the nucleus tractus solitarius at the obex level produced a 40% decrease of the PNA frequency. Destruction restricted to the lateral part of the ventral respiratory area (VRA1) including the ambiguus nucleus induced a 60% decrease of the integrated PNA amplitude followed by a 40% increase of PNA frequency. These latter effects were also observed after destruction inside the infra solitary reticular formation (ISRF). No effect was observed after destruction in other brain structures. We concluded that ISRF and VRA1 form a single ventral bulbar respiratory area. This area controls respiration in a way different from that of the dorsal respiratory area (DRA).

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.

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

SummaryWe 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.

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.

Prenatal development of central rhythm generation

Foetal breathing in mice results from prenatal activity of the two coupled hindbrain oscillators considered to be responsible for respiratory rhythm generation after birth: the pre-Bötzinger complex (preBötC) is active shortly before the onset of foetal breathing; the parafacial respiratory group (e-pF in embryo) starts activity one day earlier. Transcription factors have been identified that are essential to specify neural progenitors and lineages forming each of these oscillators during early development of the neural tube: Hoxa1, Egr2 (Krox20), Phox2b, Lbx1 and Atoh1 for the e-pF; Dbx1 and Evx1 for the preBötC which eventually grow contralateral axons requiring expression of Robo3. Inactivation of the genes encoding these factors leads to mis-specification of these neurons and distinct breathing abnormalities: apneic patterns and loss of central chemosensitivity for the e-pF (central congenital hypoventilation syndrome, CCHS, in humans), complete loss of breathing for the preBötC, right-left desynchronized breathing in Robo3 mutants. Mutations affecting development in more rostral (pontine) respiratory territories change the shape of the inspiratory drive without affecting the rhythm. Other (primordial) embryonic oscillators start in the mouse three days before the e-pF, to generate low frequency (LF) rhythms that are probably required for activity-dependent development of neurones at embryonic stages; in the foetus, however, they are actively silenced to avoid detrimental interaction with the on-going respiratory rhythm. Altogether, these observations provide a strong support to the previously proposed hypothesis that the functional organization of the respiratory generator is specified at early stages of development and is dual in nature, comprising two serially non-homologous oscillators.