[Serotonin receptor 1A-modulated dephosphorylation of glycine receptor α3: a new molecular mechanism of breathing control for compensation of opioid-induced respiratory depression without loss of analgesia]. / Die von Serotoninrezeptor 1A modulierte Dephosphorylierung des Glyzinrezeptors α3: Ein neuer molekularer Mechanismus der Atmungskontrolle zur Kompensation opioidinduzierter Atemdepression ohne Verlust der Analgesie.

To control the breathing rhythm the medullary respiratory network generates periodic salvo activities for inspiration, post-inspiration and expiration. These are under permanent modulatory control by serotonergic neurons of the raphe which governs the degree of phosphorylation of the inhibitory glycine receptor α3. The specific activation of serotonin receptor type 1A (5-HTR(1A)), which is strongly expressed in the respiratory neurons, functions via inhibition of adenylate cyclase and the resulting reduction of the intracellular cAMP level and a gradual dephosphorylation of the glycine receptor type α3 (GlyRα3). This 5-HTR(1A)-GlyRα3 signal pathway is independent of the µ-opioidergic transduction pathway and via a synaptic inhibition caused by an increase in GlyRα3 stimulates a disinhibition of some target neurons not only from excitatory but also from inhibitory neurons. Our physiological investigations show that this 5-HTR(1A)-GlyRα3 modulation allows treatment of respiratory depression due to opioids without affecting the desired analgesic effects of opioids. The molecular mechanism presented here opens new pharmacological possibilities to treat opioid-induced respiratory depression and respiratory disorders due to disturbed inhibitory synaptic transmission, such as hyperekplexia.

Which techniques are relevant in the surgical management of the nasal tip?

In this article, we give an overview of safe and consistent techniques for nasal tip surgery. A change in the morphology of the tip has an impact on the profile of the face. A knowledge of facial proportions is therefore important when performing rhinoplastic surgery. We prefer an external approach because it provides the best overview and because the possibility of bimanual working makes manipulation of the lower lateral cartilages easier. The techniques described here can be used to alter the nasal tip in a controllable way.

A combined blockade of glycine and calcium-dependent potassium channels abolishes the respiratory rhythm.

In order to test whether glycinergic inhibition is essential for the in vivo respiratory rhythm, we analysed the discharge properties of neurones in the medullary respiratory network after blockade of glycine receptors in the in situ perfused brainstem preparation of mature wild type and oscillator mice with a deficient glycine receptor. In wild type mice, selective blockade of glycine receptors with low concentrations of strychnine (0.03-0.3 microM) provoked considerable changes in neuronal discharge characteristics: The cycle phase relationship of inspiratory, post-inspiratory and expiratory specific patterns of membrane potential changes was altered profoundly. Inspiratory, post-inspiratory and expiratory neurones developed a propensity for fast voltage oscillations that were accompanied by multiple burst discharges. These burst discharges were followed by "after-burst" hyperpolarisations that were capable of triggering secondary burst discharges. Blockade of glycine receptors and the "big" Ca2+-dependent K+-conductance by charybdotoxin (3.3 nM) resulted in loss of the respiratory rhythm, whilst only tonic discharge activity remained. In contrast, rhythmic activity was only weakened, but preserved after the "small" Ca2+-dependent activated K+ conductance was blocked with apamin (8 nM). Also low concentrations of pentobarbital sodium (6 mg/kg) abolished rhythmic respiratory activity after blockade of glycine receptors in the wild type mice and in glycine receptor deficient oscillator mice. The data imply that failure of glycine receptors provokes enhanced bursting behaviour of respiratory neurones, whilst the additional blockade of BKCa channels by charybdotoxin or with pentobarbital abolishes the respiratory rhythm.

Histaminergic modulation of the intact respiratory network of adult mice.

Histaminergic modulation of neuronal activity in the respiratory network was investigated under normoxic and hypoxic conditions in the working heart-brainstem preparation of adult mice. Systemic application of histamine, as well as the H-1 and H-3 receptor agonists 6-[2-(4-imidazolyl)ethylamino]- N-(4-trifluoromethylphenyl) heptanecarboxamide (HTMT) and imetit, 0.5-10 micro M, significantly increased the frequency of respiratory burst discharges. Dimaprit, an H-2 receptor agonist, had no effect on respiratory activity. To test for ongoing histaminergic modulation we applied the histamine receptor antagonists pyrilamine (H-1); cimetidine (H-2) and thioperamide (H-3), each 0.5-10 micro M. Only the H-1 receptor antagonist had significant effects, viz. reduction of respiratory frequency and depression of burst amplitude. Underlying effects of histamine receptor activation were identified at the cellular level. Intracellular recordings showed that histamine mediated an increase in synaptic drive potentials in inspiratory neurones while augmentation of inhibitory and excitatory synaptic activity was observed in expiratory neurones. The augmented synaptic depolarisation of inspiratory neurones was blocked by the H-1 receptor antagonist. Histaminergic modulation is also involved in the hypoxic response of the respiratory network. Blockade of H-1 receptors significantly attenuated secondary depression of the biphasic hypoxic responses, while hypoxic augmentation was not affected. We conclude that histamine is a functional neuromodulator, which is tonically active in the respiratory network and is activated further during hypoxia. The data indicate that histaminergic neuromodulation acts predominantly via H-1 receptors.

The respiratory rhythm in mutant oscillator mice.

Since glycinergic inhibition is important for respiratory rhythm generation in mature mammals, we tested the hypothesis that the loss of glycine receptors during postnatal development (P17-P23) of homozygous mutant oscillator mice (spd(ot)/spd(ot)) may result in serious impairment of respiratory rhythm. We measured breathing in a plethysmographic recording chamber on conscious oscillator mice and used an in situ perfused brainstem preparation to record phrenic nerve activity, as well as membrane properties of respiratory neurones. The deletion of glycinergic inhibition did not result in failure of respiratory rhythm: homozygous mutant oscillator mice continue to generate a disturbed respiratory rhythm until death. Postsynaptic activity and membrane potential trajectories of respiratory neurones revealed a persistence of GABAergic inhibition and changes in respiratory rhythm and pattern generation.

Reorganisation of respiratory network activity after loss of glycinergic inhibition.

gamma-Aminobutyric acid (GABA)-ergic and glycinergic inhibition is believed to play a major role in the respiratory network. In the present study we tested whether specific blockade of glycinergic inhibition resulted in changes in respiratory network interaction and function. Using the working heart-brainstem preparation from adult mice, we recorded phrenic nerve activity and the activity of different types of respiratory neurones located in the ventrolateral medulla. Strychnine (0.03-0.3 microM) was given systemically to block glycine receptors (Gly-R). During exposure to strychnine, post-inspiratory (PI) neurones shifted their onset of discharge into the inspiratory phase. As a consequence, the post-inspiratory phase failed and the rhythm changed from a three-phase cycle (inspiration, post-inspiration, expiration, with a frequency of about. 0.24 Hz) to a faster, two-phased cycle (inspiration expiration, frequency about 0.41 Hz). Inspiratory and expiratory neurones altered their augmenting membrane potential pattern to a rapidly peaking pattern. Smaller voltage oscillations at approximately 10 Hz and consisting of excitatory and inhibitory postsynaptic potential sequences occurred during the expiratory interval. Due to their high frequency and low amplitude, such oscillations would be inadequate for lung ventilation. We conclude that, under physiological conditions, glycinergic inhibition does indeed play a major role in the generation of a normal respiratory rhythm in adult mice. After failure of glycinergic inhibition a faster respiratory rhythm seems to operate through reciprocal GABAergic inhibition between inspiratory and expiratory neurones, while phase switching is organised by activation of intrinsic membrane properties.

Neurotransmitters and neuromodulators controlling the hypoxic respiratory response in anaesthetized cats.

1. The contributions of neurotransmitters and neuromodulators to the responses of the respiratory network to acute hypoxia were analysed in anaesthetized cats. 2. Samples of extracellular fluid were collected at 1-1.5 min time intervals by microdialysis in the medullary region of ventral respiratory group neurones and analysed for their content of glutamate, gamma-aminobutyric acid (GABA), serotonin and adenosine by high performance liquid chromatography. Phrenic nerve activity was correlated with these measurements. 3. Levels of glutamate and GABA increased transiently during early periods of hypoxia, coinciding with augmented phrenic nerve activity and then fell below control during central apnoea. Serotonin and adenosine increased slowly and steadily with onset of hypoxic depression of phrenic nerve activity. 4. The possibility that serotonin contributes to hypoxic respiratory depression was tested by microinjecting the 5-HT-1A receptor agonist 8-OH-DPAT into the medullary region that is important for rhythmogenesis. Hypoxic activation of respiratory neurones and phrenic nerve activity were suppressed. Microinjections of NAN-190, a 5-HT-1A receptor blocker, enhanced hypoxic augmentation resulting in apneustic prolongation of inspiratory bursts. 5. The results reveal a temporal sequence in the release of neurotransmitters and neuromodulators and suggest a specific role for each of them in the sequential development of hypoxic respiratory disturbances.

Blockade of synaptic inhibition within the pre-Bötzinger complex in the cat suppresses respiratory rhythm generation in vivo.

1. The role of synaptic inhibition in respiratory rhythm generation was analysed by microinjections of GABAA and glycine receptor antagonists into the bilateral pre-Botzinger complex (PBC) of anaesthetized cats. Central respiratory activity was monitored by phrenic nerve recordings. 2. Bilateral injections of bicuculline (50 or 100 microM) irreversibly slowed respiratory frequency and induced apneustic patterns. 3. Bilateral injections of strychnine (50 or 100 microM) greatly reduced phrenic burst amplitudes leading to increased burst frequency or irreversibly blocked rhythmic phrenic discharges. After unilateral tetrodotoxin (TTX) blockade in the PBC, strychnine injection into the contralateral PBC blocked rhythmic phrenic discharges. 4. Bilateral blockade of both GABAergic and glycinergic inhibition abolished rhythmic burst discharges and only tonic phrenic activity remained. Such tonic activity was blocked only by TTX (1 microM). 5. Potentiation of synaptic inhibition by the serotonin 1A receptor agonist 8-hydroxydipropylaminotetralin (8-OH-DPAT; 50 microM) restored rhythmic activity only when given shortly after strychnine and bicuculline applications. It was, however, ineffective after blockade of synaptic inhibition was complete. 6. The study demonstrates the significance of synaptic inhibition in the process of respiratory generation in the adult cat in vivo.

cAMP-dependent protein kinase modulates expiratory neurons in vivo.

The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) second-messenger system influences neuronal excitability by modulating voltage-regulated and transmitter-activated channels. In this study we investigated the influence of the cAMP-PKA system on the excitability of expiratory (E) neurons in the caudal medulla of anesthetized, paralyzed, and artificially ventilated adult cats. We intracellularly injected the PKA inhibitors cAMP-dependent PKA inhibitor 5-22 amide (Walsh inhibitory peptide) and Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS), the PKA activator Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Sp-cAMPS), and the adenylyl cyclase activator forskolin and measured membrane potential, neuronal input resistance, and synaptic membrane currents. Inhibition of cAMP-PKA activity by Walsh inhibitory peptide or Rp-cAMPS injections hyperpolarized neurons, decreased input resistance, and depressed spontaneous bursts of action potentials. Action potential duration was shortened and afterhyperpolarizations were increased. Inhibitory synaptic currents increased significantly. Stimulation of cAMP-PKA activity by Sp-cAMPS or forskolin depolarization neurons and increased input resistance. Spontaneous inhibitory synaptic currents were reduced and excitatory synaptic currents were increased. Rp-cAMPs depressed stimulus-evoked excitatory postsynaptic potentials and currents, whereas Sp-cAMPS increased them. Sp-cAMPS also blocked postsynaptic inhibition of E neurons by 8-hydroxy-dipropylaminotetralin, a serotonin-1A (5-HT-1A) receptor agonist that depresses neuronal cAMP-PKA activity. To determine the predominant effect of G protein-mediated neuromodulation of E neurons, we injected guanosine-5'-O-(3-thiotriphosphate) tetralithium salt (GTP-gamma-S), an activator of both stimulatory and inhibitory G proteins. GTP-gamma-S hyperpolarized E neurons, reduced input resistance, and increased action potential afterhyperpolarization. We conclude that the intracellular cAMP-PKA messenger system play an important role in the activity-dependent modulation of excitability in E neurons of the caudal medulla. In addition, the cAMP-PKA pathway itself is downregulated during activation of 5-HT-1A receptors.

Nucleus raphe obscurus evokes 5-HT-1A receptor-mediated modulation of respiratory neurons.

We analysed in vivo the synaptic mechanisms underlying serotonin-mediated depression of expiratory neuronal discharges and phrenic nerve activity. We report that nucleus raphe obscurus stimulation not only abolishes phrenic nerve activity, but also hyperpolarizes the membrane potential, depresses periodic synaptic drive potentials and thus action potential discharges in caudal medullary expiratory neurons. These effects originate from pre- and post-synaptic inhibitory processes that involve 5-HT-1A receptor activation.

Treatment of apneustic respiratory disturbance with a serotonin-receptor agonist.

Apneusis is a disturbance of respiratory rhythm characterized by severely prolonged inspiratory effort. It may occur after damage to the respiratory network within the lower brain stem and pons from an overdose of central nervous system depressants, blockade of glutamate receptors, asphyxia, hypoxia, or ischemia. Experimental studies conducted on laboratory mammals, such as anesthetized cats and rats, suggest that apneusis results mostly from depression of glutamatergic synaptic processes that are necessary for activation of inhibitory mechanisms that terminate inspiration. The impairment of synaptic transmission leads to prolonged inspiratory efforts and apneustic discharges of brainstem respiratory neurons. Apneustic patterns can be consistently converted to normal by administration of serotonin type 1A (5-HT1A) receptor agonists. This observation encouraged a treatment of severe apneusis with buspirone, an agonist for 5-HT1A receptors, in a child after neurosurgery for an astrocytoma in the pons and medulla oblongata. Oral administration of buspirone produced a prompt and highly effective remission of apneusis without side effects. Treatment with 5-HT1A agonists, therefore, might offer a novel and effective pharmacotherapy against apneustic disturbances of breathing.

Hypoxic response of hypoglossal motoneurones in the in vivo cat.

1. In current and voltage clamp, the effects of hypoxia were studied on resting and synaptic properties of hypoglossal motoneurones in barbiturate-anaesthetized adult cats. 2. Twenty-nine hypoglossal motoneurones with a mean membrane potential of -55 mV responded rapidly to acute hypoxia with a persistent membrane depolarization of about +17 mV. This depolarization correlated with the development of a persistent inward current of 0.3 nA at holding potentials close to resting membrane potential. 3. Superior laryngeal nerve (SLN) stimulation-evoked EPSPs were reduced in amplitude by, on average, 46% while IPSP amplitude was reduced by 31% SLN stimulation-evoked EPSCs were reduced by 50-70%. 4. Extracellular application of adenosine (10 mM) hyperpolarized hypoglossal motoneurones by, on average, 5.6 mV, from a control value of -62 mV. SLN stimulation-evoked EPSPs decreased by 18% and IPSPs decreased by 46% during adenosine application. 5. Extracellular application of the KATP channel blocker glibenclamide led to a blockade of a persistent outward current and a significant increase of SLN stimulation-evoked EPSCs. 6. We conclude that hypoglossal motoneurones have a very low tolerance to hypoxia. They appear to be under metabolic stress even in normoxia and their capacity to activate protective potassium currents is limited when compared with other brainstem neurones. This may help to explain the rapid disturbance of hypoglossal function during energy depletion.

Intracellular signal pathways controlling respiratory neurons.

Medullary respiratory neurons are influenced by a variety of neuromodulators, but there is a lack of information about the specific intracellular signal pathways involved. In this report we describe the modulatory effects of the cyclic adenosine-triphosphate (cAMP)-dependent protein kinase and of protein kinase C pathways on voltage- and ligand-controlled ionic conductances and demonstrate their functional significance in regulating the excitability of medullary respiratory neurons of the vivo cat. Evidence is presented that PKA and PKC pathways are persistently activated. PKA regulates current flow through persistently activated and GABAB receptor-controlled potassium channels as well as GABAA receptor-controlled chloride channels. PKC also depresses persistent potassium currents but it potentiates excitatory and inhibitory synaptic currents. The clinical significance of these intracellular signal pathways is demonstrated in a case of a child suffering from apneustic breathing, who was successfully treated with a 5HT-1A receptor agonist.

ATP-sensitive K+ channels are functional in expiratory neurones of normoxic cats.

1. We analysed spontaneously active expiratory neurones (n = 48) of anaesthetized cats for the presence of ATP-sensitive K+ (KATP) channels. 2. Intracellular injection of ATP reversibly depolarized neurones during all phases of the respiratory cycle. During expiration, membrane potential depolarized by an average of 1.5 +/- 0.1 mV leading to a 25% increase of discharge frequency. During inspiration, ATP induced a 1.8 +/- 0.2 mV depolarization, which was accompanied by a maximum of 20% increase of input resistance (Rn). 3. Extracellular application of diazoxide, an agonist of KATP channels, resulted in reversible membrane hyperpolarization in 68% of neurones (n = 19). This hyperpolarization (2.5 mV during expiration and 3.1 mV during inspiration) was accompanied by a 22% decrease in Rn. 4. Extracellular application of tolbutamide and glibenclamide, two antagonists of KATP channels, evoked reversible depolarizations in 76% of neurones (n = 21). The depolarization was relatively constant throughout the respiratory cycle (1.4 mV during expiration and 2.3 mV during inspiration). Rn increased by 22%. 5. The same sulphonylureas also changed the steepness of membrane depolarization when neurones escaped spontaneous synaptic inhibition during postinspiration. Extracellularly applied tolbutamide and glibenclamide increased the steepness of depolarization by 21%, while diazoxide reduced it by 20%. 6. Antagonism of drugs was verified by simultaneous extra- and intracellular application of diazoxide and glibenclamide, respectively. 7. During voltage clamp at holding potential at -60 to -67 mV, intracellular or extracellular application of tolbutamide and glibenclamide blocked a persistent outward current. 8. We conclude that KATP channels are functional in expiratory neurones of adult cats and contribute to the control of excitability even during normoxia.

5-HT2 receptor-controlled modulation of medullary respiratory neurones in the cat.

1. The effects of the 5-HT2 receptor agonist alpha-methyl-5-HT were studied on the membrane of expiratory (E2) and post-inspiratory (PI) neurones, by intracellular recordings in the caudal medulla of anaesthetized cats. 2. Ionophoresis of alpha-Me-5-HT depolarized membrane potential and increased action potential frequency in a majority of neurones tested. Depolarization of neurones by alpha-Me-5-HT was accompanied by increased input resistance throughout all phases of the respiratory cycle. These effects were antagonized by ionophoresis of cinanserin, a receptor-blocking agent with high affinity for 5-HT2 receptors. 3. E2 neurones were voltage clamped to measure membrane current changes induced by alpha-Me-5-HT ionophoresis. alpha-Me-5-HT induced a net inward current by reducing inspiratory-phase outward currents and increasing expiratory-phase inward currents. These changes were equivalent with steady membrane depolarization, decreased inspiratory phase membrane hyperpolarization and increased expiratory drive potential recorded from the same neurones in current clamp. 4. The effects of alpha-Me-5-HT are consistent with activation of 5-HT2 receptors on E2 and PI neurones leading to blockade of synaptically activated and persistent conductances to potassium ions.

Serotonin 1A-receptor activation suppresses respiratory apneusis in the cat.

Malfunction of inhibitory synaptic processes in the brainstem result in abnormal prolonged inspiration (apneusis). Since we previously found that the serotonin (5-hydroxytryptamine; 5-HT) 5-HT1A receptor agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) shortens inspiratory discharges, we tested its ability to suppress apneusis. We recorded phrenic nerve activity and the membrane potential of medullary expiratory (E-2) and postinspiratory (PI) neurons in 14 anaesthetized, paralyzed, artificially ventilated cats. Systemic hypoxia or i.v. injection of pentobarbital sodium or the N-methyl-D-aspartate (NMDA) receptor blocker ketamine induced apneustic phrenic nerve discharges, delayed depolarization to threshold of E-2 neurons and prolonged hyperpolarization in PI neurons. 8-OH-DPAT (10-40 micrograms/kg i.v.) produced partial to complete restoration of normal phrenic nerve discharges and membrane potential.

5-HT-1A receptor-mediated modulation of medullary expiratory neurones in the cat.

The involvement of the 5-HT-1A receptor in serotoninergic responses of stage 2 expiratory (E-2) neurones was investigated in pentobarbitone-anaesthetized, mechanically ventilated cats. The specific agonist of the 5-HT-1A receptor, 8-hydroxy-diproplaminotetralin (8-OH-DPAT), administered systemically or by ionophoresis directly on to the neurones, had a clear depressant effect. Administration of 8-OH-DPAT at doses of 10-50 micrograms kg-1 (I.V.) increased the membrane hyperpolarizations of E-2 neurones during the inspiratory and postinspiratory phases, and shortened their duration of activity in association with shortening of phrenic nerve activity. Discharges of E-2 neurones were also less intense. At doses of 50-90 micrograms kg-1, 8-OH-DPAT reduced or abolished inspiratory hyperpolarizations, and reduced expiratory depolarizations of membrane potential and discharge in parallel with inhibition of phrenic nerve discharges. The effects of the larger doses were reversed by I.V. injection of NAN-190, an antagonist at the 5-HT-1A receptor. Dose-dependent effects on the membrane potential and discharge of E-2 neurones, but not on phrenic nerve activity, were also seen by ionophoretic administration of 8-OH-DPAT on to E-2 neurones. At low currents, ejection of 8-OH-DPAT hyperpolarized the neurones without affecting the duration of inspiratory hyperpolarization and expiratory depolarization. This hyperpolarization depressed the intensity and the duration of expiratory discharges. Ejection with larger currents hyperpolarized the E-2 neurones further, and depressed expiratory depolarization leading to blockade of expiratory discharges. The effects on membrane potential were accompanied by decreased neuronal input resistance. This depressed the excitability of E-2 neurones as tested by discharge evoked by intracellular current injection. The amplitudes of action potentials decreased in parallel with the changes in input resistance. The effects were attributed to a postsynaptic effect of 8-OH-DPAT leading to a gradually developing inhibition by activation of 5-HT-1A receptors. Hyperventilatory apnoea depressed on-going synaptic activity and unmasked the effect of ionophoretically applied 8-OH-DPAT. The responses of the E-2 neurone were enhanced, as evidenced by increased membrane hyperpolarization and greater reduction of input resistance. Both responses faded appreciably, indicating receptor desensitization. The degree and rate of apparent desensitization depended on the dose/ejecting current. The greater sensitivity and faster desensitization to 8-OH-DPAT were attributed to the hyperventilatory alkalinization of the extracellular fluid, which might influence agonist binding to 5HT-1A receptors and/or receptor properties.

Inhibition of caudal medullary expiratory neurones by retrofacial inspiratory neurones in the cat.

1. Comparisons between the spike discharge of inspiratory neurons within the retrofacial area (RFN), and the membrane potential of expiratory neurones within the caudal medulla were made in pentobarbitone-anaesthetized, vagotomized, artificially ventilated cats. Spike-triggered averaging (STA) of synaptic potentials, triggered by the discharge of inspiratory RFN neurones, was utilized to test for synaptic connectivity. 2. Eighty-nine neurons with respiratory-phased discharge patterns were recorded in the vicinity of the RFN. Fifty-four neurones discharged at or slightly before the onset of the inspiratory burst activity of the phrenic nerve and continued firing throughout inspiration. Two continued to fire during post-inspiration. Forty-five of fifty-four inspiratory RFN neurones exhibited incrementing discharge patterns, six discharged with a plateau pattern, while only three neurones had a decrementing discharge pattern. 3. The membrane potential trajectories of caudal expiratory neurones revealed a typical wave of early inspiratory hyperpolarization. Occasionally, a second wave of hyperpolarization occurred during late inspiration, in conjunction with increased phrenic nerve activity. 4. Spike-triggered averaging revealed averaged inhibitory postsynaptic potentials (IPSPs), indicative of inhibitory synaptic connections, between eight and sixty-three pairs of RFN inspiratory and caudal expiratory neurones. 5. Inhibitory postsynaptic potentials detected by STA exhibited a relatively long latency and a slow time course. The IPSPs began, on average, 3.8 ms after an RFN action potential. The rise times, half-widths and durations of IPSPs were longer than expected for a monosynaptic somal input from myelinated axons of inspiratory RFN neurones. It is suggested that an inhibitory relay neurone in the immediate vicinity of the expiratory neurones is activated by a collateral of the RFN inspiratory neurone. 6. Retrofacial inspiratory neurones were antidromically activated only when high-intensity electrical stimulation was applied in the vicinity of caudal expiratory neurones. 7. The averaged IPSPs were preceded by diphasic and triphasic 'spike potentials'. The averaged spike potentials were highly entrained to the action potentials of RFN inspiratory neurones which triggered IPSPs. The spike potentials may be terminal potentials recorded from axons of RFN inspiratory neurones. 8. Evidence for convergence of synaptic inputs was obtained from STA tests in a caudal expiratory neurone receiving IPSPs from four RFN neurones. 9. The functional significance of this observation is discussed. We conclude that RFN inspiratory neurones exert a moderate inhibitory influence and act conjointly with other types of medullary inspiratory neurones.