Newborn Rat Brainstem-spinal Cord Preparations Research Articles

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

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

An in vitro experimental model for analysis of central control of sympathetic nerve activity

Newborn rat brainstem-spinal cord preparations are useful for in vitro analysis of various brainstem functions including respiratory activity. When studying the central control of sympathetic nerve activity (SNA), it is important to record peripheral outputs of the SNA. We developed an in vitro preparation in which neuronal connections between the cardiovascular center in the medulla and SNA peripheral outputs are preserved. Zero- to 1-day-old rats were deeply anesthetized with isoflurane, and the brainstem and spinal cord were isolated with a partial right thoracic cage to record sympathetic nerve discharge from the right thoracic sympathetic nerve trunk (T9-T11). SNA in this preparation was strongly modulated by inspiratory activity. Single-shot electrical stimulation of the ipsilateral rostral ventrolateral medulla (RVLM) induced a transient increase of SNA. Bath application of angiotensin II induced an increase of SNA, and local ipsilateral microinjection of angiotensin II to the RVLM induced a transient increase of SNA. This preparation allows analysis of the central control of the SNA in vitro.

Role of rostral medulla in serotonin-induced changes of respiratory rhythm in newborn rat brainstem–spinal cord preparations

Abnormalities of the serotonin (5-hydroxytryptamine, 5-HT) system may induce respiratory disorders. We examined which regions in the rostral medulla are important for the effect of 5-HT on the frequency of respiratory-like nerve (fR-like) activity by transecting the preparations at different levels near the facial nucleus (nVII) in newborn rat brainstem–spinal cord preparations. The fR-like activity at the fourth cervical ventral root (C4) of the Pons–medulla–spinal cord preparations in 2–3-day-old rats (n=25) was monitored at 26°C, and the change in fR-like activity in response to application of 10μM 5-HT before and after transection was compared among three groups, in which nVII was retained (group A, n=10), partially retained (group B, n=7), or eliminated (group C, n=8) by the transection. Before transection, the resting fR-like activity (set to 100%) and stimulant effect of 5-HT (+101–143%) were similar among the groups. After transection, resting fR-like activity increased in all groups, but the facilitatory effects of 5-HT on the fR-like activity were abolished in groups A and C (fR-like activity of −4% and +7%, respectively). In group B, 5-HT became inhibitory (fR-like activity of −28%). In conclusion, a distinct part of the rostral medulla in the absence of pontine influences may mediate the inhibitory effects of 5-HT on the respiratory rhythm.

Coadministration of the AMPAKINE CX717 with Propofol Reduces Respiratory Depression and Fatal Apneas

Propofol (2,6-diisopropylphenol) is used for the induction and maintenance of anesthesia in human and veterinary medicine. Propofol's disadvantages include the induction of respiratory depression and apnea. Here, the authors report a clinically feasible pharmacological solution for reducing propofol-induced respiratory depression via a mechanism that does not interfere with anesthesia. Specifically, they test the hypothesis that the AMPAKINE CX717, which has been proven metabolically stable and safe for human use, can prevent and rescue from propofol-induced severe apnea. The actions of propofol and the AMPAKINE CX717 were measured via (1) ventral root recordings from newborn rat brainstem-spinal cord preparations, (2) phrenic nerve recordings from an adult mouse in situ working heart-brainstem preparation, and (3) plethysmographic recordings from unrestrained newborn and adult rats. In vitro, respiratory depression caused by propofol (2 μM, n = 11, mean ± SEM, 41 ± 5% of control frequency, 63 ± 5% of control duration) was alleviated by CX717 (n = 4, 50-150 μM). In situ, a decrease in respiratory frequency (44 ± 9% of control), phrenic burst duration (66 ± 7% of control), and amplitude (78 ± 5% of control) caused by propofol (2 μM, n = 5) was alleviated by coadministration of CX717 (50 μM, n = 5). In vivo, pre- or coadministration of CX717 (20-25mg/kg) with propofol markedly reduced propofol-induced respiratory depression (n = 7; 20mg/kg) and propofol-induced lethal apnea (n = 6; 30 mg/kg). Administration of CX717 before or in conjunction with propofol provides an increased safety margin against profound apnea and death.

Effects of sevoflurane on respiratory rhythm oscillators in the medulla oblongata

Using in vitro newborn rat brainstem-spinal cord preparations with and without the parafacial respiratory group (pFRG), we examined the effects of the volatile anaesthetic sevoflurane on the respiratory rhythm oscillators of the pFRG and the preBötzinger complex (preBötC). Our study indicated that sevoflurane depressed pre-inspiratory neurons (Pre-Is) in the pFRG via gamma-aminobutyric acid-A (GABA(A))ergic and glycinergic inhibition and that it depressed preBötC inspiratory neurons via GABA(A)ergic but not via glycinergic inhibition. We also found that sevoflurane had stimulant effects on the respiratory rhythm oscillators. Our results shed light on respiratory rhythm generation. In all preparations (n=16) in which Pre-Is activity was recorded, inspiratory-related cervical motor output remained after application of 0.47 mM sevoflurane, despite the disappearance of the burst activity of Pre-Is. This finding shows that Pre-Is are not essential for respiratory rhythm generation and suggests that sevoflurane, when applied at a proper concentration, might offer a pharmacological means to eliminate pFRG function while preserving preBötC activity.

Ventrolateral lesions at the ponto-medullary junction and the effects of noradrenaline on respiratory rhythm in rat brainstem–spinal cord preparations

Aims We examined whether responses of respiratory frequency ( f R) to noradrenaline (NA) were eliminated by mechanical lesions in the ventrolateral area at the ponto-medullary junction in preparations of newborn rat pons–medulla–spinal cord (PMS). Main methods Preparations obtained from 2- to 4-day-old rats were superfused with artificial cerebrospinal fluid that was equilibrated with oxygenated (95% O 2 plus 5% CO 2) gas, and f R was monitored at the C 4 ventral root at 24 °C. Bilateral lesions were made in the ventrolateral area between the VIth cranial nerve root and the anterior inferior cerebellar artery in PMS ( n = 11). The resting f R and response to exogenous NA (7 μM) were compared with those of medulla–spinal cord (MS) preparations ( n = 6). Immunohistochemistry of PMS preparations was performed to detect tyrosine hydroxylase (TH)-positive neurons at the ponto-medullary junction. Key findings PMS preparations with the lesions had (1) a significantly higher resting f R but (2) significantly less f R facilitation after NA application than those of intact PMS preparations, and (3) significantly lower resting f R and (4) significantly less f R reduction after NA application than those of MS preparations. TH-positive neurons were detected in the region from the rostral dorsolateral to the caudal ventrolateral pons (the A5 area), as well as in the ventral area near the facial nucleus. Significance Results suggest that ventrolateral area at ponto-medullary junction plays a significant role in exogenous NA-induced f R changes under the influence of pons-induced tonic f R inhibition in newborn rat brainstem–spinal cord preparations.

Opioids prolong and anoxia shortens delay between onset of preinspiratory (pFRG) and inspiratory (preBötC) network bursting in newborn rat brainstems

Differential responses to opioids established the hypothesis that pre/postinspiratory (Pre-I) neurons of the parafacial respiratory group (pFRG) and inspiratory (Insp) neurons of the pre-Bötzinger complex (preBötC) constitute a dual brainstem respiratory center. For further analysis of pFRG/preBötC interactions, we studied in newborn rat brainstem-spinal cord preparations opioid and anoxia effects on histologically identified pFRG-driven "type-I" Insp preBötC neurons and Pre-I neurons from three distinct respiratory brainstem regions. The micro-opioid [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) slowed inspiratory-related cervical nerve bursts quantally, whereas anoxia induced nonquantal slowing and repetitive cervical bursts. DAMGO had no effect on membrane potential or input resistance of Pre-I neurons, while anoxia hyperpolarized them (approximately 5 mV) and decreased their resistance (approximately 30%). DAMGO prolonged the preinspiratory phase of Pre-I neuron bursting, whereas anoxia caused a shift to postinspiratory (48%) or inspiratory (22%) activity and silenced further 30% of cells. Pre-I neuron responses were not correlated with their rostrocaudal location or morphology. Neither DAMGO nor anoxia changed membrane potential of type-I neurons, but decreased their input resistance by 33% and 21%, respectively. The opposite DAMGO- and anoxia-evoked phase shifts of Pre-I neuron activity were reflected by corresponding shifts of pre/postinspiratory drive potentials in type-I neurons and, partly, by voltage-sensitive dye-imaged medullary neuronal population activities. The findings suggest that opioids presynaptically delay activation of type-I neurons as the target of drive from the pFRG to the preBötC. Contrary, anoxia seems to partly synchronize the pFRG and preBötC rhythm generators. This may enhance inspiratory and postinspiratory medullary activities for triggering multiple inspiratory motor bursts.

2P392 Two modes of respiratory rhythm generation in the newborn rat brainstem-spinal cord preparation in vitro(44. Neuro-biophysics,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P392 Two modes of respiratory rhythm generation in the newborn rat brainstem-spinal cord preparation in vitro(44. Neuro-biophysics,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

A Novel Functional Neuron Group for Respiratory Rhythm Generation in the Ventral Medulla

We visualized respiratory neuron activity covering the entire ventral medulla using optical recordings in a newborn rat brainstem-spinal cord preparation stained with voltage-sensitive dye. We measured optical signals from several seconds before to several seconds after the inspiratory phase using the inspiratory motor nerve discharge as the trigger signal; we averaged the optical signals of 50-150 respiratory cycles to obtain an optical image correlating particularly to inspiratory activity. The optical images we obtained from the ventral approach indicated that neuron activity first appeared during the respiratory cycle in the limited region of the rostral ventrolateral medulla (RVLM), preceding the onset of inspiratory activity by approximately 500 msec. During the inspiratory phase, plateau activity appeared in the more caudal ventrolateral medulla at the level of the most rostral roots of the XIIth nerve. Comparison with electrophysiological recordings from respiratory neurons in the RVLM suggested that the optical signals preceding the inspiratory burst reflect preinspiratory neuron activity in this area. This RVLM area was determined to be ventrolateral to the facial nucleus and close to the ventral surface. We referred to this functional neuron group as the para-facial respiratory group (pFRG). Partial, bilateral electrical lesioning of the pFRG significantly reduced the respiratory frequency, together with changes in the spatiotemporal pattern of respiratory neuron activity. Our findings suggest that the pFRG comprises a neuronal population that is involved in the primary respiratory rhythm generation in the rostrocaudally extending respiratory neuron network of the medulla.

Salicylate action on medullary inspiratory neuron activity in a brainstem-spinal cord preparation from newborn rats.

Salicylate affects central respiratory control. The inspiratory neurons are the most important component of the medullary respiratory control center because they modulate the final motor output via the phrenic nerve. We investigated changes in burst rate, intraburst firing frequency, and membrane properties of inspiratory neurons in the isolated brainstem after the administration of salicylate. Newborn rat brainstem-spinal cord preparations were superfused with salicylate. Whole-cell recordings were performed from inspiratory neurons. Application of 1 mM salicylate caused an increase in the inspiratory neuronal burst rate from 6.9 +/- 1.6 bursts/min to 8.2 +/- 1.9 bursts/min (P < 0.05). The inspiratory neuron burst rate decreased from 8.3 +/- 0.7 bursts/min to 4.5 +/- 1.1 bursts/min after the application of 10 mM salicylate (P < 0.01). The depressant effect of 10 mM salicylate was antagonized by the gamma-aminobutyric acid (GABA) receptor antagonist bicuculline (1 microM). Resting membrane potential and intraburst firing frequency did not change with the application of salicylate and bicuculline even when the burst rate did change. We conclude that the effects of salicylate on the medullary inspiratory neurons are mainly due to a presynaptic action. GABAergic mechanisms are probably involved in the salicylate-induced central respiratory depression.

Opioid action on respiratory neuron activity of the isolated respiratory network in newborn rats.

Underlying mechanisms behind opioid-induced respiratory depression are not fully understood. The authors investigated changes in burst rate, intraburst firing frequency, membrane properties, as well as presynaptic and postsynaptic events of respiratory neurons in the isolated brainstem after administration of opioid receptor agonists. Newborn rat brainstem-spinal cord preparations were used and superfused with mu-, kappa-, and delta-opioid receptor agonists. Whole cell recordings were performed from three major classes of respiratory neurons (inspiratory, preinspiratory, and expiratory). Mu- and kappa-opioid receptor agonists reduced the spontaneous burst activity of inspiratory neurons and the C4 nerve activity. Forty-two percent of the inspiratory neurons were hyperpolarized and decreased in membrane resistance during opioid-induced respiratory depression. Furthermore, under synaptic block by tetrodotoxin perfusion, similar changes of inspiratory neuronal membrane properties occurred after application of mu- and kappa-opioid receptor agonists. In contrast, resting membrane potential and membrane resistance of preinspiratory and majority of expiratory neurons were unchanged by opioid receptor agonists, even during tetrodotoxin perfusion. Simultaneous recordings of inspiratory and preinspiratory neuronal activities confirmed the selective inhibition of inspiratory neurons caused by mu- and kappa-opioid receptor agonists. Application of opioids reduced the slope of rising of excitatory postsynaptic potentials evoked by contralateral medulla stimulation, resulting in a prolongation of the latency of successive first action potential responses. Mu- and kappa-opioid receptor agonists caused reduction of final motor outputs by mainly inhibiting medullary inspiratory neuron network. This inhibition of inspiratory neurons seems to be a result of both a presynaptic and postsynaptic inhibition. The central respiratory rhythm as reflected by the preinspiratory neuron burst rate was essentially unaltered by the agonists.

5-HT acting on 5-HT(1/2) receptors does not participate in the in vitro hypoxic respiratory depression.

The involvement of serotoninergic mechanisms in the central respiratory depression produced by hypoxia was studied in the newborn rat brainstem-spinal cord preparation. The respiratory frequency measured by the C4 ventral root activity was recorded. 5-HT (30 microM) superfusion elicited a rapid increase in respiratory frequency, prevented by a treatment with methysergide (a 5-HT(1/2) receptor antagonist) (40 microM). To investigate the possible participation of 5-HT in hypoxic respiratory depression, this concentration of methysergide was added to the bathing medium during hypoxia. Methysergide did not modify the decrease in respiratory frequency produced by hypoxia. In order to ensure that other 5-HT subtype receptors were not involved in hypoxic respiratory depression, 5-HT was added to the bath during hypoxic-methysergide tests; no effect on respiratory frequency was observed. These results suggest that in the newborn rat brainstem-spinal cord preparation, serotoninergic mechanisms are not involved in the elaboration of the in vitro respiratory response to hypoxia.

The adrenergic modulation of firings of respiratory rhythm-generating neurons in medulla-spinal cord preparation from newborn rat.

We analysed the modulation of respiratory neurons by adrenaline or noradrenaline (NA) in a newborn rat brainstem-spinal cord preparation. Adrenaline or NA caused a dose-dependent depression of the respiratory rhythm and induced C4 spinal tonic discharges. The inhibitory effect of adrenaline (ED50=0.5 microM) on the respiratory rhythm was stronger than NA (ED50=5 microM). The adrenaline respiratory rhythm depression was partially blocked by the alpha1-antagonist prazosin or by the alpha2-antagonist yohimbine. The C4 tonic discharge elicited by adrenaline was blocked by the alpha1-antagonist prazosin. The direct effects of adrenaline on pre-inspiratory (Pre-I) neurons were examined in a synaptic blockade solution (low Ca), and fifty-six percent of Pre-I neurons were found to continue firing. In low-Ca solution, Pre-I neurons were excited (n=29 of 39) or depressed (n=5 of 39) by adrenaline, and excited by alpha1-agonist phenylephrine or depressed by alpha2-agonist clonidine. These results suggest that the respiratory rhythm depression under intact network conditions is mediated by some other inhibitory system. The inhibitory effect of adrenaline on the respiratory rhythm was partially blocked by the GABA(A)-antagonists bicuculline or picrotoxin, but not by the GABA(B)-antagonist phaclofen. The present results suggest that: (1) respiratory rhythm generation is more sensitive to adrenaline than NA through alpha-adrenergic action of adrenaline; (2) the activity of Pre-I neurons could be directly regulated by excitation via alpha1-receptors and inhibition via alpha2-receptors; and (3) the depression of the respiratory rhythm by adrenaline is partly mediated by GABA(A)ergic neurons.

2412 Autonomic nerve responses and emotion in humans

Involvement of cAMP in the generation of respiratory rhythm was studied in newborn rat brainstem-spinal cord preparations. The respiratory rhythm was monitored by C4 inspiratory activity and/or pre-inspiratory (Pre-I) activity of neurons in the rostral ventrolateral medulla; previously suggested to be primary rhythm generating neurons which have pacemaker properties. The effects of four cAMP-increasing agents (forskolin, IBMX, Db-cAMP, and 8-Br-cAMP) on this neuronal activity were examined. Perfusion with forskolin (3–10 μM) increased the burst rate of C4 inspiratory activity in 20 of 32 preparations, but in 8 of those the increase was preceded by transient depression. The facilitation of the respiratory rhythm was greater whenever the burst rate before forskolin treatment was lower. The Pre-I neuron burst rate, which was recorded together with C4 activity, predominantly increased with forskolin treatment. The effects of IBMX, Db-cAMP and 8-Br-cAMP were similar to those of forskolin, but they were slightly less potent. Long-lasting depression of the respiratory rhythm (C4 and Pre-I activity) by clonidine, which might decrease intracellular cAMP level viaμ2-receptors, was reversed by forskolin. To investigate the direct effects of the cAMP-increasing agents on Pre-I neurons, Pre-I activity was isolated by blocking the chemical synaptic transmission by incubation in a low Ca solution (0.2 mM Ca2+, 5 mM Mg2+). Forskolin (5–10 μM), IBMX (5–10 μM), Db-cAMP (0.2–0.4 mM), and 8-Br-cAMP (0.4–0.75 mM) all enhanced the burst rate of isolated Pre-I neurons. Results suggest that cAMP is important in respiratory rhythm generation; this may be due to its regulation of the intrinsic burst generating properties of Pre-I neurons.

Tachykinins and central respiratory activity: an in vitro study on the newborn rat

The newborn rat brainstem-spinal cord preparation was used to study the effects of tachykinins on the activity of the respiratory rhythm generator in vitro and to characterize the receptors involved. Substance P and tachykinin NK 1 and NK 3 receptor agonists induced a concentration-dependent increase in the respiratory frequency (10 −9−10 −7 M), whereas the respiratory frequency was only slightly affected by the tachykinin NK 2 receptor agonist. Pre-treatments with tachykinin NK 1 receptor antagonists (SR140333, ( S)1-{2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin- n-3-yl]ethyl}-4-phenyl-1-azoniabicyclo[2,2,2]octane chloride; G pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-(S-S)Pro-Leu(spiro-γ-lactam)-Trp-NH 2) reduced the substance P-induced increases in the respiratory frequency but the tachykinin NK 2 receptor antagonists (SR48968, (( S)- N-methyl- N-[4-(4-acetylamino-4-phenylpiperidine)-2-(3,4-dichlorophenyl)butyl]benzamide); MEN 10376, Asp-Tyr- d-Trp-Val- d-Trp- d-Trp-Lys-NH 2) had no effect; the increase in the respiratory frequency induced by the tachykinin NK 3 receptor agonist was not affected by a pre-treatment with tachykinin NK 1 and NK 2 receptor antagonists. These results indicate that tachykinin NK 1 and NK 3 receptors may be involved in the control of the respiratory frequency.

Perinatal developmental changes in respiratory activity of medullary and spinal neurons: an in vitro study on fetal and newborn rats

Experiments were performed in vitro on fetal and newborn rat brainstem-spinal cord preparations to analyse the perinatal developmental changes in inspiratory motor output. The amplitude of the inspiratory bursts of the whole C4 ventral root (global extracellular recording), the firing patterns of 80 medullary inspiratory neurons (unitary extracellular recording) and the firing and membrane properties of 71 respiratory neurons in the C 4 ventral horn (whole-cell recording) were analysed at embryonic day 18 (E18), 21 (E21) and post natal days 0 to 3 (P0–3). At E18, the amplitude of the C 4 bursts was weak and variable from one respiratory cycle to the next, as well as the discharge pattern of most of the medullary inspiratory neurons. C 4 motoneurons were immature, very excitable and displaying variable inspiratory discharges, but already able to deliver sustained bursts of potentials when depolarised. At E21 and P0–3, the amplitude of the C 4 bursts was increased and stable, most of the medullary inspiratory neurons already were able to generate a stable firing pattern and C 4 motoneurons showed maturational changes in terms of the resting potential, spike amplitude and input membrane resistance. This work suggests that the short period extending from E18 to E21 is a critical maturational period for the medullary respiratory network which becomes able to elaborate a stable respiratory motor output.

Effects of cAMP on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rat

Involvement of cAMP in the generation of respiratory rhythm was studied in newborn rat brainstem-spinal cord preparations. The respiratory rhythm was monitored by C4 inspiratory activity and/or pre-inspiratory (Pre-I) activity of neurons in the rostral ventrolateral medulla; previously suggested to be primary rhythm generating neurons which have pacemaker properties. The effects of four cAMP-increasing agents (forskolin, IBMX, Db-cAMP, and 8-Br-cAMP) on this neuronal activity were examined. Perfusion with forskolin (3–10 μM) increased the burst rate of C4 inspiratory activity in 20 of 32 preparations, but in 8 of those the increase was preceded by transient depression. The facilitation of the respiratory rhythm was greater whenever the burst rate before forskolin treatment was lower. The Pre-I neuron burst rate, which was recorded together with C4 activity, predominantly increased with forskolin treatment. The effects of IBMX, Db-cAMP and 8-Br-cAMP were similar to those of forskolin, but they were slightly less potent. Long-lasting depression of the respiratory rhythm (C4 and Pre-I activity) by clonidine, which might decrease intracellular cAMP level viaμ 2-receptors, was reversed by forskolin. To investigate the direct effects of the cAMP-increasing agents on Pre-I neurons, Pre-I activity was isolated by blocking the chemical synaptic transmission by incubation in a low Ca solution (0.2 mM Ca 2+, 5 mM Mg 2+). Forskolin (5–10 μM), IBMX (5–10 μM), Db-cAMP (0.2–0.4 mM), and 8-Br-cAMP (0.4–0.75 mM) all enhanced the burst rate of isolated Pre-I neurons. Results suggest that cAMP is important in respiratory rhythm generation; this may be due to its regulation of the intrinsic burst generating properties of Pre-I neurons.

Serotonin and cervical respiratory motoneurones: intracellular study in the newborn rat brainstem-spinal cord preparation

Previous experiments performed in the in vitro newborn rat brainstem-spinal cord preparation reported that the addition of serotonin (5-HT, 30-50 microM) to the bathing medium induced increases in the respiratory frequency and a large tonic discharge on all the cervical ventral roots. The aim of the present work was to define whether the 5-HT-induced tonic discharge involved respiratory or non-respiratory motoneurones. Intracellular recordings demonstrated that cervical (C2) motoneurones (n = 27) were depolarized by 5-HT but that the 5-HT-induced tonic discharge was mainly due to recruitment of silent motoneurones (n = 18) which fired permanently (15/18; 17 +/- 3 Hz) under 5-HT. The respiratory motoneurones (n = 9) retained a phasic inspiratory discharge (5/9), even if some (4/9) occasionally exhibited a few spikes during expiration. Therefore, it is concluded that the 5-HT-induced tonic discharge is unlikely to have functional significance in respiration.