- Email: [email protected]
Serotonergic inhibition of phrenic motoneuron activity: an in vitro study in neonatal rat
Neuroscience Letters 230 (1997) 29–32
Serotonergic inhibition of phrenic motoneuron activity: an in vitro study in neonatal rat Eric Di Pasquale a, Amy Lindsay b, Jack Feldman b, Roger Monteau a ,*, Ge´rard Hilaire a a
UPRESA CNRS 6034, Biologie des Rythmes et du De´veloppement, Faculte´ des Sciences de St Je´roˆme, 13397 Marseille, Cedex 20, France b Systems Neurobiology Laboratory, Department of Physiological Science, UCLA, Box 951527, Los Angeles, CA 90095–1527, USA Received 24 February 1997; accepted 18 June 1997
Abstract In vitro experiments were conducted on neonatal rat brainstem-spinal cord preparations to test the hypothesis of an inhibitory modulation of phrenic activity by serotonin (5-HT) via non-5-HT2A receptors [Lindsay, A.D. and Feldman, J.L., Modulation of respiratory activity of neonatal rat phrenic motoneurones by serotonin, J. Physiol., 461 (1993) 213–233]. The changes induced by 5-HT and related agents on phrenic root discharges and membrane currents in identified phrenic motoneurons were analysed after blockade of spinal 5-HT2A receptors. Spinal application of 5-HT1B (but not 5-HT1A) receptor agonists depressed the phrenic activity and the effect was prevented by pretreatment with 5-HT1B (but not 5-HT1A, 5-HT2A and 5-HT3) receptor antagonists. Results from phrenic motoneuron whole cell recordings do not reject a presynaptic location of the 5-HT receptors responsible for this depression. 1997 Elsevier Science Ireland Ltd. Keywords: Phrenic motoneuron; Serotonin; Serotonergic receptors; Respiratory network; Neonatal rat
The isolated brainstem-spinal cord preparations of neonatal rats elaborate a respiratory-like activity on phrenic roots [20] which is modulated by serotonin (5-HT). This widely distributed neurotransmitter and neuromodulator increases the activity of the central rhythm generator via 5-HT1A receptors [15] and the excitability of phrenic motoneurons via spinal postsynaptic 5-HT2A receptors [11,15– 17]. The hypothesis has been put forward however, that besides the spinal 5-HT2A facilitation, 5-HT might also depress the transmission of the central drive to phrenic motoneurons via non-5-HT2A receptors [11]. To test this hypothesis, we analysed the changes induced by 5-HT and related agents on global and unitary phrenic activity after blockade of spinal 5-HT2A receptors with a potent and selective 5-HT2A receptor antagonist (SR 46349B [15,19]). Results confirm that activation of spinal non-5-HT2A receptors depresses the phrenic activity and suggest presynaptic 5-HT1B receptors as the source of this inhibition. Brainstem and spinal cord of 0–3-day-old rats were isolated in an in vitro chamber, superfused with artificial cere* Corresponding author. Tel.: +33 91 288197; fax: +33 91 288333; e-mail: [email protected]
brospinal fluid (aCSF, in mM: NaCl 129, KCl 3.35, CaCl2 1.26, MgCl2 1.15, NaHCO 3 21.0, NaH2 PO4 0.58, glucose 30.0; pH 7.4), warmed to 27 ± 1°C, and equilibrated with 95% O2/5% CO2 [1]. A partition was placed at the level of C1 to bathe the brainstem with normal aCSF and the cervical cord with aCSF containing drugs. The global activity of C4 phrenic roots was recorded with suction electrode and the unitary activity of identified phrenic motoneurons with patch-clamp microelectrode [1] (antidromic activation following ipsilateral C4 ventral root stimulation). The following 5-HT related agents [3,5,8,21] were dissolved in aCSF and applied by superfusion to the spinal cord for 10 min (except 5-HT, 4–5 min): 5-HT (Sigma), (±)-8hydroxy-2-(di-N-propylamino) tetralin hydrobromide (8OH-DPAT, RBI) and 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN-190, RBI) as 5-HT1A receptor agonist and antagonist respectively, 5methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1-H-indol-succinate (RU24969, Roussel-UCLAF) and 7-trifluoromethyl4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a] quinoxaline, 1:2 maleate (CGS-12066B, RBI), 2-[5-[3-(4-methylsulfonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine (L-694,247, Tocris Cookson) as 5-HT1B receptor ago-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0469-2
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E. Di Pasquale et al. / Neuroscience Letters 230 (1997) 29–32
nists, S(−)-1-(1H-indol-4-yloxy)-3-[(1-methylethyl)amino]2-propanol ((−)pindolol, RBI) as 5-HT1B receptor antagonist, {trans-4-([3(Z)3-(2-dimethylaminoethyl)oxyimino-3 (2-fluorophenyl)propen1-yl]phenol hemifumarate} (SR 46349B, Sanofi-Recherche) as 5-HT2A receptor antagonist and granisetron (Beecham Pharmaceuticals) as 5-HT3 receptor antagonist. In some experiments, drugs were applied locally via broken patch microelectrodes (placed close to or just below the ventral surface of the spinal cord) and pressure pulse ejection (ejected volumes 0.5–5 ml). Only one preparation was used for each series of drug applications and experiments were repeated using standardised protocols. Changes in amplitude of the integrated phrenic discharge were expressed in % of control amplitude measured under normal aCSF prior drug application (100%). Results are given as mean ± SEM and differences
between means were taken to be significant at P value less than 0.05. As already reported [15–17], application of aCSF containing 5-HT (30 mM, 4–5 min) to the spinal cord induced a tonic discharge superimposed on the phrenic bursts elaborated by the neonatal rat brainstem-spinal cord preparation (Fig. 1A, upper trace). In all the experiments reported below, the tonic activity was prevented by a pretreatment with the 5-HT2A receptor antagonist SR 46349B (20 mM, [15]). Thereafter, 5-HT no longer induced a tonic discharge but significantly depressed the amplitude of the phrenic bursts to 64 ± 10% of the control value (n = 12; Fig. 1B). Similar effects were obtained with the 5-HT1B receptor agonists (Fig. 1C–D) CGS-12066B (77 ± 13% when applied at 40 mM, n = 5), RU24969 (66 ± 26% at 20 mM, n = 10, 68 ± 18% at 100 mM, n = 4; no significant effect at 2 mM,
Fig. 1. Depression of the phrenic bursts by serotonin. Schematic representation of the in vitro brainstem-spinal cord preparation (ventral surface upward) showing a suction electrode on the C4 ventral root, the raw (lower trace) and integrated (upper trace) phrenic burst and the level of the partition placed to bathe the brainstem with normal aCSF and the spinal cord with aCSF containing 5-HT related agents (horizontal arrow). (A and B) Applying 5-HT (30 mM, 4 min, horizontal black bar) elicited a tonic discharge superimposed on the phrenic bursts (A); occurrence of the tonic discharge was prevented by pretreatment with the 5-HT2A receptor antagonist SR 46349B (B; 20 mM, horizontal dotted line); the thin dotted line in B indicates the mean amplitude level of the integrated phrenic bursts during the SR 46349B pretreatment; thereafter, 5-HT depressed the amplitude of the phrenic bursts below the dotted line. (C, D and E) The histograms (phrenic integral amplitude in arbitrary unit, Amp (a.u.), versus time) represent the average of 10 phrenic integrated bursts during the SR 46349B pretreatment (thin line) and under aCSF containing drugs (heavy line). In C and D, the 5-HT1B receptor agonists RU24969 (in C, 20 mM) and CGS12066B (in D, 40 mM) both depressed the integrated phrenic bursts. In E, the 5-HT2A receptor antagonist SR 46349B and the 5-HT1B receptor antagonist (−)pindolol were applied for 10 min. Thereafter aCSF containing RU24969 no longer depressed the phrenic bursts. (F) Local application RU24969 within the right C4 ventral horn by pressure ejection (arrow) depressed the amplitude of the right (R) phrenic nerve discharge while the left (L) was not affected (the dotted line defines the mean amplitude of 10 inspiratory bursts preceding the ejection).
E. Di Pasquale et al. / Neuroscience Letters 230 (1997) 29–32
n = 4) and L-694,247 (59 ± 7% at 20 mM, n = 5). The phrenic burst depression by RU24969 was prevented by pretreatment with the 5-HT1B receptor antagonist (−)pindolol (20 mM, n = 3, Fig. 1E). Local application of RU24969 via a micropipette inserted into the right C4 cervical ventral horn depressed the ipsilateral phrenic bursts to 63 ± 13% of control without affecting the contralateral phrenic discharges (n = 8, 2–20 mM). The phrenic bursts were not depressed by spinal superfusion of the 5-HT1A receptor agonist 8-OHDPAT (20 mM, n = 4) and the phrenic burst depression by RU24969 was not prevented by pretreatment with the 5HT1A receptor antagonist NAN-190 (20 mM, n = 3) and the 5-HT3 receptor antagonist granisetron (20 mM, n = 3). Then, implication of 5-HT1A, 5-HT2A and 5-HT3 receptors can be ruled out. Involvement of 5-HT1B receptors sounds likely since phrenic bursts were depressed and protected by the 5-HT1B receptor agonists and antagonist, respectively. A definitive conclusion cannot be reached, however, because (i) highly selective 5-HT1B agents are lacking, (ii) high concentrations of drugs were used in this study so that the specificity of our 5-HT1B agents can be questioned and (iii) finally, ‘the multiplicity of 5-HT receptors subtypes... has far exceeded most of the predictions that might have been made on the basis of pharmacological data’ [18]. In 15 phrenic motoneurons, the resting membrane potential and input membrane resistance ranged 60 mV and 150 MQ. During the phrenic bursts, they depolarised by 7–20 mV (current-clamp recording) with an inward central synaptic current which was maximal at the beginning of the phrenic burst (600–900 pA, voltage clamp recording, n = 6) and then decayed slowly through the burst (Fig. 2A1). In three motoneurons, RU24969-containing aCSF (20 mM) was applied and did not change neither resting potential nor input membrane resistance but depressed the depolarisation during the burst and the corresponding inward central synaptic current by around 50% (Fig. 2A2). Because the transmission of the central drive to the phrenic motoneurons is mediated by excitatory amino acid [12], RU24969 might depress the phrenic discharge by decreasing the responsiveness of the glutamate postsynaptic receptors. To test this possibility, the depolarisation induced in phrenic motoneurons by exogenous glutamate was compared between normal aCSF and RU24969 containing aCSF. Since the depolarisation of phrenic motoneurons could reflect both their direct response to glutamate and indirect effects relayed by spinal interneurons, TTX (1 mM) was applied to block any indirect effects. The following protocol was used: (i) analysis of three control responses to glutamate ejection in the vicinity of the neuron in normal aCSF, (ii) 3 responses after SR 46349B pretreatment, (iii) three responses after TTX treatment and finally, (iv) at least three responses under RU24969 (20 mM). In 5/6 phrenic motoneurons that satisfied all these criteria, RU24969 did not attenuate the response to glutamate. These results concerning electrophysiological properties of phrenic motoneurons are consistent with previously pub-
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lished data [1,11]. Effects of RU24969 do not support a postsynaptic location of the 5-HT receptors and the hypothesis that they are located at the presynaptic level cannot be ruled out [11]. At least three different locations within the C4 ventral horn may be suggested. Firstly, 5-HT receptors on serotonergic fibres could diminish an ongoing release of endogenous 5-HT [5], leading to a withdrawal of a tonic postsynaptic facilitation on phrenic motoneurons. Secondly, 5-HT receptors on spinal interneurons could reduce a polysynaptic component of the central drive (see [1,14]). Thirdly, 5-HT receptors located on the axon terminals of
Fig. 2. RU24969 depresses the inspiratory drive currents in phrenic motoneurons without affecting responsiveness of glutamate receptors. Whole cell patch-clamp recordings in three different phrenic motoneurons identified by antidromic activation. A: the motoneuron was clamped at −72 mV to avoid contamination of inward central synaptic drive current by action potentials. Histograms show integrated phrenic bursts (at the top, arbitrary unit, a.u.) and central synaptic drive currents (at the bottom, membrane current, pA) occurring in five successive cycles (thin lines, individual traces, heavy line, average trace). (A1) Control after SR 46349B pretreatment. (A2) Effect of RU24969; note the decrease in central synaptic drive current and integrated phrenic bursts. (B and C): current clamp recording in two different phrenic motoneurons (upper trace) and contralateral phrenic burst (lower trace). A pipette containing glutamate was positioned in the C4 ventral horn to eject glutamate (pressure pulse) in the vicinity of the recorded motoneuron. (B1 and C1) Normal aCSF. (B2 and C2) After SR 46349B, RU24969 and TTX treatments (no spiking). (B1) The motoneuron was depolarised by ejection of glutamate within the C4 ventral horn (vertical arrow) and fired a sustained burst of potential. (B2) The direct response of the motoneuron to glutamate was not affected by SR 46349B, RU24969 and TTX treatments. (C1) This other motoneuron showed subthreshold depolarisation during each phrenic bursts and remained silent; ejections of glutamate by short (20 ms, thin arrow on the left) and long pressure pulses (100 ms, large arrow on the right) induced weak and strong depolarisations (with firing), respectively. (C2) Both responses to short (thin arrow on the right) and long (large arrow on the left) pressure pulses persisted after SR 46349B, RU24969 and TTX treatments.
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the medullary neurons impinging on the phrenic motoneurons could modulate the monosynaptic transmission of the central drive [14]. None of the three hypothesis can be cancelled yet but the third sounds attractive. Our results obtained on brainstem-spinal cord preparation from neonatal rats confirm that activation of spinal non-5HT2A receptors depresses the amplitude of the phrenic discharges [11]. They suggest that the involved 5-HT receptors (1) may be located at the presynaptic level [11] and (2) may belong to the 5-HT1B subtype but the complexity of the 5HT mechanisms and receptor subtype classification does not allow a definite conclusion [3,5,8,18,21]. The in vitro results may reflect, at least in part, the in vivo organisation since 5HT affects the phrenic activity in vivo [6,7,9,10,13,14]. Therefore, 5-HT might modulate phrenic motoneuron firing via activation of spinal postsynaptic 5-HT2A [11,15–17] and presynaptic 5-HT1B receptors. The 5-HT depression of the phrenic bursts may be seen as a protective mechanism against overstimulation of the diaphragm [11] and/or may intervene when the inspiratory central drive onto the motoneurons has to be gated, i.e. in situations where they have to subserve non-respiratory functions (i.e. thermoregulation, vomiting, defecation, yawning, etc. [4,14]) or during REM sleep [2]. This work was supported by grants from the NIH (NS24742), Sanofi-Recherche S.A and the CNRS (URA1832). G. Hilaire and J.L. Feldman were supported by fellowships from ‘Conseil Regional Provence Alpes Coˆte d’Azur’ and ‘Route des Hautes Technologies’. The authors acknowledge the highly useful assistance of Mrs. A.M. Lajard with the photography. [1] Di Pasquale, E., Tell, F., Monteau, R. and Hilaire, G., Perinatal developmental changes in respiratory activity of medullary and spinal neurons: an in vitro study on fetal and newborn rats, Dev. Brain Res., 91 (1996) 121–130. [2] Feldman, J.L. and Smith, J.C., Neural control of respiratory pattern in mammals: an overview. In J.A. Dempsey and A.I. Pack. (Eds.), Lung Biology in Health and Disease, Vol 79: Regulation of breathing, M. Dekker, New York, 1994, pp. 39–69. [3] Glennon, R.A. and Dukat, M., Serotonin receptors and their ligands: a lack of selective agents, Pharmacol. Biochem. Behav., 40 (1991) 1009–1017. [4] Grelot, L., Milano, S., Portillo, F., Miller, A.D. and Bianchi, A.L., Membrane potential changes of phrenic motoneurons during fictive vomiting, coughing and swallowing in the decerebrate cat, J. Neurophysiol., 68 (1992) 2110–2119.
[5] Hammon, M. and Gozlan, H., Les re´cepteurs centraux a` la se´rotonine, Me´d. Sci., 9 (1993) 21–30. [6] Holtman, J.R., Dick, T.E. and Berger, A.J., Involvement of serotonin in the excitation of phrenic motoneurones evoked by stimulation of the raphe obscurus, J. Neurosci., 6 (1986) 1185–1193. [7] Holtman, J.R., Dick, T.E. and Berger, A.J., Serotonin-mediated excitation of recurrent laryngeal and phrenic motoneurons evoked by stimulation of the raphe obscurus, Brain Res., 417 (1987) 12–20. [8] Hoyer, D. Agonists and antagonists at 5-HT receptors subtypes. In P.B. Bradley, S.L. Handley, S.J. Cooper, N.M. Barnes and J.H. Coote (Eds.), Serotonin, CNS Receptors and Brain Function, Pergamon Press, Oxford, 1991, pp. 29–47. [9] Lalley, P.M., Responses of phrenic motoneurones of the cat to stimulation of medullary raphe nuclei, J. Physiol., 380 (1986) 349–371. [10] Lalley, P.M., Serotonergic and non-serotonergic responses of phrenic motoneurones to raphe stimulation in the cat, J. Physiol., 380 (1986) 373–385. [11] Lindsay, A.D. and Feldman, J.L., Modulation of respiratory activity of neonatal rat phrenic motoneurones by serotonin, J. Physiol., 461 (1993) 213–233. [12] Liu, G., Feldman, J.L. and Smith, J.C., Excitatory amino acid mediated transmission of inspiratory drive to phrenic motoneurons, J. Neurophysiol., 64 (1990) 423–436. [13] Mitchell, G.S., Sloan, H.E., Jiang, C., Miletic, V., Hayashi, F. and Lipski, J., 5-hydroxytryptophan (5-HTP) augments spontaneous and evoked phrenic motoneuron discharge in spinalized rats, Neurosci. Lett., 141 (1992) 75–78. [14] Monteau, R. and Hilaire, G., Spinal respiratory motoneurons, Progr. Neurobiol., 37 (1991) 83–144. [15] Monteau, R., Di Pasquale, E. and Hilaire, G., Further evidence that various 5-HT receptor subtypes modulates central respiratory activity: in vitro studies with SR 46349B, Eur. J. Pharmacol., 259 (1994) 71–74. [16] Morin, D., Hennequin, S., Monteau, R. and Hilaire, G., Serotonergic influences on central respiratory activity: an in vitro study in the newborn rat, Brain Res., 535 (1990) 281–287. [17] Morin, D., Monteau, R. and Hilaire, G., Compared effects of serotonin on cervical and hypoglossal inspiratory activities: an in vitro study in the newborn rat, J. Physiol., 451 (1992) 605–629. [18] Peroutka, S., Molecular biology of serotonin (5-HT) receptors, Synapse, 18 (1994) 241–260. [19] Rinaldi-Carmona, M., Congy, C., Santucci, V., Simian, J., Gautret, B., Neliat, G., Labeeuw, B., Le Fur, G., Soubrie, P. and Breliere, J.C., Biochemical and pharmacological properties of SR 46349B, a new potent and selective 5-hydroxytryptamine-2 receptor antagonist, J. Pharmacol. Exp. Ther., 262 (1992) 759–768. [20] Suzue, T., Respiratory rhythm generation in the in vitro brainstemspinal cord preparation of the newborn rat, J. Physiol., 354 (1984) 173–183. [21] Zifa, E. and Fillion, G., 5-Hydroxytryptamine receptors, Pharmacol. Rev., 44 (1992) 401–457.
Serotonergic inhibition of phrenic motoneuron activity: an in vitro study in neonatal rat Eric Di Pasquale a, Amy Lindsay b, Jack Feldman b, Roger Monteau a ,*, Ge´rard Hilaire a a
UPRESA CNRS 6034, Biologie des Rythmes et du De´veloppement, Faculte´ des Sciences de St Je´roˆme, 13397 Marseille, Cedex 20, France b Systems Neurobiology Laboratory, Department of Physiological Science, UCLA, Box 951527, Los Angeles, CA 90095–1527, USA Received 24 February 1997; accepted 18 June 1997
Abstract In vitro experiments were conducted on neonatal rat brainstem-spinal cord preparations to test the hypothesis of an inhibitory modulation of phrenic activity by serotonin (5-HT) via non-5-HT2A receptors [Lindsay, A.D. and Feldman, J.L., Modulation of respiratory activity of neonatal rat phrenic motoneurones by serotonin, J. Physiol., 461 (1993) 213–233]. The changes induced by 5-HT and related agents on phrenic root discharges and membrane currents in identified phrenic motoneurons were analysed after blockade of spinal 5-HT2A receptors. Spinal application of 5-HT1B (but not 5-HT1A) receptor agonists depressed the phrenic activity and the effect was prevented by pretreatment with 5-HT1B (but not 5-HT1A, 5-HT2A and 5-HT3) receptor antagonists. Results from phrenic motoneuron whole cell recordings do not reject a presynaptic location of the 5-HT receptors responsible for this depression. 1997 Elsevier Science Ireland Ltd. Keywords: Phrenic motoneuron; Serotonin; Serotonergic receptors; Respiratory network; Neonatal rat
The isolated brainstem-spinal cord preparations of neonatal rats elaborate a respiratory-like activity on phrenic roots [20] which is modulated by serotonin (5-HT). This widely distributed neurotransmitter and neuromodulator increases the activity of the central rhythm generator via 5-HT1A receptors [15] and the excitability of phrenic motoneurons via spinal postsynaptic 5-HT2A receptors [11,15– 17]. The hypothesis has been put forward however, that besides the spinal 5-HT2A facilitation, 5-HT might also depress the transmission of the central drive to phrenic motoneurons via non-5-HT2A receptors [11]. To test this hypothesis, we analysed the changes induced by 5-HT and related agents on global and unitary phrenic activity after blockade of spinal 5-HT2A receptors with a potent and selective 5-HT2A receptor antagonist (SR 46349B [15,19]). Results confirm that activation of spinal non-5-HT2A receptors depresses the phrenic activity and suggest presynaptic 5-HT1B receptors as the source of this inhibition. Brainstem and spinal cord of 0–3-day-old rats were isolated in an in vitro chamber, superfused with artificial cere* Corresponding author. Tel.: +33 91 288197; fax: +33 91 288333; e-mail: [email protected]
brospinal fluid (aCSF, in mM: NaCl 129, KCl 3.35, CaCl2 1.26, MgCl2 1.15, NaHCO 3 21.0, NaH2 PO4 0.58, glucose 30.0; pH 7.4), warmed to 27 ± 1°C, and equilibrated with 95% O2/5% CO2 [1]. A partition was placed at the level of C1 to bathe the brainstem with normal aCSF and the cervical cord with aCSF containing drugs. The global activity of C4 phrenic roots was recorded with suction electrode and the unitary activity of identified phrenic motoneurons with patch-clamp microelectrode [1] (antidromic activation following ipsilateral C4 ventral root stimulation). The following 5-HT related agents [3,5,8,21] were dissolved in aCSF and applied by superfusion to the spinal cord for 10 min (except 5-HT, 4–5 min): 5-HT (Sigma), (±)-8hydroxy-2-(di-N-propylamino) tetralin hydrobromide (8OH-DPAT, RBI) and 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN-190, RBI) as 5-HT1A receptor agonist and antagonist respectively, 5methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1-H-indol-succinate (RU24969, Roussel-UCLAF) and 7-trifluoromethyl4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a] quinoxaline, 1:2 maleate (CGS-12066B, RBI), 2-[5-[3-(4-methylsulfonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine (L-694,247, Tocris Cookson) as 5-HT1B receptor ago-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0469-2
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E. Di Pasquale et al. / Neuroscience Letters 230 (1997) 29–32
nists, S(−)-1-(1H-indol-4-yloxy)-3-[(1-methylethyl)amino]2-propanol ((−)pindolol, RBI) as 5-HT1B receptor antagonist, {trans-4-([3(Z)3-(2-dimethylaminoethyl)oxyimino-3 (2-fluorophenyl)propen1-yl]phenol hemifumarate} (SR 46349B, Sanofi-Recherche) as 5-HT2A receptor antagonist and granisetron (Beecham Pharmaceuticals) as 5-HT3 receptor antagonist. In some experiments, drugs were applied locally via broken patch microelectrodes (placed close to or just below the ventral surface of the spinal cord) and pressure pulse ejection (ejected volumes 0.5–5 ml). Only one preparation was used for each series of drug applications and experiments were repeated using standardised protocols. Changes in amplitude of the integrated phrenic discharge were expressed in % of control amplitude measured under normal aCSF prior drug application (100%). Results are given as mean ± SEM and differences
between means were taken to be significant at P value less than 0.05. As already reported [15–17], application of aCSF containing 5-HT (30 mM, 4–5 min) to the spinal cord induced a tonic discharge superimposed on the phrenic bursts elaborated by the neonatal rat brainstem-spinal cord preparation (Fig. 1A, upper trace). In all the experiments reported below, the tonic activity was prevented by a pretreatment with the 5-HT2A receptor antagonist SR 46349B (20 mM, [15]). Thereafter, 5-HT no longer induced a tonic discharge but significantly depressed the amplitude of the phrenic bursts to 64 ± 10% of the control value (n = 12; Fig. 1B). Similar effects were obtained with the 5-HT1B receptor agonists (Fig. 1C–D) CGS-12066B (77 ± 13% when applied at 40 mM, n = 5), RU24969 (66 ± 26% at 20 mM, n = 10, 68 ± 18% at 100 mM, n = 4; no significant effect at 2 mM,
Fig. 1. Depression of the phrenic bursts by serotonin. Schematic representation of the in vitro brainstem-spinal cord preparation (ventral surface upward) showing a suction electrode on the C4 ventral root, the raw (lower trace) and integrated (upper trace) phrenic burst and the level of the partition placed to bathe the brainstem with normal aCSF and the spinal cord with aCSF containing 5-HT related agents (horizontal arrow). (A and B) Applying 5-HT (30 mM, 4 min, horizontal black bar) elicited a tonic discharge superimposed on the phrenic bursts (A); occurrence of the tonic discharge was prevented by pretreatment with the 5-HT2A receptor antagonist SR 46349B (B; 20 mM, horizontal dotted line); the thin dotted line in B indicates the mean amplitude level of the integrated phrenic bursts during the SR 46349B pretreatment; thereafter, 5-HT depressed the amplitude of the phrenic bursts below the dotted line. (C, D and E) The histograms (phrenic integral amplitude in arbitrary unit, Amp (a.u.), versus time) represent the average of 10 phrenic integrated bursts during the SR 46349B pretreatment (thin line) and under aCSF containing drugs (heavy line). In C and D, the 5-HT1B receptor agonists RU24969 (in C, 20 mM) and CGS12066B (in D, 40 mM) both depressed the integrated phrenic bursts. In E, the 5-HT2A receptor antagonist SR 46349B and the 5-HT1B receptor antagonist (−)pindolol were applied for 10 min. Thereafter aCSF containing RU24969 no longer depressed the phrenic bursts. (F) Local application RU24969 within the right C4 ventral horn by pressure ejection (arrow) depressed the amplitude of the right (R) phrenic nerve discharge while the left (L) was not affected (the dotted line defines the mean amplitude of 10 inspiratory bursts preceding the ejection).
E. Di Pasquale et al. / Neuroscience Letters 230 (1997) 29–32
n = 4) and L-694,247 (59 ± 7% at 20 mM, n = 5). The phrenic burst depression by RU24969 was prevented by pretreatment with the 5-HT1B receptor antagonist (−)pindolol (20 mM, n = 3, Fig. 1E). Local application of RU24969 via a micropipette inserted into the right C4 cervical ventral horn depressed the ipsilateral phrenic bursts to 63 ± 13% of control without affecting the contralateral phrenic discharges (n = 8, 2–20 mM). The phrenic bursts were not depressed by spinal superfusion of the 5-HT1A receptor agonist 8-OHDPAT (20 mM, n = 4) and the phrenic burst depression by RU24969 was not prevented by pretreatment with the 5HT1A receptor antagonist NAN-190 (20 mM, n = 3) and the 5-HT3 receptor antagonist granisetron (20 mM, n = 3). Then, implication of 5-HT1A, 5-HT2A and 5-HT3 receptors can be ruled out. Involvement of 5-HT1B receptors sounds likely since phrenic bursts were depressed and protected by the 5-HT1B receptor agonists and antagonist, respectively. A definitive conclusion cannot be reached, however, because (i) highly selective 5-HT1B agents are lacking, (ii) high concentrations of drugs were used in this study so that the specificity of our 5-HT1B agents can be questioned and (iii) finally, ‘the multiplicity of 5-HT receptors subtypes... has far exceeded most of the predictions that might have been made on the basis of pharmacological data’ [18]. In 15 phrenic motoneurons, the resting membrane potential and input membrane resistance ranged 60 mV and 150 MQ. During the phrenic bursts, they depolarised by 7–20 mV (current-clamp recording) with an inward central synaptic current which was maximal at the beginning of the phrenic burst (600–900 pA, voltage clamp recording, n = 6) and then decayed slowly through the burst (Fig. 2A1). In three motoneurons, RU24969-containing aCSF (20 mM) was applied and did not change neither resting potential nor input membrane resistance but depressed the depolarisation during the burst and the corresponding inward central synaptic current by around 50% (Fig. 2A2). Because the transmission of the central drive to the phrenic motoneurons is mediated by excitatory amino acid [12], RU24969 might depress the phrenic discharge by decreasing the responsiveness of the glutamate postsynaptic receptors. To test this possibility, the depolarisation induced in phrenic motoneurons by exogenous glutamate was compared between normal aCSF and RU24969 containing aCSF. Since the depolarisation of phrenic motoneurons could reflect both their direct response to glutamate and indirect effects relayed by spinal interneurons, TTX (1 mM) was applied to block any indirect effects. The following protocol was used: (i) analysis of three control responses to glutamate ejection in the vicinity of the neuron in normal aCSF, (ii) 3 responses after SR 46349B pretreatment, (iii) three responses after TTX treatment and finally, (iv) at least three responses under RU24969 (20 mM). In 5/6 phrenic motoneurons that satisfied all these criteria, RU24969 did not attenuate the response to glutamate. These results concerning electrophysiological properties of phrenic motoneurons are consistent with previously pub-
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lished data [1,11]. Effects of RU24969 do not support a postsynaptic location of the 5-HT receptors and the hypothesis that they are located at the presynaptic level cannot be ruled out [11]. At least three different locations within the C4 ventral horn may be suggested. Firstly, 5-HT receptors on serotonergic fibres could diminish an ongoing release of endogenous 5-HT [5], leading to a withdrawal of a tonic postsynaptic facilitation on phrenic motoneurons. Secondly, 5-HT receptors on spinal interneurons could reduce a polysynaptic component of the central drive (see [1,14]). Thirdly, 5-HT receptors located on the axon terminals of
Fig. 2. RU24969 depresses the inspiratory drive currents in phrenic motoneurons without affecting responsiveness of glutamate receptors. Whole cell patch-clamp recordings in three different phrenic motoneurons identified by antidromic activation. A: the motoneuron was clamped at −72 mV to avoid contamination of inward central synaptic drive current by action potentials. Histograms show integrated phrenic bursts (at the top, arbitrary unit, a.u.) and central synaptic drive currents (at the bottom, membrane current, pA) occurring in five successive cycles (thin lines, individual traces, heavy line, average trace). (A1) Control after SR 46349B pretreatment. (A2) Effect of RU24969; note the decrease in central synaptic drive current and integrated phrenic bursts. (B and C): current clamp recording in two different phrenic motoneurons (upper trace) and contralateral phrenic burst (lower trace). A pipette containing glutamate was positioned in the C4 ventral horn to eject glutamate (pressure pulse) in the vicinity of the recorded motoneuron. (B1 and C1) Normal aCSF. (B2 and C2) After SR 46349B, RU24969 and TTX treatments (no spiking). (B1) The motoneuron was depolarised by ejection of glutamate within the C4 ventral horn (vertical arrow) and fired a sustained burst of potential. (B2) The direct response of the motoneuron to glutamate was not affected by SR 46349B, RU24969 and TTX treatments. (C1) This other motoneuron showed subthreshold depolarisation during each phrenic bursts and remained silent; ejections of glutamate by short (20 ms, thin arrow on the left) and long pressure pulses (100 ms, large arrow on the right) induced weak and strong depolarisations (with firing), respectively. (C2) Both responses to short (thin arrow on the right) and long (large arrow on the left) pressure pulses persisted after SR 46349B, RU24969 and TTX treatments.
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the medullary neurons impinging on the phrenic motoneurons could modulate the monosynaptic transmission of the central drive [14]. None of the three hypothesis can be cancelled yet but the third sounds attractive. Our results obtained on brainstem-spinal cord preparation from neonatal rats confirm that activation of spinal non-5HT2A receptors depresses the amplitude of the phrenic discharges [11]. They suggest that the involved 5-HT receptors (1) may be located at the presynaptic level [11] and (2) may belong to the 5-HT1B subtype but the complexity of the 5HT mechanisms and receptor subtype classification does not allow a definite conclusion [3,5,8,18,21]. The in vitro results may reflect, at least in part, the in vivo organisation since 5HT affects the phrenic activity in vivo [6,7,9,10,13,14]. Therefore, 5-HT might modulate phrenic motoneuron firing via activation of spinal postsynaptic 5-HT2A [11,15–17] and presynaptic 5-HT1B receptors. The 5-HT depression of the phrenic bursts may be seen as a protective mechanism against overstimulation of the diaphragm [11] and/or may intervene when the inspiratory central drive onto the motoneurons has to be gated, i.e. in situations where they have to subserve non-respiratory functions (i.e. thermoregulation, vomiting, defecation, yawning, etc. [4,14]) or during REM sleep [2]. This work was supported by grants from the NIH (NS24742), Sanofi-Recherche S.A and the CNRS (URA1832). G. Hilaire and J.L. Feldman were supported by fellowships from ‘Conseil Regional Provence Alpes Coˆte d’Azur’ and ‘Route des Hautes Technologies’. The authors acknowledge the highly useful assistance of Mrs. A.M. Lajard with the photography. [1] Di Pasquale, E., Tell, F., Monteau, R. and Hilaire, G., Perinatal developmental changes in respiratory activity of medullary and spinal neurons: an in vitro study on fetal and newborn rats, Dev. Brain Res., 91 (1996) 121–130. [2] Feldman, J.L. and Smith, J.C., Neural control of respiratory pattern in mammals: an overview. In J.A. Dempsey and A.I. Pack. (Eds.), Lung Biology in Health and Disease, Vol 79: Regulation of breathing, M. Dekker, New York, 1994, pp. 39–69. [3] Glennon, R.A. and Dukat, M., Serotonin receptors and their ligands: a lack of selective agents, Pharmacol. Biochem. Behav., 40 (1991) 1009–1017. [4] Grelot, L., Milano, S., Portillo, F., Miller, A.D. and Bianchi, A.L., Membrane potential changes of phrenic motoneurons during fictive vomiting, coughing and swallowing in the decerebrate cat, J. Neurophysiol., 68 (1992) 2110–2119.
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