Neuroscience Letters, 111 (1990) 127-132
127
Elsevier Scientific Publishers Ireland Ltd. NSL 06749
Differential effects of serotonin on respiratory activity of hypoglossal and cervical motoneurons: an in vitro study on the newborn rat R. Monteau,
D . M o r i n , S. H e n n e q u i n a n d G . H i l a i r e
Dbparternent de Physiologie et Neurophysiologie, Equipe Biologie des Rythmes et du D~veloppement, Facultk des Sciences et Techniques St. Jkr6me, Marseilles (France)
(Received 31 May 1989; Revised version received 24 November 1989; Accepted 24 November 1989) Key words: Respiration; Serotonin; Hypoglossal nerve; Newborn; Rat; In vitro
Newborn rat respiratory activity was recorded on hypoglossal nerve and ventral cervical roots during in vitro experiments performed on superfused brainstem spinal cord preparations. The addition of serotonin (5-HT) to the bathing medium increased the respiratory frequency and selectively depressed the hypoglossal activity. Any decreases in the amplitude of cervical recordings were always restricted and reversible, whereas the hypoglossal activity was abolished. Furthermore, on cervical roots, 5-HT induced a tonic activity superimposed on the respiratory one, which was never observed with the hypoglossal nerve. When 5-HT was applied on isolated hemispinal cord, a tonic activity could still be elicited. These results indicate that serotonin (i) modulates the activity of neurons involved in the generation of respiratory rhythm, (ii) depresses the activity of hypoglossal motoneurons, and (iii) evokes tonic activity in cervical motoneurons, probably as the result of direct spinal effects.
A n a l y s i s o f r e s p i r a t o r y c o n t r o l systems can be facilitated b y the d e v e l o p m e n t o f in vitro p r e p a r a t i o n s o f isolated m a m m a l i a n brainstems. A viable in vitro n e w b o r n rat p r e p a r a t i o n was recently d e v e l o p e d [19]. As drugs can be a p p l i e d to the b r a i n s t e m by superfusion in c o n t r o l l e d c o n c e n t r a t i o n s , this p r e p a r a t i o n is very useful for investigating the a c t i o n o f p u t a t i v e n e u r o t r a n s m i t t e r s o n central r e s p i r a t o r y activity [3, 12, 18]. F u r t h e r m o r e , with this p r e p a r a t i o n it is possible to s i m u l t a n e o u s l y r e c o r d the r h y t h m i c r e s p i r a t o r y m o t o r o u t p u t f r o m cranial (hypoglossal) a n d cervical (C4) ventral roots. In the p a s t years, it has been d e m o n s t r a t e d t h a t m a n y agents cause differential changes in the activity o f the phrenic a n d h y p o g l o s s a l nerves. A s a b a l a n c e between the activities o f muscles o f the u p p e r a i r w a y s a n d the d i a p h r a g m is r e q u i r e d to a v o i d o b s t r u c t i o n o f the u p p e r a i r w a y s [16], a n e u r o t r a n s m i t t e r system Correspondence.. R. Monteau, D6partement de Physiologic et Neurophysiologie, Equipe 'Biologie des Rythmes et du D6veloppement', Facult6 des Sciences et Techniques St. J6r6me, Avenue Escadrille Normandie-Niemen, 13397 Marseille C6dex 13, France.
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
128
which differentially affects hypoglossal and phrenic activities can be suspected of being involved in airway obstruction such as apnea in prematurely born infants. Centrally applied serotonin (5-HT) is known to induce respiratory effects in both the adult [11] and the newborn rat [12]. The aim of the present study was to compare the effects of serotonin on hypoglossal and phrenic activities. The brainstem and cervical spinal cord of newborn rats (0-3 days old) were dissected, placed in a 2 ml chamber filled with artificial cerebrospinal solution and fixed with the ventral surface upwards. Nerve root electrical activities were recorded using suction electrodes. Complete experimental procedures have been described elsewhere [3]. Signals were filtered (5-3000 Hz), amplified (Neurolog system, Digitimer), and displayed on an oscilloscope and a paper recorder (Gould TA 2000). The signals were sampled by an A/D converter coupled with a microcomputer and integrated on line
An ,
C4
C41~
XII
C C4
t
......
m"
5-HT
,
D XII
C4
i;
b
Xll C4
XII i
Xll 1
see
~
II 5-HT
1mln
Fig. 1. Effects induced by 5-HT superfusion. A: schematic drawing of the ventral surface of the preparation and the recording electrodes on the hypoglossal nerve (XII) and a cervical ventral root (C4). B: simultaneous recordings of both motor outputs; superfusion with 5-HT begins at the arrow. C: from top to bottom, recordings of both discharges before, during and after superfusion with 5-HT. D: effects of 5-HT (superfusion at the horizontal bar) on the integrated discharges of both nerves. Note the initial increase in frequency, the tonic activity on the C4 recording, and the suppression of the hypoglossal discharge. B-D originate from different experiments.
129 or stored on files. Serotonin (Sigma) was dissolved in the bathing medium and applied by superfusion. All the results reported herein were obtained using serotonin at a concentration of 30/,M. As previously reported [3, 12, 18, 19], the resting respiratory frequency of the isolated preparation was slow (5.2 4- 0.6 m i n - l, mean _ S.E.M.). Changes in cranial and/ or hypoglossal activities were analyzed when the preparation was superfused with a medium containing 5-HT for 6 min in 23 experiments. Recordings were made from either a cervical root alone (11/23), a hypoglossal nerve alone (4/23) or both cervical and hypoglossal nerve simultaneously (8/23). Fig. 1 shows the typical effects of 5-HT whereas Fig. 2 illustrates averaged effects. Within a few minutes the respiratory frequency increased and reached a maximum of 8.3 4- 0.6 m i n - l (different from control value: P < 0.01). Then, the frequency slightly decreased although 5-HT was still present (Figs. I B, D and 2A) but remained above the control level. During the initial period of increased frequency, the amplitude of the respiratory motor output (estimated from the integrated discharge) was generally reduced, but this was more pronounced in the case of the hypoglossal (Figs. 1D and 2C, more than 50% of decrease after 2 rain) than in that of the cervical activity (less than 20%). The two patterns then clearly diverged. On the cervical recordings, a tonic discharge was superimposed on the respiratory activity (60% of experiments, mean latency 2 rain) as long as the preparation was superfused with 5-HT (Fig. 1B-D). On the hypoglossai recordings, no such tonic activity was ever observed and the amplitude of the respiratory activity continued to decrease (Figs. 1 and 2). In most experiments (10/12), the hypoglossal discharge was totally suppressed either during superfusion with 5-HT (i.e. within 6 min), or 1-2 min later. In two experiments, the hypoglossal activity was merely reduced by the first superfusion with 5-HT, but was completely abolished by a second superfusion performed 15 min later. When returned to normal bathing medium, the respiratory rhythm returned to control values. Although the tonic activity disappeared within a few minutes on the cervical roots (Fig. 1D), the hypoglossal activity A
B
Frequency variation (%)
C
A m p l i t u d e variation (%)
Amplitude variation (%)
14~ 120 100
2
4
6
8
[l~l~,='~ : NORMAL
10
12
14 ~
2 : 5-HT
4
6
8
10
12
14
2
4
6
8
I0
12
14
Time (min)
Fig. 2. Mean effectsof 5-HT on the frequencyand amplitudeof the respiratoryactivities.Averagedeffects on the respiratory frequency(A, n=23), the amplitude of the integrated cervical discharge (B, n= 19), and the amplitude of the integrated hypoglossaldischarge (C, n= 12) when changing normal bathing medium(hatched areas) by mediumcontaining5-HT (black areas) are expressedin %of control values.
130
was never o b s e r v e d to recover even 30 m i n u t e s after being r e t u r n e d to the n o r m a l medium. A d d i t i o n a l experiments were p e r f o r m e d to investigate w h e t h e r a n y spinal effects o f 5 - H T were involved in the genesis o f the tonic discharge. T y p i c a l results are shown in Fig. 3. S i m u l t a n e o u s b i l a t e r a l recordings o n cervical ventral r o o t s were p e r f o r m e d a n d the p r e p a r a t i o n was superfused with 5 - H T (for 2 min) to e v o k e tonic activity. A f t e r recovery u n d e r n o r m a l m e d i u m , a hemisection o f the spinal c o r d was p e r f o r m e d at Cl level to suppress the r e s p i r a t o r y drive f r o m the m e d u l l a r y centres to the m o t o neurons. S u p e r f u s i o n with 5 - H T still e v o k e d a tonic activity on b o t h sides. A f t e r being r e t u r n e d to n o r m a l m e d i u m , the spinal c o r d was split a l o n g the midline, in o r d e r to isolate the left side. Superfusion with 5 - H T e v o k e d a n o r m a l response on the intact side a n d a tonic activity on the ventral r o o t s o f the isolated h e m i s p i n a l cord. The b u l b o s p i n a l p a t h w a y s m e d i a t i n g the r e s p i r a t o r y drive were therefore n o t involved in
R1
R2 5-HT
R2 5-HT
RlW, iAmlmB R21
5-HT
Fig. 3. Origin of the tonic C 4 activity. On the left, schematic view of the experimental set-up; on the right, cervical C4 discharges recorded on both sides of the spinal cord. Horizontal bar: superfusion with 5-HT for 2 min. Top panel: in the intact preparation, 5-HT induced tonic activity on both sides. Mid panel: hemisection of the spinal cord performed at C~ level suppressed the spontaneous respiratory activity on the side of the lesion but 5-HT still induced a tonic activity. Bottom panel: after a sagittal section performed on the midline, 5-HT still induced tonic activity on the ventral root of the isolated left spinal segment.
131
the tonic discharge and the possibility that 5-HT had direct effects at the spinal level must be considered. The results reported here demonstrate that superfusion of the isolated brainstem of newborn rats with a medium containing 5-HT induced: (i) an initial increase in respiratory frequency, (ii) a tonic discharge from cervical ventral roots, (iii) the suppression of, or a drastic reduction, in the hypoglossal activity. The observed increase in respiratory frequency is in agreement with previous results [12], but the other effects have never been reported previously. The effects of 5-HT on respiratory activity in the adult have been well documented but are still controversial since both excitatory and inhibitory central and peripheral effects have been reported. At the central level, 5-HT is present in the cranial nuclei and specially in the hypogiossal nuclei [14, 15] as well as in the vicinity of the respiratory neurons [4, 15, 17]. Some neurons are excited and others are inhibited by 5-HT applied iontophoretically [1, 2]. Likewise, activation of the raphe nuclei induces excitatory [6, 8] as well as inhibitory [8] effects. 5-HT weakly affects the phrenic motoneurons [9] although 5-HT terminations are present in the nucleus [5]. Lastly, 5-HTP injected either intravenously or centrally inhibits or excites respiration respectively [ll]. 5-HT neurons develop in the rat brain after 8-9 days of gestation [13], and are sensitive at birth to agents affecting 5-HT synthesis, reuptake and degradation [10]. In isolated newborn rat preparation, the changes in frequency induced by 5-HT may be attributed to medullary structures whereas the changes in amplitude might be due to medullary and/or spinal effects. The results clearly indicated that the tonic activity induced by 5-HT was at least partly of spinal origin. In view of the weak effects of 5-HT on phrenic motoneurons [9] the tonic activity observed might result from activation of non-respiratory cervical motoneurons. As the tonic activity was sometimes followed by a slow rhythmic bursting pattern, motoneurons participating in the locomotor activity of the forelimb might be involved [18]. The increases in respiratory frequency reported above occurred after a short latency (less than 1 min), which suggests that superficial rather than deep structures were involved (because of the diffusion time). Hence, it is unlikely that 5-HT may act directly on deeply located respiratory neurons, even if they are sensitive to 5-HT [1, 2],and an indirect action through structures located near the surface is to be expected. Many agents have been reported to depress more hypoglossal than phrenic activity (see Iscoe [7] for review) but, as far as we know, the effects on hypoglossal motoneurons of directly applied 5-HT or stimulation of the raphe nuclei have never been looked for. Further studies will be necessary to analyse the differential action of 5-HT on the hypoglossal and phrenic motor outputs before we can conclude as to whether endogenous 5-HT is involved in the inhibition of hypoglossal activity which might be responsible for obstructive apnea. The authors gratefully acknowledge the excellent technical assistance of A.M. Lajard and M. Manneville. English revision by Dr. Jessica Blanc. This work was supported by the CNRS (URA 205) and the INSERM (Grant 886006).
132 1 Champagnat, J., Denavit-Saubie, M., Henry, J.L. and Leviel, V., Catecholaminergic depressant effects on bulbar respiratory mechanisms, Brain Res., 160 (1979) 574i8. 2 Fallert, M., B6hmer, G., Dinse, H.R.O., Sommer, T.J. and Bittner, A., Microelectrophoretic application of putative neurotransmitters onto various types of bulbar respiratory neurons. Arch. Ital. Biol. 117 (1979) 1 12. 3 Hilaire, G., Monteau, R. and Errchidi, S., Possible modulation of the medullary respiratory rhythm generator by the noradrenergic A5 area: an in vitro study in the newborn rat, Brain Res., 485 (1989) 325-332. 4 Holtman, J.R., lmmunohistochemical localization of serotonin- and substance P-containing fibers around respiratory muscle motoneurons in the nucleus ambiguus of the cat, Neuroscience, 26 (1988) 169 -178. 5 Holtman, J.R., Norman, W.P., Skirboll, L., Dretchen, K.L., Cuello, C., Visser, T.J., H6kfelt, T. and Gillis, R.A., Evidence for 5-hydroxytryptamine, substance P and thyrotropin-releasing hormone in neurons innervating the phrenic motor nucleus, J. Neurosci., 4 (1984) 1064-1071. 6 Holtman, J.R., Anastasi, N.C., Norman, W.P. and Dretchen, K.L., Effect of electrical and chemical stimulation of the raphe obscurus on phrenic nerve activity in the cat, Brain Res., 362 (1986) 214--220. 7 lscoe, S.D., Central control of the upper airways. In O.P. Mathew and G. Sant'Ambogio (Eds.), Respiratory function of the upper airway. Dekker, New York, 1988, pp. 125 191. 8 Lalley, P.M., Responses of phrenic motoneurons of the cat to stimulation of medullary raphe nuclei, J. Physiol. (Lond.), 380 (1986) 349 371. 9 Lalley, P.M., Serotoninergic and non-serotoninergic responses of phrenic motoneurones to raphe stimulation in the cat, J. Physiol. (Lond.), 380 (1986) 373- 385. 10 Loizou, L.A., The postnatal ontogeny of monoamine-containing neurones in the central nervous system of the albino rat. Brain Res., 40 (1972) 395-418. 11 Millhorn, D.E., Eldridge, F.L., Waldrop, T.G. and Klinger, L.E., Centrally and peripherally administered 5-HTP have opposite effects on respiration, Brain Res., 264 (1983) 349 354, 12 Murakoshi, T., Suzue, T. and Tamai, S., A pharmacological study on respiratory rhythm in the isolated brainstem-spinal cord preparation of the newborn rat, Br. J. Pharmacol., 86 (1985) 95 104. 13 Olson, L. and Seiger, A., Early ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations~ Z. Anat. Entwickl. Gesch., 137 (1972) 301--316. t4 Palkovits, M., Brownstein, M. and Saavedra, J.M., Serotonin content of the brain stem nuclei in the rat, Brain Res., 80 (1974) 237 249. 15 Rea, M.A., Aprison, M.H. and Fetten, DL., Catecholamines and serotonin in the caudal medulla of the rat: combined neurochemical-histofluorescence study, Brain Res. Bull., 9 (1982) 227 236. 16 Remmers, J.E., deGroot, W.T., Sauerland, E.K. and Anch, A.M., Pathogenesis of upper airway occlusion during sleep, J. Appl. Physiol., 44 (1978) 931 938. 17 Saether, K., Hilaire, G. and Monteau, R., Dorsal and ventral respiratory groups of neurons in the medulla of the rat, Brain Res., 419 (1987) 87-96. 18 Smith, J.C. and Feldman, J.L., In vitro brainstem-spinal cord preparation for study of motor systems for mammalian respiration and locomotion, J. Neurosci. Methods, 21 (1987) 321 333. 19 Suzue, T., Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat, J. Physiol. (Lond.), 354 (1984) 173 183.
127
Elsevier Scientific Publishers Ireland Ltd. NSL 06749
Differential effects of serotonin on respiratory activity of hypoglossal and cervical motoneurons: an in vitro study on the newborn rat R. Monteau,
D . M o r i n , S. H e n n e q u i n a n d G . H i l a i r e
Dbparternent de Physiologie et Neurophysiologie, Equipe Biologie des Rythmes et du D~veloppement, Facultk des Sciences et Techniques St. Jkr6me, Marseilles (France)
(Received 31 May 1989; Revised version received 24 November 1989; Accepted 24 November 1989) Key words: Respiration; Serotonin; Hypoglossal nerve; Newborn; Rat; In vitro
Newborn rat respiratory activity was recorded on hypoglossal nerve and ventral cervical roots during in vitro experiments performed on superfused brainstem spinal cord preparations. The addition of serotonin (5-HT) to the bathing medium increased the respiratory frequency and selectively depressed the hypoglossal activity. Any decreases in the amplitude of cervical recordings were always restricted and reversible, whereas the hypoglossal activity was abolished. Furthermore, on cervical roots, 5-HT induced a tonic activity superimposed on the respiratory one, which was never observed with the hypoglossal nerve. When 5-HT was applied on isolated hemispinal cord, a tonic activity could still be elicited. These results indicate that serotonin (i) modulates the activity of neurons involved in the generation of respiratory rhythm, (ii) depresses the activity of hypoglossal motoneurons, and (iii) evokes tonic activity in cervical motoneurons, probably as the result of direct spinal effects.
A n a l y s i s o f r e s p i r a t o r y c o n t r o l systems can be facilitated b y the d e v e l o p m e n t o f in vitro p r e p a r a t i o n s o f isolated m a m m a l i a n brainstems. A viable in vitro n e w b o r n rat p r e p a r a t i o n was recently d e v e l o p e d [19]. As drugs can be a p p l i e d to the b r a i n s t e m by superfusion in c o n t r o l l e d c o n c e n t r a t i o n s , this p r e p a r a t i o n is very useful for investigating the a c t i o n o f p u t a t i v e n e u r o t r a n s m i t t e r s o n central r e s p i r a t o r y activity [3, 12, 18]. F u r t h e r m o r e , with this p r e p a r a t i o n it is possible to s i m u l t a n e o u s l y r e c o r d the r h y t h m i c r e s p i r a t o r y m o t o r o u t p u t f r o m cranial (hypoglossal) a n d cervical (C4) ventral roots. In the p a s t years, it has been d e m o n s t r a t e d t h a t m a n y agents cause differential changes in the activity o f the phrenic a n d h y p o g l o s s a l nerves. A s a b a l a n c e between the activities o f muscles o f the u p p e r a i r w a y s a n d the d i a p h r a g m is r e q u i r e d to a v o i d o b s t r u c t i o n o f the u p p e r a i r w a y s [16], a n e u r o t r a n s m i t t e r system Correspondence.. R. Monteau, D6partement de Physiologic et Neurophysiologie, Equipe 'Biologie des Rythmes et du D6veloppement', Facult6 des Sciences et Techniques St. J6r6me, Avenue Escadrille Normandie-Niemen, 13397 Marseille C6dex 13, France.
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
128
which differentially affects hypoglossal and phrenic activities can be suspected of being involved in airway obstruction such as apnea in prematurely born infants. Centrally applied serotonin (5-HT) is known to induce respiratory effects in both the adult [11] and the newborn rat [12]. The aim of the present study was to compare the effects of serotonin on hypoglossal and phrenic activities. The brainstem and cervical spinal cord of newborn rats (0-3 days old) were dissected, placed in a 2 ml chamber filled with artificial cerebrospinal solution and fixed with the ventral surface upwards. Nerve root electrical activities were recorded using suction electrodes. Complete experimental procedures have been described elsewhere [3]. Signals were filtered (5-3000 Hz), amplified (Neurolog system, Digitimer), and displayed on an oscilloscope and a paper recorder (Gould TA 2000). The signals were sampled by an A/D converter coupled with a microcomputer and integrated on line
An ,
C4
C41~
XII
C C4
t
......
m"
5-HT
,
D XII
C4
i;
b
Xll C4
XII i
Xll 1
see
~
II 5-HT
1mln
Fig. 1. Effects induced by 5-HT superfusion. A: schematic drawing of the ventral surface of the preparation and the recording electrodes on the hypoglossal nerve (XII) and a cervical ventral root (C4). B: simultaneous recordings of both motor outputs; superfusion with 5-HT begins at the arrow. C: from top to bottom, recordings of both discharges before, during and after superfusion with 5-HT. D: effects of 5-HT (superfusion at the horizontal bar) on the integrated discharges of both nerves. Note the initial increase in frequency, the tonic activity on the C4 recording, and the suppression of the hypoglossal discharge. B-D originate from different experiments.
129 or stored on files. Serotonin (Sigma) was dissolved in the bathing medium and applied by superfusion. All the results reported herein were obtained using serotonin at a concentration of 30/,M. As previously reported [3, 12, 18, 19], the resting respiratory frequency of the isolated preparation was slow (5.2 4- 0.6 m i n - l, mean _ S.E.M.). Changes in cranial and/ or hypoglossal activities were analyzed when the preparation was superfused with a medium containing 5-HT for 6 min in 23 experiments. Recordings were made from either a cervical root alone (11/23), a hypoglossal nerve alone (4/23) or both cervical and hypoglossal nerve simultaneously (8/23). Fig. 1 shows the typical effects of 5-HT whereas Fig. 2 illustrates averaged effects. Within a few minutes the respiratory frequency increased and reached a maximum of 8.3 4- 0.6 m i n - l (different from control value: P < 0.01). Then, the frequency slightly decreased although 5-HT was still present (Figs. I B, D and 2A) but remained above the control level. During the initial period of increased frequency, the amplitude of the respiratory motor output (estimated from the integrated discharge) was generally reduced, but this was more pronounced in the case of the hypoglossal (Figs. 1D and 2C, more than 50% of decrease after 2 rain) than in that of the cervical activity (less than 20%). The two patterns then clearly diverged. On the cervical recordings, a tonic discharge was superimposed on the respiratory activity (60% of experiments, mean latency 2 rain) as long as the preparation was superfused with 5-HT (Fig. 1B-D). On the hypoglossai recordings, no such tonic activity was ever observed and the amplitude of the respiratory activity continued to decrease (Figs. 1 and 2). In most experiments (10/12), the hypoglossal discharge was totally suppressed either during superfusion with 5-HT (i.e. within 6 min), or 1-2 min later. In two experiments, the hypoglossal activity was merely reduced by the first superfusion with 5-HT, but was completely abolished by a second superfusion performed 15 min later. When returned to normal bathing medium, the respiratory rhythm returned to control values. Although the tonic activity disappeared within a few minutes on the cervical roots (Fig. 1D), the hypoglossal activity A
B
Frequency variation (%)
C
A m p l i t u d e variation (%)
Amplitude variation (%)
14~ 120 100
2
4
6
8
[l~l~,='~ : NORMAL
10
12
14 ~
2 : 5-HT
4
6
8
10
12
14
2
4
6
8
I0
12
14
Time (min)
Fig. 2. Mean effectsof 5-HT on the frequencyand amplitudeof the respiratoryactivities.Averagedeffects on the respiratory frequency(A, n=23), the amplitude of the integrated cervical discharge (B, n= 19), and the amplitude of the integrated hypoglossaldischarge (C, n= 12) when changing normal bathing medium(hatched areas) by mediumcontaining5-HT (black areas) are expressedin %of control values.
130
was never o b s e r v e d to recover even 30 m i n u t e s after being r e t u r n e d to the n o r m a l medium. A d d i t i o n a l experiments were p e r f o r m e d to investigate w h e t h e r a n y spinal effects o f 5 - H T were involved in the genesis o f the tonic discharge. T y p i c a l results are shown in Fig. 3. S i m u l t a n e o u s b i l a t e r a l recordings o n cervical ventral r o o t s were p e r f o r m e d a n d the p r e p a r a t i o n was superfused with 5 - H T (for 2 min) to e v o k e tonic activity. A f t e r recovery u n d e r n o r m a l m e d i u m , a hemisection o f the spinal c o r d was p e r f o r m e d at Cl level to suppress the r e s p i r a t o r y drive f r o m the m e d u l l a r y centres to the m o t o neurons. S u p e r f u s i o n with 5 - H T still e v o k e d a tonic activity on b o t h sides. A f t e r being r e t u r n e d to n o r m a l m e d i u m , the spinal c o r d was split a l o n g the midline, in o r d e r to isolate the left side. Superfusion with 5 - H T e v o k e d a n o r m a l response on the intact side a n d a tonic activity on the ventral r o o t s o f the isolated h e m i s p i n a l cord. The b u l b o s p i n a l p a t h w a y s m e d i a t i n g the r e s p i r a t o r y drive were therefore n o t involved in
R1
R2 5-HT
R2 5-HT
RlW, iAmlmB R21
5-HT
Fig. 3. Origin of the tonic C 4 activity. On the left, schematic view of the experimental set-up; on the right, cervical C4 discharges recorded on both sides of the spinal cord. Horizontal bar: superfusion with 5-HT for 2 min. Top panel: in the intact preparation, 5-HT induced tonic activity on both sides. Mid panel: hemisection of the spinal cord performed at C~ level suppressed the spontaneous respiratory activity on the side of the lesion but 5-HT still induced a tonic activity. Bottom panel: after a sagittal section performed on the midline, 5-HT still induced tonic activity on the ventral root of the isolated left spinal segment.
131
the tonic discharge and the possibility that 5-HT had direct effects at the spinal level must be considered. The results reported here demonstrate that superfusion of the isolated brainstem of newborn rats with a medium containing 5-HT induced: (i) an initial increase in respiratory frequency, (ii) a tonic discharge from cervical ventral roots, (iii) the suppression of, or a drastic reduction, in the hypoglossal activity. The observed increase in respiratory frequency is in agreement with previous results [12], but the other effects have never been reported previously. The effects of 5-HT on respiratory activity in the adult have been well documented but are still controversial since both excitatory and inhibitory central and peripheral effects have been reported. At the central level, 5-HT is present in the cranial nuclei and specially in the hypogiossal nuclei [14, 15] as well as in the vicinity of the respiratory neurons [4, 15, 17]. Some neurons are excited and others are inhibited by 5-HT applied iontophoretically [1, 2]. Likewise, activation of the raphe nuclei induces excitatory [6, 8] as well as inhibitory [8] effects. 5-HT weakly affects the phrenic motoneurons [9] although 5-HT terminations are present in the nucleus [5]. Lastly, 5-HTP injected either intravenously or centrally inhibits or excites respiration respectively [ll]. 5-HT neurons develop in the rat brain after 8-9 days of gestation [13], and are sensitive at birth to agents affecting 5-HT synthesis, reuptake and degradation [10]. In isolated newborn rat preparation, the changes in frequency induced by 5-HT may be attributed to medullary structures whereas the changes in amplitude might be due to medullary and/or spinal effects. The results clearly indicated that the tonic activity induced by 5-HT was at least partly of spinal origin. In view of the weak effects of 5-HT on phrenic motoneurons [9] the tonic activity observed might result from activation of non-respiratory cervical motoneurons. As the tonic activity was sometimes followed by a slow rhythmic bursting pattern, motoneurons participating in the locomotor activity of the forelimb might be involved [18]. The increases in respiratory frequency reported above occurred after a short latency (less than 1 min), which suggests that superficial rather than deep structures were involved (because of the diffusion time). Hence, it is unlikely that 5-HT may act directly on deeply located respiratory neurons, even if they are sensitive to 5-HT [1, 2],and an indirect action through structures located near the surface is to be expected. Many agents have been reported to depress more hypoglossal than phrenic activity (see Iscoe [7] for review) but, as far as we know, the effects on hypoglossal motoneurons of directly applied 5-HT or stimulation of the raphe nuclei have never been looked for. Further studies will be necessary to analyse the differential action of 5-HT on the hypoglossal and phrenic motor outputs before we can conclude as to whether endogenous 5-HT is involved in the inhibition of hypoglossal activity which might be responsible for obstructive apnea. The authors gratefully acknowledge the excellent technical assistance of A.M. Lajard and M. Manneville. English revision by Dr. Jessica Blanc. This work was supported by the CNRS (URA 205) and the INSERM (Grant 886006).
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