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Effect of substance P on respiratory rhythm and pre-inspiratory neurons in the ventrolateral structure of rostral medulla oblongata: an in vitro study
272
l~ratn Rc.~ear~fi. ~,t)~Jit~92) 2?2 _'/4 :~: 1992 Elsevier Science Publishers B.V. All rights reserved 01itl,~,-~gt;~;.'~E'.SO~.it!
BRES 18380
Effect of substance P on respiratory rhythm and pre-inspiratory neurons in the ventrolateral structure of rostral medulla oblongata: an in vitro study Yuji Yamamoto
a, Hiroshi Onimaru b and Ikuo Homma b
The Nobel Institute for Neurophysiology, Karolinska lnstitutet and Department of Pediatrics, Karolinska Hospital, Stockholm (Sweden) and b Department of Physiology, Showa University School of Medicine, Tokyo (Japan)
(Accepted 28 July 1992)
Key words: In vitro; Substance P; Rat; Respiration; Rhythm generation
The pre-inspiratory (Pre-I) neurons which fire in the pre- and usually also during the post-inspiratory phase are located in the ventrolateral structures of the rostral medulla. They are suggested as primary rhythm generating neurons for respiration. These have been studied in isolated brainstem-spinal cord preparations from newborn 0-5-day-old rats. We have found that application of substance P (SP) enhanced the respiratory rhythm as measured by C4 ventral root and pre-I neuronal activities. Furthermore, the effect of SP was dependent on basal respiratory rate. An increase of the Pre-I and C 4 burst rate by SP was clearer when the basal respiratory rhythm was somewhat lower. Moreover. long lasting depression of respiratory rate after the application of the alpha 2-agonist clonidine was reversed by SP. On the other hand, an inhibitory effect appeared in preparations with a higher basal respiratory rate, while the Pre-I burst rate tended to increase during SP perfusion. During chemical synaptic transmission blockade by perfusion with low Ca z+, high Mg 2+ solution, a pre-I burst retained or completely blocked was found to be enhanced or reactivated by SP perfusion. The results suggest a direct postsynaptic action of SP, which could strongly stimulate burst generating properties of Pre-I neurons.
INTRODUCTION The basal mechanisms generating respiratory rhythm s e e m to b e l o c a t e d within t h e m e d u l l a o b l o n g a t a ~°'u, b u t t h e exact m e c h a n i s m for r h y t h m g e n e r a t i o n h a s not yet b e e n g e n e r a l l y a g r e e d u p o n . O u r p r e v i o u s study, however, has shown t h a t focal b l o c k o f t h e s t r u c t u r e closely a s s o c i a t e d with n. p a r a g i g a n t o c e l l u l a r i s lateralis, l o c a t e d directly b e l o w t h e v e n t r a l surface of t h e b r a i n s t e m c a u s e d a c o m p l e t e c e s s a t i o n o f r e s p i r a t i o n 5. F u r t h e r s t u d i e s in vivo have also shown t h a t this a r e a m i g h t have a r e s p i r a t o r y i n t e g r a t i v e f u n c t i o n f r o m p e r i p h e r a l a n d c e n t r a l c h e m o r e c e p t o r s , muscles, joints a n d skin 9'13'21. O n the o t h e r h a n d , a r e c e n t study f r o m an in vitro p r e p a r a t i o n o f t h e n e w b o r n rat, which was first d e s c r i b e d by Suzue 24, has d e m o n s t r a t e d t h e prese n c e o f d i f f e r e n t r e s p i r a t i o n - r e l a t e d n e u r o n s in this
a r e a 17-19'22. It has b e e n shown t h a t a g r o u p of n e u r o n s which fire at t h e p r e - i n s p i r a t o r y a n d p o s t - i n s p i r a t o r y p h a s e ( P r e - I n e u r o n ) r e t a i n t h e i r activities even after b l o c k a d e o f c h e m i c a l s y n a p t i c t r a n s m i s s i o n ~. This finding suggests t h a t t h e s e n e u r o n s could have intrinsic burst generating properties. Different neurotransmitt e r s / m o d u l a t o r s have b e e n a p p l i e d in such in vitro p r e p a r a t i o n s o f n e w b o r n rat. O n e of the n e u r o m o d u l a tors t h a t s t i m u l a t e r e s p i r a t o r y rate was f o u n d to be s u b s t a n c e P (SP) 15. T h e s t i m u l a t i n g effect o f SP on b r e a t h i n g has b e e n s t u d i e d in d i f f e r e n t l a b o r a t o ries 7'8"26. F u r t h e r m o r e , the action of SP was r e c e n t l y r e l a t e d to the c h e m o c e p t i v e p a t h w a y 23~27. R e c e n t studies using in vitro p r e p a r a t i o n s have d e m o n s t r a t e d t h e p o s s i b l e i n v o l v e m e n t o f a d r e n a l i n e in the r e s p i r a t o r y r h y t h m g e n e r a t i o n by t h e i r action on P r e - I n e u r o n s 2. H o w e v e r , the action o f SP on these n e u r o n s still re-
Correspondence: Y. Yamamoto, The Nobel Institute for Neurophysiology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden. Fax: (46) (8) 349544.
273 mains unclear. In the present study we have made an attempt to investigate the possible action of SP on Pre-I neurons.
;L..
MATERIALS AND METHODS B 50 nM Sp (6 min)
The methods used in the present study have been described previously in detail TM. The isolated brainstem and cervical spinal cord of newborn Wistar rats (0-5 days old, n = 25) were obtained under deep ether anesthesia. The brainstem was rostrally decerebrated between the sixth cranial nerve root and the lower border of the trapezoid body. The isolated preparation was perfused continuously in the chamber with the solution containing (mM): NaCI 124, KC1 5.0, KH2PO 4 1.2, CaCI 2 2.4, MgSO 4 1.3, NaHCO 3 26, glucose 30, equilibrated with 95% O 2 and 5% CO2; at 25-26°C, at pH 7.4. In order to block the synaptic transmission, the Mg 2+ concentration was increased to 5 mM and Ca 2+ concentration reduced to 0.2 mM 19. Clonidine, SP and spantide ([o-Arg~,D-Trp7'9,Leull]-SP)were obtained from Sigma (St Louis, MD, USA). Drugs were dissolved in the bathing medium and applied by superfusion for 6-10 min. Respiratory activity was monitored by recording the C 4 or C 5 ventral root. The unit activity was recorded extracellularly from the ventrolateral aspect of the rostral medulla by a glass electrode filled with 2% Pontamine sky blue in 0.5 M sodium-acetate (5-20 MI2). Respiration-related neuronal activity was recorded 50-300 p,m from the ventral surface 3. Values are mean +_standard deviation (S.D.). Statistical analysis was made by paired t-test.
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RESULTS As previously demonstrated by Onimaru and Homma 17 we have found the following types of neurons in the ventrolateral surface structure of the rostral medulla oblongata; (1) inspiratory neurons, (2) pre-inspiratory (Pre-I) neurons which fire in pre- and usually also during post-inspiratory phases, (3) tonically firing neurons inhibited during inspiration and (4) tonically firing neurons. Application of SP (10-100 nM) through the perfusate caused an increase of inspiratory burst rate in the C a (or C 5) root as well as a Pre-I neuronal burst rate in 12 of the 19 preparations (Fig. 1A,B). Fig. 2 shows the relationship between control C a (or C 5) burst rate and change of the burst rate induced by SP. In preparations with comparatively low burst rate, SP could also increase the burst rate up to a certain level. On the other hand, the C 4 (or C 5) burst rate in 6 preparations, in which the control burst rate was comparatively high, was decreased with application of SP (Fig. 2). In these cases, the Pre-I burst rate tended to be still increased. Effects of 10 nM SP on C a and Pre-I burst rate are shown in Table I. As shown in Fig. 1 and Table I, the burst rate of Pre-I neurons tended to be larger than those of C4 (or C5), because in some preparations every Pre-I burst was not followed by a C 4 burst. Application of an alpha-2 agonist clonidine ( 5 - 1 0 / z M ) caused a long lasting depression of C 4 root activity as well as Pre-I neuron burst rate as previously
Fig. 1. Effects of SP on Pre-I and C 5 inspiratory activity. A: Pre-I (upper trace) and C 5 (lower trace) activity in control bath solution. Average burst rate calculated from the number of bursts recorded for 3 min was 6.7 cycles/min for Pre-I and 4.0 cycles/min for C 5. B: activity 6 min after perfusion with 50 nM SP. Note an increase in Pre-I burst rate (7.3 cycles/min), and C 5 burst rate was 4.3 cycles/min. C: activity 22 min after washing out SP. The burst rate was 6.7 cycles/min for Pre-I and 4.0 cycles/min for C s. D: activity 5 min washing out after pretreatment (9 min) with 5 /~M clonidine. Note marked reduction in Pre-I (3.3 cycles/min) and C 5 (3.3 cycles/min) burst rate. E: activity 5 min after perfusion with 10 nM SP. Note recovery of Pre-I (5.5 cycles/min) and C s (5.5 cycles/min) activity from long lasting depression. Calibration 0.1 mV and 5 s.
demonstrated by Arata 1. Application of SP caused a recovery of the respiratory rate from the depression after clonidine treatment (Figs. 1D,E and 2). Consistent results were obtained in all 5 preparations examined. Apart from the respiratory effect of SP, we have confirmed that higher concentrations (0.1-1/~M) of SP cause an increased tonic motor activity at the cervical spinal level (Fig. 3) as described by Murakoshi 15. To investigate the direct effects of SP on Pre-I neurons, single Pre-I neuronal activity (n = 9) was isolated by blocking the chemical synaptic transmission by incubating the preparate with a low Ca 2+, high Mg 2+ solution (Low Ca2+). In a low Ca z+ solution, 3 of these Pre-I neurons retained the rhythmic burst, 3 neurons were silent and other 3 neurons fired tonically. Application of SP (10 nM) increased the burst rate (6.9 _+ 1.9 to 12.2 +_ 6.0 cycles/min, n = 3) of Pre-I neurons that
274 300 -
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Fig. 2. Relationship between control C 4 (or C s) burst rate (cycles/min) and percentage of the burst rate during SP against control (before SP). Open triangles, 100 n M SP; open squares, 50 nM; open circles, 10 nM; filled circles, 10 n M SP after clonidine (5 or 10 /zM). Note clearer exitatory effect of SP at lower control burst rate and inhibitory effect at higher burst rate.
retained their rhythmicity in low C a 2+ solution (Fig. 4). Two of the three Pre-I neurons that lost their activity in a low Ca 2+ solution could be reactivated by application of SP (Fig. 5). Addition of SP into the perfusion medium increased the firing rate in one and decreased the firing rate in two of the three Pre-I neurons that were firing tonically in low Ca: ÷ solution. To define the importance of endogenous SP on respiratory rhythm we have examined the effect of spantide, an SP antagonist. The SP antagonist (50 nM to 1 ~M) perfused in normal Ringer solution caused mainly a decreased C 4 burst rate (Table II).
01 mV
Fig. 3. Effects of high concentration of SP on Pre-I and C4 activity, A: Pre-I (upper trace) and C 4 (lower trace) activity in controlbath solution. B: activity 4 rain after perfusion with 100 nM SP. Note increases both in tonic motor activity of C 4 and in Pre-I burst rate. C: activity 10 rain after washing out SP. Calibration: 0.1 mV and 10 s.
the Pre-I and C 4 burst rate by SP was clearer when basal respiratory rhythm was somewhat lower, as suggested by Murakoshi ~5. Furthermore, long lasting deA
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DISCUSSION
The present study suggests that the effect of SP depends on basal respiratory rate. An enhancement of
C 10 nM SP (7 min)
TABLE I Effects of SP (10 nM) on respiratory rhythm (C 4 or C 5 burst rate and Pre-1 burst rate) Values are mean_+ S.D. before (control) and in the presence of SP. + = exhibiting an increase of burst rate; - = exhibiting a decrease of burst rate. Responses
+ Total
No.
7 3 10
C4, C 5 burst rate
Pre-I burst rate
(cycles/ min)
(cycles/ min)
Control
SP
Control
SP
5.2+1.0 7.4_+1.8 5.9-+1.6
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6.6_+1.1 7.4+1.4 6.9+1.2
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• P < 0.05, * * P < 0.01, * ** P < 0.001 by paired t-test.
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Fig. 4. Effects of SP on Pre-I activity in low Ca 2~, high Mg 2~ solution. A: Pre-I (upper trace) and C4 (lower trace) activity in control bath solution. B: activity 150 min after incubation i n low Ca 2÷, high Mg 2+ solution. Note rhythmic Pre-I activity retained. C: activity 10 min after washing out SP in low Ca2~i high Mg 2÷ solution. Calibration: 0.1 mV and 10 s.
275 A Control
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C4 Fig. 5. Effects of SP on Pre-I activity in low Ca 2+, high Mg 2+ solution. A: Pre-I and C4 activity in control bath solution. B: activity 34 min after incubation in low Ca 2+, high Mg 2+ solution. Note disappearance of Pre-I firing. C: activity 6 min after incubation with 10/xM SP. Note reactivation of Pre-I burst.
pression of respiratory rate after the application of the alpha-2 agonist clonidine was reversed by SP. On the other hand, a decrease of the burst rate was observed in preparations with a higher basal respiratory rate, while the Pre-I burst rate tended to increase during SP perfusion. This inhibitory effect might be explained by the failure of triggering inspiratory burst generation by Pre-I neurons at a higher Pre-I burst rate and it may be related to the threshold of the inspiratory burst generating network. In previous studies ~8-z°, Pre-I neurons that possess intrinsic burst generating properties were hypothesized as primary rhythm generating neurons in the rostral ventrolateral medulla oblongata. During perfusion of low Ca 2+, high Mg 2+ solution, a Pre-I burst activity was found to be enhanced or reactivated by SP perfusion. The results suggest a direct postsynaptic action of SP, which could strongly stimulate burst generating properties of Pre-I neurons. The present results may be comparable with those from
T A B L E II
Effects of SP antagonist (spantide, 50 n M - 1 ixM) on respiratory rhythm (C 4 or C5 burst rate) - = exhibiting a decrease of burst rate; + = exhibiting an increase of burst rate; - ---, + = exhibiting a transient decrease followed by an increase of burst rate; + ---, - = exhibiting a transient increase followed by a decrease of burst rate. Changes in burst rate were expressed in percentage of control.
Responses
No.
Control burst rate (cycles / min)
% Change
-
5
5.0 + 1.5
- 39%
- ~ + + + ~-
3 1 1
8.0+1.3 2.7 8.0
-22%--,35% +59% +25%~-55%
adult rat in vivo experiments by Chen et al. 7'8, They have shown that microinjections of SP into the ventrolateral medulla induce a stimulatory effect on ventilation and that iontophoretic application of SP to respiration-related neurons in this area cause mainly excitatory responses in these neurons. It has been demonstrated that adrenaline (or noradrenaline) could inhibit respiratory rhythm by acting on alpha-2 receptors 2J°. Arata et al. have further suggested that the second messenger system (cAMP) might mediate Pre-I neuronal rhythm regulation 4. It has also been suggested that clonidine may decrease the level of intracellular cyclic AMP via activation of alpha-2 receptors 16'25. Therefore, long lasting depression of the respiratory rate after clonidine treatment may be due to reduction of intracellular cyclic AMP level. Indeed, application of cyclic AMP elevating agents caused recovery of the respiratory rhythm from the depression 4. The effect of SP seen in the present study might therefore be due to a similar mechanism, via a second messenger system. A S P antagonist administered to inhibit endogenous SP activity has a varying response on C a activity. However, in some of the experiments, the SP antagonist markedly inhibited the respiratory rate. Local application of the SP antagonist in the ventrolateral medulla of the adult rats during in vivo experiments 6 has been shown to induce a marked ventilatory depression, suggesting a role of SP in the maintenance of normal respiratory activity. However, the effect of the SP antagonist on respiratory rhythm was not conclusive in the present study because of its agonistic properties, such as the transient enhancement of respiration followed by inhibition. Apart from the respiratory effect of SP, we have found that SP at a higher concentration (0.1-1 /zM) could stimulate cervical motoneuron activity, which would cause an increased tonic C 4 activity (see also ref. 15). Our previous study has demonstrated that a direct application of SP onto the ventral surface of medulla causes an enhanced locomotor activity in rabbit pups 26. In the present study we have obtained further suggestion that SP might stimulate locomotor activity at the cervical motoneurons as well. However, further studies will be needed to differentiate the motoneurons affected by SP. It has been demonstrated that 5-HT could also induce tonic activity at the spinal cord level, but this effect was not respiratory in nature 14. In summary, we have found that SP enhanced the respiratory rhythm measured by C a ventral root and Pre-I neuronal activity. Spontaneous or drug-induced depression of respiratory rate was strongly reversed by SP. Furthermore, Pre-I neuronal activity was enhanced
276 M., van Lunteren, [:. and yon Eulcr. ( . , l)~ sl;uc[ur~s in thc
or reactivated by SP during chemical synaptic transmission blockade. Acknowledgements. This investigation was supported in part by Research Grants from Swedish Medical Research Council 9074 and 5234. REFERENCES 1 Arata, A., Onimaru, H. and Homma, I., Distribution of PNMTimmunoreactive neurons and effects of adrenaline on respiratory rhythm in the medulla of newborn rat, Neurosci. Res. Suppl., 9 (1989) S-37. 2 Arata, A., Onimaru, H. and Homma, I., A possible role of adrenaline on respiratory rhythm generation in the medulla of newborn rat in vitro, Neurosci. Res. Suppl., 11 (1990)S-22. 3 Arata, A., Onimaru, H. and Homma, I., Respiration-related neurons in the ventral medulla of newborn rats in vitro, Brain Res. Bull., 24 (1990) 5999-6004. 4 Arata, A., Onimaru, H. and Homma, I., Involvement of cAMP in respiratory rhythm generation in the medulla of newborn rat in vitro, Neurosci. Res. Suppl., 14 (1991) S-24. 5 Budzinska, K., von Euler, C., Kao, F.F., Pantaleo, T. and Yamamoto, Y., Effects of graded focal cold block in rostral areas of the medulla, Acta Physiol. Scand., 124 (1985) 329-340. 6 Chen, Z., Hedner, J. and Hedner, T., Hypoventilation and apnoea induced by the substance P antagonist [D-Pro z, D-Trp7'9)-SP in the ventrolateral rat medulla, Acta Physiol. Scand., 134 (1990) 153-154. 7 Chen, Z., Hedner, J. and Hedner, T., Local effects of substance P on respiratory regulation in the medulla oblongata, Acta Physiol. Scand., 68 (1990) 693-699. 8 Chen, Z., Hedner, J. and Hedner, T., Antagonistic effects of somatostatin and substance P on respiratory regulation in the rat ventrolateral medulla, Brain Res., 556 (1991) 13-21. 9 Dillon, G.H., Welseh, D.E. and Waldrop, T.G., Modulation of respiratory reflexes by an excitatory amino acid mechanism in the ventrolateral medulla, Resp. Physiol., 85 (1991) 55-72. 10 Errchidi, S., Monteau, R. and Hilaire, G., Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: an in vitro study, J. Physiol., 443 (1991) 477-498. 11 von Euler, C., On the central pattern generator for the basic breathing rhythmicity, J. Appl. Physiol., 55 (1983) 1647-1659. 12 von Euler, C., Brain stem mechanisms for generation and control of the breathing pattern. In N.S. Cherniack and J.G. Widdicombe (Eds.), Handbook of Physiology, Section 2 (The Respiratory Systern), American Physiological Society, Bethesda, MD, 1986, pp. 1-67.
13 Mitra, J., Prabhakar, N.R., Pantaleo, T., Yamamoto, Y., Runold,
14
15
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18
19
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22
23
24
25
26
27
region of nucleus paragigantocellularis (nPG) integr~le and mediate ventilatory drive inputs?, Soc. Neurosci ~4h~'tr.. I v. parl I (t986) 304. Morin, D., Monteau, R. and Hilaire, G.. Serotonin and cervical respiratory motoneurons: intracellular study m the newborn ral brainstem-spinal cord preparation. F~p. Brain Re~.. g~ (19tH) 229-232. 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 .L Pharrnacol., ,q6 (1985) 95-104. Okada, M., Mine, K. and Fujiwara, M., Relationship ot calcium and adenylate cyclase messenger systems in rat brain synaptosomes, Brain Res., 501 (1989) 23-31. Onimaru, H. and Homma, l., Respiratory rhythm generator neurons in medulla of brainstem-spinal cord preparation from newborn rat, Brain Res., 403 (1987) 380-384. Onimaru, H., Arata, A. and Homma, !., Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat, Brain Res., 445 (1988) 314-324. Onimaru, H., Arata, A. and Homma, 1., Firing properties of respiratory rhythm generating neurons in the absence of synaptic transmission in rat medulla in vitro, Exp. Brain Re,s:, 76 (1980) 530-536. Onimaru, H., Arata, A. and Homma, 1., Inhibitory synaptic inputs to the respiratory rhythm generator in the medulla isolated from newborn rats, Pfliigers Arch, 417 (1990) 425-432. Seller, H., K6ning, S. and Czachurski, J.i Chemosensitivity of synapthoexcitatory neurons in the rostroventrolateral medUlla of the cat, Pfliigers Arch., 416 (1990) 735-741. Smith, J.C., Greer, J.J., Liu, G. and Feldman, J,L., Neural mechanisms generating respiratory pattern in mammalian brainstem-spinal cord in vitro. I. Spatiotemporal patterns of motor and medullary neuron activity, J. Neuropkysiol., 64 (1990) 1149-1169. Srinivasan, M., Goiny, M., Brodin, E., Pantaleo, T. and Yamamoto, Y., Enhanced in vivo release of substance P in the nucleus tractus solitarii during hypoxia in the rabbit: role of peripheral input, Brain Res., 546 (1991) 211-216. Suzue, T., Respiratory rhythm generation in the in vitro brainstem-spinal cord preparation of the neonatal rat, J. Physiol., 354 (1984) 173-183. Uhlen, S. and Wikberg, J.E.S., Inhibition of cyclic AMP production by a2-adrenoceptor stimulation in the guinea-pig spinal cord slices, Pharmaeol. Toxicol., 63 (1988) 178-182. Yamamoto, Y., Lagercrantz, H. and yon Euler, C, Effects of substance P and TRH on ventilation and pattern of breathing in newborn rabbits, Acta Physiol. Scand., t13 (1981) 541-543. Yamamoto, Y. and Lagererantz, H., Some effects of substance P on central respiratory control in rabbit pups, Acta Physiol. Scand.. 124 (1985) 449-455.
l~ratn Rc.~ear~fi. ~,t)~Jit~92) 2?2 _'/4 :~: 1992 Elsevier Science Publishers B.V. All rights reserved 01itl,~,-~gt;~;.'~E'.SO~.it!
BRES 18380
Effect of substance P on respiratory rhythm and pre-inspiratory neurons in the ventrolateral structure of rostral medulla oblongata: an in vitro study Yuji Yamamoto
a, Hiroshi Onimaru b and Ikuo Homma b
The Nobel Institute for Neurophysiology, Karolinska lnstitutet and Department of Pediatrics, Karolinska Hospital, Stockholm (Sweden) and b Department of Physiology, Showa University School of Medicine, Tokyo (Japan)
(Accepted 28 July 1992)
Key words: In vitro; Substance P; Rat; Respiration; Rhythm generation
The pre-inspiratory (Pre-I) neurons which fire in the pre- and usually also during the post-inspiratory phase are located in the ventrolateral structures of the rostral medulla. They are suggested as primary rhythm generating neurons for respiration. These have been studied in isolated brainstem-spinal cord preparations from newborn 0-5-day-old rats. We have found that application of substance P (SP) enhanced the respiratory rhythm as measured by C4 ventral root and pre-I neuronal activities. Furthermore, the effect of SP was dependent on basal respiratory rate. An increase of the Pre-I and C 4 burst rate by SP was clearer when the basal respiratory rhythm was somewhat lower. Moreover. long lasting depression of respiratory rate after the application of the alpha 2-agonist clonidine was reversed by SP. On the other hand, an inhibitory effect appeared in preparations with a higher basal respiratory rate, while the Pre-I burst rate tended to increase during SP perfusion. During chemical synaptic transmission blockade by perfusion with low Ca z+, high Mg 2+ solution, a pre-I burst retained or completely blocked was found to be enhanced or reactivated by SP perfusion. The results suggest a direct postsynaptic action of SP, which could strongly stimulate burst generating properties of Pre-I neurons.
INTRODUCTION The basal mechanisms generating respiratory rhythm s e e m to b e l o c a t e d within t h e m e d u l l a o b l o n g a t a ~°'u, b u t t h e exact m e c h a n i s m for r h y t h m g e n e r a t i o n h a s not yet b e e n g e n e r a l l y a g r e e d u p o n . O u r p r e v i o u s study, however, has shown t h a t focal b l o c k o f t h e s t r u c t u r e closely a s s o c i a t e d with n. p a r a g i g a n t o c e l l u l a r i s lateralis, l o c a t e d directly b e l o w t h e v e n t r a l surface of t h e b r a i n s t e m c a u s e d a c o m p l e t e c e s s a t i o n o f r e s p i r a t i o n 5. F u r t h e r s t u d i e s in vivo have also shown t h a t this a r e a m i g h t have a r e s p i r a t o r y i n t e g r a t i v e f u n c t i o n f r o m p e r i p h e r a l a n d c e n t r a l c h e m o r e c e p t o r s , muscles, joints a n d skin 9'13'21. O n the o t h e r h a n d , a r e c e n t study f r o m an in vitro p r e p a r a t i o n o f t h e n e w b o r n rat, which was first d e s c r i b e d by Suzue 24, has d e m o n s t r a t e d t h e prese n c e o f d i f f e r e n t r e s p i r a t i o n - r e l a t e d n e u r o n s in this
a r e a 17-19'22. It has b e e n shown t h a t a g r o u p of n e u r o n s which fire at t h e p r e - i n s p i r a t o r y a n d p o s t - i n s p i r a t o r y p h a s e ( P r e - I n e u r o n ) r e t a i n t h e i r activities even after b l o c k a d e o f c h e m i c a l s y n a p t i c t r a n s m i s s i o n ~. This finding suggests t h a t t h e s e n e u r o n s could have intrinsic burst generating properties. Different neurotransmitt e r s / m o d u l a t o r s have b e e n a p p l i e d in such in vitro p r e p a r a t i o n s o f n e w b o r n rat. O n e of the n e u r o m o d u l a tors t h a t s t i m u l a t e r e s p i r a t o r y rate was f o u n d to be s u b s t a n c e P (SP) 15. T h e s t i m u l a t i n g effect o f SP on b r e a t h i n g has b e e n s t u d i e d in d i f f e r e n t l a b o r a t o ries 7'8"26. F u r t h e r m o r e , the action of SP was r e c e n t l y r e l a t e d to the c h e m o c e p t i v e p a t h w a y 23~27. R e c e n t studies using in vitro p r e p a r a t i o n s have d e m o n s t r a t e d t h e p o s s i b l e i n v o l v e m e n t o f a d r e n a l i n e in the r e s p i r a t o r y r h y t h m g e n e r a t i o n by t h e i r action on P r e - I n e u r o n s 2. H o w e v e r , the action o f SP on these n e u r o n s still re-
Correspondence: Y. Yamamoto, The Nobel Institute for Neurophysiology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden. Fax: (46) (8) 349544.
273 mains unclear. In the present study we have made an attempt to investigate the possible action of SP on Pre-I neurons.
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MATERIALS AND METHODS B 50 nM Sp (6 min)
The methods used in the present study have been described previously in detail TM. The isolated brainstem and cervical spinal cord of newborn Wistar rats (0-5 days old, n = 25) were obtained under deep ether anesthesia. The brainstem was rostrally decerebrated between the sixth cranial nerve root and the lower border of the trapezoid body. The isolated preparation was perfused continuously in the chamber with the solution containing (mM): NaCI 124, KC1 5.0, KH2PO 4 1.2, CaCI 2 2.4, MgSO 4 1.3, NaHCO 3 26, glucose 30, equilibrated with 95% O 2 and 5% CO2; at 25-26°C, at pH 7.4. In order to block the synaptic transmission, the Mg 2+ concentration was increased to 5 mM and Ca 2+ concentration reduced to 0.2 mM 19. Clonidine, SP and spantide ([o-Arg~,D-Trp7'9,Leull]-SP)were obtained from Sigma (St Louis, MD, USA). Drugs were dissolved in the bathing medium and applied by superfusion for 6-10 min. Respiratory activity was monitored by recording the C 4 or C 5 ventral root. The unit activity was recorded extracellularly from the ventrolateral aspect of the rostral medulla by a glass electrode filled with 2% Pontamine sky blue in 0.5 M sodium-acetate (5-20 MI2). Respiration-related neuronal activity was recorded 50-300 p,m from the ventral surface 3. Values are mean +_standard deviation (S.D.). Statistical analysis was made by paired t-test.
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RESULTS As previously demonstrated by Onimaru and Homma 17 we have found the following types of neurons in the ventrolateral surface structure of the rostral medulla oblongata; (1) inspiratory neurons, (2) pre-inspiratory (Pre-I) neurons which fire in pre- and usually also during post-inspiratory phases, (3) tonically firing neurons inhibited during inspiration and (4) tonically firing neurons. Application of SP (10-100 nM) through the perfusate caused an increase of inspiratory burst rate in the C a (or C 5) root as well as a Pre-I neuronal burst rate in 12 of the 19 preparations (Fig. 1A,B). Fig. 2 shows the relationship between control C a (or C 5) burst rate and change of the burst rate induced by SP. In preparations with comparatively low burst rate, SP could also increase the burst rate up to a certain level. On the other hand, the C 4 (or C 5) burst rate in 6 preparations, in which the control burst rate was comparatively high, was decreased with application of SP (Fig. 2). In these cases, the Pre-I burst rate tended to be still increased. Effects of 10 nM SP on C a and Pre-I burst rate are shown in Table I. As shown in Fig. 1 and Table I, the burst rate of Pre-I neurons tended to be larger than those of C4 (or C5), because in some preparations every Pre-I burst was not followed by a C 4 burst. Application of an alpha-2 agonist clonidine ( 5 - 1 0 / z M ) caused a long lasting depression of C 4 root activity as well as Pre-I neuron burst rate as previously
Fig. 1. Effects of SP on Pre-I and C 5 inspiratory activity. A: Pre-I (upper trace) and C 5 (lower trace) activity in control bath solution. Average burst rate calculated from the number of bursts recorded for 3 min was 6.7 cycles/min for Pre-I and 4.0 cycles/min for C 5. B: activity 6 min after perfusion with 50 nM SP. Note an increase in Pre-I burst rate (7.3 cycles/min), and C 5 burst rate was 4.3 cycles/min. C: activity 22 min after washing out SP. The burst rate was 6.7 cycles/min for Pre-I and 4.0 cycles/min for C s. D: activity 5 min washing out after pretreatment (9 min) with 5 /~M clonidine. Note marked reduction in Pre-I (3.3 cycles/min) and C 5 (3.3 cycles/min) burst rate. E: activity 5 min after perfusion with 10 nM SP. Note recovery of Pre-I (5.5 cycles/min) and C s (5.5 cycles/min) activity from long lasting depression. Calibration 0.1 mV and 5 s.
demonstrated by Arata 1. Application of SP caused a recovery of the respiratory rate from the depression after clonidine treatment (Figs. 1D,E and 2). Consistent results were obtained in all 5 preparations examined. Apart from the respiratory effect of SP, we have confirmed that higher concentrations (0.1-1/~M) of SP cause an increased tonic motor activity at the cervical spinal level (Fig. 3) as described by Murakoshi 15. To investigate the direct effects of SP on Pre-I neurons, single Pre-I neuronal activity (n = 9) was isolated by blocking the chemical synaptic transmission by incubating the preparate with a low Ca 2+, high Mg 2+ solution (Low Ca2+). In a low Ca z+ solution, 3 of these Pre-I neurons retained the rhythmic burst, 3 neurons were silent and other 3 neurons fired tonically. Application of SP (10 nM) increased the burst rate (6.9 _+ 1.9 to 12.2 +_ 6.0 cycles/min, n = 3) of Pre-I neurons that
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retained their rhythmicity in low C a 2+ solution (Fig. 4). Two of the three Pre-I neurons that lost their activity in a low Ca 2+ solution could be reactivated by application of SP (Fig. 5). Addition of SP into the perfusion medium increased the firing rate in one and decreased the firing rate in two of the three Pre-I neurons that were firing tonically in low Ca: ÷ solution. To define the importance of endogenous SP on respiratory rhythm we have examined the effect of spantide, an SP antagonist. The SP antagonist (50 nM to 1 ~M) perfused in normal Ringer solution caused mainly a decreased C 4 burst rate (Table II).
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Fig. 3. Effects of high concentration of SP on Pre-I and C4 activity, A: Pre-I (upper trace) and C 4 (lower trace) activity in controlbath solution. B: activity 4 rain after perfusion with 100 nM SP. Note increases both in tonic motor activity of C 4 and in Pre-I burst rate. C: activity 10 rain after washing out SP. Calibration: 0.1 mV and 10 s.
the Pre-I and C 4 burst rate by SP was clearer when basal respiratory rhythm was somewhat lower, as suggested by Murakoshi ~5. Furthermore, long lasting deA
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DISCUSSION
The present study suggests that the effect of SP depends on basal respiratory rate. An enhancement of
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TABLE I Effects of SP (10 nM) on respiratory rhythm (C 4 or C 5 burst rate and Pre-1 burst rate) Values are mean_+ S.D. before (control) and in the presence of SP. + = exhibiting an increase of burst rate; - = exhibiting a decrease of burst rate. Responses
+ Total
No.
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C4, C 5 burst rate
Pre-I burst rate
(cycles/ min)
(cycles/ min)
Control
SP
Control
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pression of respiratory rate after the application of the alpha-2 agonist clonidine was reversed by SP. On the other hand, a decrease of the burst rate was observed in preparations with a higher basal respiratory rate, while the Pre-I burst rate tended to increase during SP perfusion. This inhibitory effect might be explained by the failure of triggering inspiratory burst generation by Pre-I neurons at a higher Pre-I burst rate and it may be related to the threshold of the inspiratory burst generating network. In previous studies ~8-z°, Pre-I neurons that possess intrinsic burst generating properties were hypothesized as primary rhythm generating neurons in the rostral ventrolateral medulla oblongata. During perfusion of low Ca 2+, high Mg 2+ solution, a Pre-I burst activity was found to be enhanced or reactivated by SP perfusion. The results suggest a direct postsynaptic action of SP, which could strongly stimulate burst generating properties of Pre-I neurons. The present results may be comparable with those from
T A B L E II
Effects of SP antagonist (spantide, 50 n M - 1 ixM) on respiratory rhythm (C 4 or C5 burst rate) - = exhibiting a decrease of burst rate; + = exhibiting an increase of burst rate; - ---, + = exhibiting a transient decrease followed by an increase of burst rate; + ---, - = exhibiting a transient increase followed by a decrease of burst rate. Changes in burst rate were expressed in percentage of control.
Responses
No.
Control burst rate (cycles / min)
% Change
-
5
5.0 + 1.5
- 39%
- ~ + + + ~-
3 1 1
8.0+1.3 2.7 8.0
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adult rat in vivo experiments by Chen et al. 7'8, They have shown that microinjections of SP into the ventrolateral medulla induce a stimulatory effect on ventilation and that iontophoretic application of SP to respiration-related neurons in this area cause mainly excitatory responses in these neurons. It has been demonstrated that adrenaline (or noradrenaline) could inhibit respiratory rhythm by acting on alpha-2 receptors 2J°. Arata et al. have further suggested that the second messenger system (cAMP) might mediate Pre-I neuronal rhythm regulation 4. It has also been suggested that clonidine may decrease the level of intracellular cyclic AMP via activation of alpha-2 receptors 16'25. Therefore, long lasting depression of the respiratory rate after clonidine treatment may be due to reduction of intracellular cyclic AMP level. Indeed, application of cyclic AMP elevating agents caused recovery of the respiratory rhythm from the depression 4. The effect of SP seen in the present study might therefore be due to a similar mechanism, via a second messenger system. A S P antagonist administered to inhibit endogenous SP activity has a varying response on C a activity. However, in some of the experiments, the SP antagonist markedly inhibited the respiratory rate. Local application of the SP antagonist in the ventrolateral medulla of the adult rats during in vivo experiments 6 has been shown to induce a marked ventilatory depression, suggesting a role of SP in the maintenance of normal respiratory activity. However, the effect of the SP antagonist on respiratory rhythm was not conclusive in the present study because of its agonistic properties, such as the transient enhancement of respiration followed by inhibition. Apart from the respiratory effect of SP, we have found that SP at a higher concentration (0.1-1 /zM) could stimulate cervical motoneuron activity, which would cause an increased tonic C 4 activity (see also ref. 15). Our previous study has demonstrated that a direct application of SP onto the ventral surface of medulla causes an enhanced locomotor activity in rabbit pups 26. In the present study we have obtained further suggestion that SP might stimulate locomotor activity at the cervical motoneurons as well. However, further studies will be needed to differentiate the motoneurons affected by SP. It has been demonstrated that 5-HT could also induce tonic activity at the spinal cord level, but this effect was not respiratory in nature 14. In summary, we have found that SP enhanced the respiratory rhythm measured by C a ventral root and Pre-I neuronal activity. Spontaneous or drug-induced depression of respiratory rate was strongly reversed by SP. Furthermore, Pre-I neuronal activity was enhanced
276 M., van Lunteren, [:. and yon Eulcr. ( . , l)~ sl;uc[ur~s in thc
or reactivated by SP during chemical synaptic transmission blockade. Acknowledgements. This investigation was supported in part by Research Grants from Swedish Medical Research Council 9074 and 5234. REFERENCES 1 Arata, A., Onimaru, H. and Homma, I., Distribution of PNMTimmunoreactive neurons and effects of adrenaline on respiratory rhythm in the medulla of newborn rat, Neurosci. Res. Suppl., 9 (1989) S-37. 2 Arata, A., Onimaru, H. and Homma, I., A possible role of adrenaline on respiratory rhythm generation in the medulla of newborn rat in vitro, Neurosci. Res. Suppl., 11 (1990)S-22. 3 Arata, A., Onimaru, H. and Homma, I., Respiration-related neurons in the ventral medulla of newborn rats in vitro, Brain Res. Bull., 24 (1990) 5999-6004. 4 Arata, A., Onimaru, H. and Homma, I., Involvement of cAMP in respiratory rhythm generation in the medulla of newborn rat in vitro, Neurosci. Res. Suppl., 14 (1991) S-24. 5 Budzinska, K., von Euler, C., Kao, F.F., Pantaleo, T. and Yamamoto, Y., Effects of graded focal cold block in rostral areas of the medulla, Acta Physiol. Scand., 124 (1985) 329-340. 6 Chen, Z., Hedner, J. and Hedner, T., Hypoventilation and apnoea induced by the substance P antagonist [D-Pro z, D-Trp7'9)-SP in the ventrolateral rat medulla, Acta Physiol. Scand., 134 (1990) 153-154. 7 Chen, Z., Hedner, J. and Hedner, T., Local effects of substance P on respiratory regulation in the medulla oblongata, Acta Physiol. Scand., 68 (1990) 693-699. 8 Chen, Z., Hedner, J. and Hedner, T., Antagonistic effects of somatostatin and substance P on respiratory regulation in the rat ventrolateral medulla, Brain Res., 556 (1991) 13-21. 9 Dillon, G.H., Welseh, D.E. and Waldrop, T.G., Modulation of respiratory reflexes by an excitatory amino acid mechanism in the ventrolateral medulla, Resp. Physiol., 85 (1991) 55-72. 10 Errchidi, S., Monteau, R. and Hilaire, G., Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: an in vitro study, J. Physiol., 443 (1991) 477-498. 11 von Euler, C., On the central pattern generator for the basic breathing rhythmicity, J. Appl. Physiol., 55 (1983) 1647-1659. 12 von Euler, C., Brain stem mechanisms for generation and control of the breathing pattern. In N.S. Cherniack and J.G. Widdicombe (Eds.), Handbook of Physiology, Section 2 (The Respiratory Systern), American Physiological Society, Bethesda, MD, 1986, pp. 1-67.
13 Mitra, J., Prabhakar, N.R., Pantaleo, T., Yamamoto, Y., Runold,
14
15
16
17
18
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23
24
25
26
27
region of nucleus paragigantocellularis (nPG) integr~le and mediate ventilatory drive inputs?, Soc. Neurosci ~4h~'tr.. I v. parl I (t986) 304. Morin, D., Monteau, R. and Hilaire, G.. Serotonin and cervical respiratory motoneurons: intracellular study m the newborn ral brainstem-spinal cord preparation. F~p. Brain Re~.. g~ (19tH) 229-232. 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 .L Pharrnacol., ,q6 (1985) 95-104. Okada, M., Mine, K. and Fujiwara, M., Relationship ot calcium and adenylate cyclase messenger systems in rat brain synaptosomes, Brain Res., 501 (1989) 23-31. Onimaru, H. and Homma, l., Respiratory rhythm generator neurons in medulla of brainstem-spinal cord preparation from newborn rat, Brain Res., 403 (1987) 380-384. Onimaru, H., Arata, A. and Homma, !., Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat, Brain Res., 445 (1988) 314-324. Onimaru, H., Arata, A. and Homma, 1., Firing properties of respiratory rhythm generating neurons in the absence of synaptic transmission in rat medulla in vitro, Exp. Brain Re,s:, 76 (1980) 530-536. Onimaru, H., Arata, A. and Homma, 1., Inhibitory synaptic inputs to the respiratory rhythm generator in the medulla isolated from newborn rats, Pfliigers Arch, 417 (1990) 425-432. Seller, H., K6ning, S. and Czachurski, J.i Chemosensitivity of synapthoexcitatory neurons in the rostroventrolateral medUlla of the cat, Pfliigers Arch., 416 (1990) 735-741. Smith, J.C., Greer, J.J., Liu, G. and Feldman, J,L., Neural mechanisms generating respiratory pattern in mammalian brainstem-spinal cord in vitro. I. Spatiotemporal patterns of motor and medullary neuron activity, J. Neuropkysiol., 64 (1990) 1149-1169. Srinivasan, M., Goiny, M., Brodin, E., Pantaleo, T. and Yamamoto, Y., Enhanced in vivo release of substance P in the nucleus tractus solitarii during hypoxia in the rabbit: role of peripheral input, Brain Res., 546 (1991) 211-216. Suzue, T., Respiratory rhythm generation in the in vitro brainstem-spinal cord preparation of the neonatal rat, J. Physiol., 354 (1984) 173-183. Uhlen, S. and Wikberg, J.E.S., Inhibition of cyclic AMP production by a2-adrenoceptor stimulation in the guinea-pig spinal cord slices, Pharmaeol. Toxicol., 63 (1988) 178-182. Yamamoto, Y., Lagercrantz, H. and yon Euler, C, Effects of substance P and TRH on ventilation and pattern of breathing in newborn rabbits, Acta Physiol. Scand., t13 (1981) 541-543. Yamamoto, Y. and Lagererantz, H., Some effects of substance P on central respiratory control in rabbit pups, Acta Physiol. Scand.. 124 (1985) 449-455.