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

ELSEVIER

Neuroscience Letters 172 (1994) 59-62

NEOROSCI[NC[ LETTERS

Serotonin 1A-receptor activation suppresses respiratory apneusis in the cat Peter M. Lalleyb'*, Anna-Maria Bischof?'**, Diethelm W. Richter" ~'lI. Institute of Physiology, Universi O, of Goettingen, Humboldtallee 23, 37073 G6ttingen, FRG bDepartment of Physiology, University of Wisconsin Madison, 480 Service Memorial Institutes, 1300 Universi O, Ave., Madison, WI 53706, USA Received 23 December 1993; Revised version received 10 March 1994; Accepted I I March 1994

Abstraet Malfunction of inhibitory synaptic processes in the brainstem result in abnormal prolonged inspiration (apneusis). Since we previously found that the serotonin (5-hydroxytryptamine; 5-HT) 5-HT1A receptor agonist 8-hydroxy-dipropylaminotetralin(8-OHDPAT) shortens inspiratory discharges, we tested its ability to suppress apneusis. We recorded phrenic nerve activity and the membrane potential of medullary expiratory (E-2) and postinspiratory (PI) neurons in 14 anaesthetized, paralyzed, artifically ventilated cats. Systemic hypoxia or i.v. injection of pentobarbital sodium or the N-methyl-D-aspartate (NMDA) receptor blocker ketamine induced apneustic phrenic nerve discharges, delayed depolarization to threshold of E-2 neurons and prolonged hyperpolarization in PI neurons. 8-OH-DPAT (10~,0/2g/kg i.v.) produced partial to complete restoration of normal phrenic nerve discharges and membrane potential. Key words. Apneusis; Serotonin 5-HTIA receptor; NMDA receptor; Barbiturate; Hypoxia

When the off-switch mechanisms terminating inspiration are compromised, abnormally long periods of inspiration (apneusis) occur. Such apneustic patterns have been observed during and after hypoxia [19], cerebrovascular accidents which damage the pons [17], lesioning the pontine complex [20], and administration of drugs such as pentobarbital [22] or N-methyl-D-aspartate (NMDA) receptor antagonists [6,7,16]. The serotonin (5-hydroxytryptamine; 5-HT) 5-HTIA receptor agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT, 10-50 Bg/ kg i.v.) has the opposite effect of producing shorter and more frequent inspiratory discharges in the adult cat [10]. We wished to determine, therefore, if apneusis induced by drugs or hypoxia can be reversed by 8-OH-DPAT. Our results demonstrate that apneusis is suppressed by i.v. injection of 8-OH-DPAT. Experiments were performed on 14 cats of either sex (2.5 5.0 kg) anaesthetized with pentobarbital sodium (40 mg/kg i.p. followed by 4-8 mg/h) to produce and main-

* Corresponding author. Fax: (1) (608) 262-2327. **Acknowledged for assistance. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00228-3

tain adequate levels of anaesthesia. Animals were ventilated with oxygen-enriched air. Normocapnic hypoxaemia was induced by ventilating with 10% 02 in N. which induced apneusis. Ventilatory rate and tidal volume were adjusted to maintain end-tidal CO,_ levels at 4-4.5 vol%. A pneumothorax was established bilaterally. Gallamine triethiodide (4-8 mg/kg i.v. initially, followed by 4-8 mg/kg/h) was administered for muscular paralysis. Body temperature was maintained at 37 38°C. The 5-HTIA receptor agonist 8-OH-DPAT was given intravenously in doses of 10~,0/2g/kg [10]. These doses of 8-OHDPAT produced arterial hypotension so it was sometimes necessary to maintain blood pressure with continuous (0.2-0.4 ml/min) i.v. infusion of noradrenaline (40/.tg/ml) in glucose/Ringer solution. Surgical and recording methods were described in earlier reports [19]. Intracellular recordings were obtained from expiratory (E-2; n = 8) and postinspiratory (PI; n = 3) neurons with glass micropipettes filled with 3 M KC1 (40-70 M£2 resistance). Neurons were accepted for analysis if they exhibited maximal membrane potentials of at least -50 mV. Apneusis was produced by hypoxia (n = 6), intravenous administration of the N M D A receptor antagonist

60

t! M. L a l h ' r el Jl / N c u r t ) s c i c n ~ c

k e t a m i n e (n = 5 [7]), or p e n t o b a r b i t a l sodium (n = 3). All f o r m s o f apneustic disturbances of the respiratory r h y t h m were suppressed by 8-OH-DPAT. Pentobarbital can produce apneusis by weakening the inspiratory off-switch [22]. In Fig. 1, such apneustic discharges of phrenic nerve activity were p r o d u c e d by cumulative doses (55-58 mg/kg) of p e n t o b a r b i t a l sodium. The apneustic discharges lasted over 4-34 s and occurred at rates of 2 to 9 per minute (Fig. 1A). After injection o f 8 - O H - D P A T (Fig. 1B), inspiratory bursts became augmenting in their pattern, shorter in their duration (0.81.3 sec) and occurred m o r e frequently (15 24/min). The effect o f 8 - O H - D P A T was evident over observation periods o f 30-50 minutes, when inspiratory discharges were once again significantly longer than control. This is consistent with the effect and d u r a t i o n o f 8 - O H - D P A T (1050 /tg/kg i.v.) in shortening non-apneustic inspiratory discharges [10]. In order to obtain m o r e precise i n f o r m a t i o n a b o u t the synaptic m e c h a n i s m s underlying these changes o f respiratory activity, we recorded f r o m 8 late-expiratory (E-2) neurons caudal to the obex and 3 postinspiratory (PI) neurons, 1-3 m m rostral to the obex in the ventral respiratory group. Five o f the E-2 neurons were antidromically activated by stimulation o f the cervical (C3) reticulospinal tracts, whereas the PI neurons were not responsive to vagal or reticulospinal tract stimulation. U n d e r n o r m a l conditions, E-2 neurons are hyperpolarized at the beginning o f inspiration and then slowly depolarize during the remainder of the inspiratory phase (Fig. 2B,). This pattern o f h y p e r p o l a r i z a t i o n is attributed to inhibition by early inspiratory (e-I) neurons which discharge at high frequency at the beginning o f inspiration and then decline to low discharge rates [18]. D u r i n g apneustic A

P e m o b a r b Apneusi$

B

8-OH DPAT, 10 ~Jg / kg i v

PN A

PN

0.~ rnVI I

I 8s

Fig. 1. Reversal of pentobarbital-induced apneusis by 8-hydroxy-dipro-

pylaminotetralin (8-OH-DPAT). A: apneustic discharges of phrenic nerve activity (PN; averaged form, PNA) produced by pentobarbital, cumulative dose, 55 mg/kg (40 mg/kg i.v., initial dose followed by 15 mg/kg in 3 doses given over 40 minutes). B: reversal of apneusis by 8-OH-DPAT, 10/tg/kg, injected 30 s before records in B were taken.

/,cll<,r,~ 172 ( 1094 : .5(: 62

ill]J J" PN o 2 v l l ~ l ~ " 4 i I - d i l ~

~

IIII PN o~mv

°2 I _ _ 1 •

!

B3LIJ Fig. 2. Reversal of apneustic discharge patterns of late-discharging expiratory (E-2) neurons and phrenic nerve activity by 8-OH-DPAT. A]; apneustic activity after exposure to hypoxia (10 vol% O2) is characterized by a prolonged discharge of phrenic nerve and prolonged mem-

brane potential hyperpolarization of the E-2 neuron. A2: records taken 3 min after i.v. injection of 8-OH-DPAT, 15/,tg/kg. The prolonged discharges of phrenic nerve and membrane potential hyperpolarization of the E-2 neuron are blocked. The E-2 neuron sometimes fails to reach threshold for discharge of action potentials (upper reference line): BL: control records of phrenic nerve activity and membrane potential of another E-2 neuron before ketamine. B2: apneustic patterns recorded 2 rain after injection of ketamine, 0.5 mg/kg. C: records showing marked reduction of apneusis after injecting 8-OH-DPAT, 10/tg/kg. MP, membrane potential; PN, phrenic nerve activity. A simplified model to explain apneusis and its suppression by activation of 5-HTIa receptors is shown on the lower left side. Phrenic motonem:ons (PN) receive excitatory synaptic input from bulbospinal inspiratory (1) neurons which exhibit an augmenting discharge pattern. The discharge of 1 neurons is suppressed reversibly by late-inspiratory (l-I) and subsequently irreversibly blocked by post-inspiratory (PI) neurons, t-1 and PI neurons are inhibited by e-I neurons. See [6] for a more detailed model description of respiratory rhythm generation. Apneusis might result from disfacilitation of I-I neurons or by blockade of their synaptic transmission to inspiratory neurons (see flash arrows). The prevailing effect, however, seems to be disfacilitation of M neurons resulting from a prolonged discharge of e-I neurons (horizontal arrow). This leads to a delay in the onset of firing of 1-I and PI neurons and, therefore, prolonged inspiratory phrenic nerve activity (horizontal arrow). The present data indicate that activation of 5-HT~A receptors reduces the discharge duration of e-I neurons to allow earlier discharges of M, PI and E2 neurons resulting in a normalization of the respiratory rhythm. breathing, however, discharge o f e-I neurons is prolonged resulting in persistence o f early inspiratory inhibition of E-2 neurons (Fig. 2A1, 2B2 [6,16]). At the end o f inspiration, depolarization o f E-2 neurons is slowed during the after-discharge o f phrenic nerve activity. This pattern o f depolarization is shaped by a second period of declining synaptic inhibition during postinspiration which is likely due to the activation o f postinspiratory neurons. After this phase, the threshold for excitation is reached leading to action potential discharge during the later period of the expiratory interval (Fig. 2B, [4]). H y p o x i a is often followed by depression o f synaptic potentials [t 9] and the a p p e a r a n c e o f apneustic breathing

P M. Lalley et al. / Neuroscience Letters 172 (1994) 59 ~2

patterns with prolonged phases of inspiration (Fig. 2A~). An initial augmenting phase of phrenic nerve activity is followed by prolonged plateau activity at lower amplitude which is characteristic of apneusis. At the onset of phrenic nerve activity, synaptic membrane hyperpolarization of E-2 neurons is normal, but then evolves into a secondary phase of reduced hyperpolarization during the plateau phase of phrenic nerve activity. This is followed by gradual depolarization during postinspiration and discharge of action potentials during the E-2 phase. Systemic administration of 8-OH-DPAT ( 1 5 4 0 #g/kg) produces significant anti-apneustic effects, an example of which is shown in Fig. 2A 2. The apneustic plateau-discharge of phrenic nerve activity and the concommitant wave of membrane hyperpolarization of E-2 neurons disappears so that the respiratory rhythm as well as the membrane potential trajectories of E-2 neurons become normal. These effects may be explained by the 8-OHDPAT-evoked activation of 5-HT~A receptors of e-I neurons, shortening their discharge duration and inhibition of E-2 neurons (see the schema in Fig. 2). Apneusis produced by NMDA receptor antagonists such as ketamine and MK-801 also disturbs inhibitory synaptic interactions between medullary e-I neurons, 1-I neurons and PI neurons [6,16], resulting in impairment of both the reversible inspiratory oR-switch during lateinspiration and the irreversible inspiratory off-switch during postinspiration. Injection of ketamine (0.5 1.O mg/kg) converted the control patterns of phrenic nerve discharge and membrane potential trajectories of E-2 neurons (Fig. 2B 0 to apneustic patterns (Fig. 2B2). Injection of 8-OH-DPAT (10-20 #g/kg i.v.) restored the control patterns of respiratory activity and membrane potential trajectories of E-2 neurons (Fig. 2B3). These antiapneustic effects were accompanied by slight membrane hyperpolarizations and a reduction of the action potential frequency of E-2 neurons. Apneusis produced by NMDA receptor blockade is accompanied by disturbance of the rhythm and discharge pattern of postinspiratory (PI) neurons [6,16] which are important in producing irreversible off-switching of inspiration [18]. Under control conditions (Fig. 3A), PI neurons exhibit waves of inspiratory membrane hyperpolarization which are supposed to be transmitted from early-inspiratory neurons [18]. After injection of ketamine (0.25 mg/kg i.v.), inspiratory hyperpolarization was considerably prolonged and somewhat reduced in amplitude (Fig. 3B). Such changes are consistent with the protracted, lower frequency discharge of eI neurons induced by NMDA receptor blockade [16]. After injection of 8-OH-DPAT (10 20 #g/kg) reversal of apneusis was significant although incomplete, resulting in a nearly normal respiratory pattern. This was evident from shorter waves of membrane hyperpolarization, increased postinspiratory discharges and the reduced durations of inspiratory burst activity in phrenic nerves (Fig. 3C).

61

I,]!

|~11111111111111[ ii ;

,

I

ilij

illliLii'~ ImmnuR~l [] i#', ,' Lr -50 [

li

MP~mV -60

i

'

.

/,'

2S

Fig. 3. Apneusis produced in a postinspiratory (PI) neuron by ketamine and reversal by 8-OH-DPAT. A: control membrane potential and phrenic nerve activity. MP, membrane potential; PN, phrenic nerve activity; PNA, moving average of phrenic nerve action potential frequency. B: after injection of ketamine, 0.25 mg/kg i.v., inspiratory MP is less hyperpolarized and considerably prolonged. C: after injection of 8-OH-DPAT. 10 #g/kg, inspiratory MP hyperpolarization and discharges of PN are much shorter.

Larger doses of 8-OH-DPAT produced greater hypotension and loss of cell recording. 5-HT is recognized as an important neuromodulator in the respiratory network of mammals [3,5,8,911,14,15]. Many types of respiratory neurons in the medulla receive serotoninergic input [21] and respiratory network activities are altered dramatically when concentrations of 5-HT are altered [2,12,13]. The specific effects of 5-HT depend on its binding to different subtypes of 5-HT receptors [1]. The excitability of bulbar respiratory neurons is increased when 5-HT2c receptors are activated, whereas activation of 5-HTIA receptors leads to depression of their excitability [5,8,11,14,15]. The 5-HTiA receptor-induced depression of neuronal discharges results from membrane hyperpolarization, as seen during ionophoresis of 8-OH-DPAT on single expiratory neurons. 8-OH-DPAT given systemically has similar effects on individual respiratory neurons, but in addition, influences the activity within the whole respiratory network. These effects are antagonized by i.v. injection of NAN-190, a 5-HTIA receptor blocker [10]. The present study shows that 5-HTmA receptor activation modulates respiratory rhythmogenesis by influencing the reversible and irreversible inspiratory off-switch

62

P M. Lalle)' et aL / Neuroscience Letters 172 (1994) 5q 02

mechanisms. Apneusis results from direct disturbances of the synaptic inhibition (reversible off-switch) of inspiratory neurons by l-I neurons and/or from disfacilitation of l-I neurons through the extended firing of e-I neurons (as indicated by the horizontal arrow in the scheme of Fig. 2 [6,16]). Prolonged discharges of e-! neurons is suggested by the prolonged respiratory inhibition of P! neurons (Fig. 3 [18]) and by the delayed depolarization to threshold of E-2 neurons (Fig. 2A,B [6]). The extended periods of neuronal hyperpolarization were shortened by 8-OH-DPAT because, according to our model, activation of 5-HTIA receptors shortens the discharges of e-I neurons. This may occur directly by activation of 5-HT1A receptors to increase membrane conductances of e-I neurons to potassium [1]. The shortening of the discharge duration of e-I neurons leads to disinhibition of 1-I and PI neurons (see the scheme in Fig. 2), thus allowing reversible (late-inspiratory) inhibition of inspiration and irreversible (postinspiratory) termination of inspiration [6,16]. This study shows for the first time that activation of 5-HTIA receptors on neurons of the respiratory network compensates for disturbances of inhibitory synaptic mechanisms and is capable of converting respiratory patterns from apneustic to normal in anesthetized cats. This work was supported by the Deutsche Forschungsgemeinschaft and the National Institute of Health. [1] Anwyl, R., Neurophysiological actions of 5-hydroxytryptamine in the vertebrate nervous system, Prog. Neurobiol., 35 (1990) 451 468. [2] Arita, H. and Ochiishi, M., Opposing effects of 5-hydroxytryptamine on two types of medullary inspiratory neurons with distinct firing patterns, Neurophysiology, 66 (1991) 285-292. [3] Armijo, J.A., Mediavilla, A.M. and Florez, J., Inhibition of the activity of the respiratory and vasomotor centers by centrally administered 5-hydroxy-tryptamine in cats, Rev. Esp. Fiziol., 35 (1979) 219-228. [4] Ballantyne, D. and Richter, D.W., The non-uniform character of expiratory synaptic activity in expiratory bulbospinal neurones of the cat, J. Physiol., 370 (1986) 433~,56. [5] Di Pasquate, E., Morin, D., Monteau, R, and Hilaire, G., Serotonergic modulation of the respiratory rhythm generator at birth: An in vitro study in the rat, Neurosci. Lett., 143 (1992) 91-95. [6] Feldman, J.L., Windhorst, U., Anders, K. and Richter, D.W.,

Synaptic interaction between medullary respiratory neurones during apneusis induced by NMDA-receptor blockade in cat, J. Physiol., 450 (1991) 303-323. [7] Foutz, A.S., Champagnat, J. and Denavit-Saubie, M., N-MethylD-aspartate receptors control respiratory off-switch in cat, Neurosci. Lett., 87 (1988) 221 226. [8] Kubin, L., Tojima, H., Davies, R.O. and Pack, A.I., Serotonergic excitatory drive to hypoglossal motoneurons in the decerebrate cat, Neurosci. Lett., 139 (1992) 243 248. [9] Lalley, P.M., Serotoninergic and non-serotoninergic responses of phrenic motoneurones to stimulation of raphe nuclei in the cat, J. Physiol., 380 (1986) 373-385. [10] Lalley, P.M. and Richter, D.W., 5HT-1A receptor-mediated modulation of medullary expiratory neurones in the cat, J. Physiol., in press. [1 l] 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] Lundberg, D., Mueller, R.A. and Breese, G.R., An evaluation of the mechanism by which serotoninergic activation depresses respiration, J. Pharmacol. Exp. Ther., 212 (1980) 397-404. [13] McCrimmon, D.R. and Lalley, P.M., Inhibition of respiratory neural discharges by clonidine and 5-hydroxytryptophan, J. Pharmacol. Exp. Ther., 222 (1982) 771--777. [14] Monteau, R., Morin, D., Hennequin, S. and Hilaire, G., Differential effects of serotonin on respiratory activity of hypoglossal and cervical motoneurons: An in vitro study on the newborn rat, Neurosci. Lett., 111 (1990) 127 132. [15] 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. [16] Pierrefiche, O., Foutz, A.S., Champagnat, J. and Denavit-Saubie, M., The bulbar network of respiratory neurons during apneusis induced by a blockade of NMDA receptors, Exp. Brain Res., 89 (1992) 623~39. [17] Plum, F. and Alvord, E.C., Apneustic breathing in man, Arch. Neurol., 10 (1964) 101-112. [18] Richter, D.W., Ballantyne, D. and Remmers, J.E., How is the respiratory rhythm generated? A model, News Physiol. Sci., 1 (1986) 109 112. [19] Richter, D.W., Bischoff, A., Anders, K., Betlingham, M. and Windhorst, U., Response of the medullary respiratory network of the cat to hypoxia, J. Physiol., 443 (1991) 231-256. [20] St.John, W.M., Glasser, R.L. and King, R.A., Apneustic breathing after vagotomy in cats with chronic pneumotaxic center lesions, Resp. Physiol., 12 (1971) 239-250. [21] Voss, M.D., DeCastro, D., Lipski, J., Pilowsky, P.M. and Jiang, C., Serotonin immunoreactive boutons form close appositions with respiratory neurons of the dorsal respiratory group in the cat, J. Comp. Neurol., 295 (1990) 208-218. [22] Younes, M.K., Remmers, J.E. and Baker, J., Characteristics of inspiratory inhibition by phasic volume feedback in cats, J. Appl. Physiol. Resp. Env. Exer. Physiol., 45 (t978) 80-86.