134 Neuroseience Letters, 109 (1990) 134-- 139 Elsevier Scientific Publishers Ireland Ltd

NSL 06601

Effects of N-methyl-o-aspartate (NMDA) antagonist MK-801 on breathing pattern in rats

R. Monteau, P. Gauthier, P. Rega and G. Hilaire

Dkparternent de Physiologie et Neurophysiologie, Equipe 'Biologie des Rythmes et du Ddveloppement ', Facultb des Sciences et Techniques, St. J('rfme, Marseilles (France)

(Received 8 June 1989; Revised version received 9 September 1989; Accepted 17 September 1989)

Key words: Respiration; Apneusis; N-Methyl-D-aspartate; Rat

In anaesthetized, bivagotomized and artificially ventilated rats, the respiratory effects of systemic injec- tion of MK-801, a non-competitive N-methyl-D-aspartate antagonist, were studied. In all the experiments (n = 11), the injection increased the inspiratory duration and decreased the expiratory duration. In 4 exper- iments, the inspiratory duration was drastically lengthened, resulting in an apneustic-like breathing pat- tern. These results demonstrate that apneusis is difficult but possible to induce in rats and suggest that termination of inspiration is controlled via central mechanisms in which NMDA-like receptors are involved.

According to the classical views on the genesis of rhythmic respiratory activity, the termination of the inspiratory phase of breathing is thought to involve two systems acting on the respiratory generator: the vagal afferent system and the pontine pneu- motaxic center. Elimination of these influences by bivagotomy and brainstem tran- section induces a sustained inspiratory activity named apneusis [10]. Apneusis could also be induced by discrete electrolytic lesions performed in the dorsal pons [4, 17]. It has been hypothesized that the pneumotaxic center is mainly responsible for the termination of inspiration (see ref. 5 for a review). An apneustic breathing pattern could also be evoked in vagotomized cats by systemic administration of N-methyl-D- aspartate (NMDA) antagonists able to penetrate the blood brain barrier [7, 8]. This suggests that N M D A antagonis ts may interrupt pneumotaxic inputs to the inspira-

tory te rminat ion mechanism of the respiratory generator [8]. All the above results

were obta ined in cats but apneusis has never been described in rats even when vago-

tomy and pont ine transection have been performed [12]. It was therefore proposed

to investigate whether apneusis could be pharmacological ly induced in rats, using

systemic injections of N M D A antagonist .

Correspondence: R. Monteau, D6partement de Physiologie et Neurophysiologie, Equipe Biologie des Rythmes et du D6veloppement, Facult6 des Sciences et Techniques, St. J6r6me, Avenue Escadrille Nor- mandie-Niemen, 13397 Marseilles Cedex 13, France.

0304-3940/90/$ 03.50 ~) 1990 Elsevier Scientific Publishers Ireland Ltd.

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Fig. 1. Effects of systemic MK-801 in two rats. Left panels: from top to bottom, recording of the phrenic nerve activity before, 1, 5 and 40 min after injection. Doses: 1.7 mg/kg in A; 3.5 mg/kg in B. Right panels: diagrams representing the changes in TI (filled squares) and TE (empty squares). Abscissa: time in minutes; ordinate: phase duration in milliseconds. Systemic injection was performed at time 0. In A, a low dose of MK-801 induced a short lasting moderate increase in TI; in B a higher dose caused apneusis; both doses produced similar decreases in TE.

Experiments were performed on 11 adult rats (280-410 g) anaesthetized with pen- tobarbitai (50 mg/kg, i.p.). A tracheotomy and cannulations of femoral artery and vein were performed. Both vagus nerves were prepared for section. The left phrenic nerve was dissected and cut distally. Phrenic motor output was recorded with bipolar electrodes, and then amplified and filtered. Arterial pressure was recorded. The rats were artificially ventilated with oxygen (Harvard apparatus), and paralyzed by sys- temic administration of gallamine triethiodide. All the signals (phrenic activity, inte- grated phrenic activity, arterial and tracheal pressure, ECG) were viewed on scope and paper recorder (Mingograph). After a control period, the animals were vagoto- mized. N M D A antagonist (MK-801) was dissolved in saline and administered intra- venously. A single dose of MK-801 (0.5 or 1 mg for each animal) was injected and flushed with saline while continuous recordings of the signals were performed for at least 90 min.

Under control conditions (anaesthetized, vagotomized, paralyzed rats) the durations of the two phases of the respiratory cycle were 344__+ 17 ms in the case of inspiration (TI) and 1069_ 140 ms in the case of expiration (TE), leading to a mean ratio TI/TE of 0.38 + 0.05 (individual values from 0.2 to 0.77). The N M D A antago- nist MK-801 was administered slowly (i.v.) inducing no change in arterial blood pres-

136

sure. In all experiments, all changes in respiratory parameters (TI, TE, ratio TI/TE) induced by MK-801 injections were qualitatively similar from one experiment to an- other. Within the first minute, mean TI increased (1658 + 574 ms), whereas mean TE decreased (641 + 72 ms). The ratio TI /TE increased up to 3.3 + 1.4. Fig. 1 shows two examples of individual responses. In Fig. 1A, the injection (1.7 mg/kg) can be seen to have induced a short lasting moderate increase in TI (+49%) and a decrease in TE ( - 3 4 % ) . In Fig. IB, which gives data on another rat, the injection (3.3 mg/kg) induced a decrease in TE ( - 3 2 % ) , and a drastic increase in TI (+786%). In this rat, MK-801 clearly induced an apneustic breathing pattern. The period of true apneusis

was short but TI remained longer than the control values for more than 90 min. As

shown in Fig. 2, the response does not only depend on the injected dose. Limited increases in TI were obtained with doses lower than 2 mg/kg, but a variability between rats exit since larger doses (2.5-3.5 mg/kg) induced either limited effects (4/8 experiments) or apneusis (4/8). The decrease in TE duration was the same whether apneusis was induced or not (34%_+ 12 and 33.8 % _+ 8, respectively). As shown in Fig. 3, the duration of inspiration during the apneustic period of breathing was not stable; this was particularly obvious at the beginning of the apneustic period. Long lasting inspirations (several seconds) and almost normal ones were intermingled (Fig. 3A); sometimes the amplitude of long inspirations was modulated (Fig. 3B), thus clearly indicating a failure of the ' inspiratory off-switch' mechanisms.

The results herein provide evidence that blocking NMDA-type glutamate recep- tors in rats lengthens the inspiration and shortens the expiration. In some cases, an

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Fig. 2. Dose-effect relationship on TI and TE. Abscissa, concentration of MK-801; ordinate, changes in phase duration (% of control); filled squares, TI; empty squares, TE. Left panel: effects of MK-801 injec- tion on TI and TE; experiments in which a limited increase in TI occurred. Right panel: experiments in which apneusis was evoked. Decreases in TE were similar whatever the dose in both groups of experi- ments.

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apneustic-like breathing pattern can be evoked. As far as we know, no such pattern has ever been reported previously in rats. This phenomenon requires to be discussed in relation which pharmacologically induced apneusis in cats and with the lack of apneusis induced by pontine lesions in rats. The apneusis induced by surgical lesions of the so-called pontine pneumotaxic center has been thoroughly documented in cats [5, 10]. The pneumotaxic center (parabrachialis medialis and Ktilliker-Fuse nuclei [1]) is assumed to be a major source of the inspiratory off-switch mechanisms. However the ubiquity of the 'pneumotaxic mechanism' has recently been challenged. In guinea pigs [9], focal cold block of nucleus parabrachialis induced no apneusis; in rats, after bivagotomy and pontine transection, no apneusis was observed [12]. These results do not support the classical view that 'the pontine pneumotaxic center is mainly re- sponsible for inspiratory off-switch and development of apneusis in vagotomised ani- mals' [9]. Systemic injection of MK-801 in vagotomised cats, in acute [7] or chronic [8] experiments, induced apneusis. The results herein concerning rats are comparable but some qualitative and quantitative differences exist. In cats, doses of 0.03 mg/kg effectively increase TI and doses of I mg/kg induce sustained apneusis; in rats, doses 2.5 times higher induced apneusis in only one half of the experiments. In cats, the respiratory effects of MK-801 were not entirely identical to those of surgical pontine lesions and the pharmacological tools were assumed to be more selective [8] since only TI was found to be modified. In rats, both TI and TE were changed. The most striking difference was that in rats the pharmacological approach was able to increase the duration of TI and to induce apneusis in some cases, whereas pontine lesions were not. It is therefore doubtful whether the sites of action on the medullary generator of pontine influences and NMDA antagonist are identical. The similarity between

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138

the effects of pneumotaxic lesions and MK-801 in cats has led F O U T Z et al. [8] to hypothesize that MK-801 'may interrupt pneumotaxic threshold lowering inputs to the inspiration termination mechanisms of the central pattern generator' . The results obtained on rats do not support this hypothesis. Apneusis has also been obtained in cats by cooling [3] and by neurotensin injections [13] in the region of the nucleus tractus solitarius. Thus the dorsal respiratory group [2], which is involved in the integration of many inputs, could be a possible site of N M D A antagonist action. However, conflicting reports exist concerning the dorsal respiratory group in the rat [6, 14, 15, 18]. Pharmacologically induced apneusis might result from a blockade of receptors involved in neural circuits promoting termination of inspiration. In rats (and possibly guinea pigs) the relative weight of pontine pneumotaxic inputs (if any) contributing to the inspiratory off-switch appears to be less than in cats but this is

highly questionable. The results of the present study are in agreement with previous data [16] which

suggested that apneusis might be a functional state of the respiratory generator(s) rather than the consequence of pontine lesions. The coexistence of two distinct rhythms, as shown in Fig. 3, has already been reported under very different experi- mental conditions (onset of polypnea induced by electrical stimulation of the hypoth- alamus in cats, see Fig. 7 in ref. 11), suggesting that several rhythm generators are responsible for respiratory timing. Electrical or pharmacological stimulations might affect the state of the generators (quiescent or active) and/or the relationships between them, leading to transient respiratory changes until reorganization of the network is achieved. The occurrence of transient phenomena of this kind might help to throw light on the complex mechanisms underlying respiratory rhythmicity.

The authors gratefully acknowledge the excellent technical assistance of A.M. Lajard. The advise of Drs. M. Denavit-Saubi6 and A.S. Foutz was greatly appre- ciated. English revision by Dr. Jessica Blanc. This research was supported by the C.N.R.S. (U.R.A. 205) and the I.N.S.E.R.M. (Grant 886006).

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