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Effect of catecholamines on the swallowing reflex after pressure microinjections into the lateral solitary complex of the medulla oblongata
Brain Research, 386 (1986) 69-77 Elsevier
69
BRE 12098
Effect of Catecholamines on the Swallowing Reflex After Pressure Microinjections into the Lateral Solitary Complex of the Medulla Oblongata J.P. KESSLER and A. JEAN Laboratoire de Neurobiologie Fonctionnelle, Ddpartement de Physiologie et Neurophysiologie (UA CNRS 205), Facultd des Sciences et Techniques de Saint-Jdr6me, MarseiUe (France) (Accepted 25 March 1986) Key words: Nucleus of the solitary tract - - Swallowing - - Pressure microinjection - Noradrenaline - - Dopamine - - Medullary polysynaptic reflex - - Rat
The present study was carried out to elucidate the influence of catecholamines on swallowing, a polysynaptic reflex organized by an interneuronal network localized mainly within the lateral solitary complex (LSC) of the medulla oblongata. The effects of catecholaminergic agents were investigated in the rat, on rhythmic swallowing elicited by repetitive stimulation of the superior laryngeal nerve (SLN). Catecholaminergic agents were microinjected by pressure application, through multibarrelled glass micropipettes, into the LSC including the tractus solitarius, the swallowing region of the nucleus of the solitary tract and the adjacent reticular formation. Microinjections of noradrenaline (NA, 0.1-5 nmol, 50 nl) induced a significant decrease of the number and the amplitude of the rhythmic swallows elicited by stimulation of the ipsilateral SLN. This inhibitory effect was dose-related. Microinjections of clonidine (2.5 nmol, 50 nl), dopamine (0.25-2.5 nmol, 50 nl) and apomorphine (0.5 nmol, 50 nl), also inhibited swallowing. No significant modification of swallowing was induced by control injections of the vehicle (50 nl) within the active sites. Moreover the NA-induced inhibition of swallowing, was significantly antagonized by pretreatment with the a-adrenergic blocker phentolamine applied locally in the LSC. Furthermore neither blood pressure, nor respiratory rhythm were consistently modified by the catecholaminergic microinjections, indicating that the catecholamine-induced inhibition of swallowing was not a secondary side effect originating from alteration of these functions. It can therefore be concluded that the present results demonstrate the existence within the LSC of a catecholaminergic inhibition of the swallowing reflex. This inhibitory effect likely arises from activation of specific catecholaminergic receptors and affects the swallowing structures localized within the LSC, i.e., the laryngeal swallowing afferents running in the solitary tract and/or the swallowing interneurons within the nucleus of the solitary tract.
INTRODUCTION The nucleus of the solitary tract is a structure which receives a m a j o r input from visceral afferents running in the IX and X cranial nerves 7'18'29'36. Furt h e r m o r e , this nucleus was found to be involved in the organization and the regulation of m a n y vegetative functions such as breathing and b l o o d pressure regulation as well as digestive tract motility 6'27,3°. In this connection, it was d e m o n s t r a t e d that the swallowing reflex is organized within the nucleus of the solitary tract and the a d j a c e n t reticular formation 15'16'24. I n d e e d this reflex can be elicited by activation of laryngeal afferents, running in the X cranial nerve, which were shown to t e r m i n a t e within the nu-
cleus of the solitary tract u'17'18. In addition, the interneurons involved in the triggering and the p r o g r a m ming of swallowing are localized within the lateral portion of the nucleus of the solitary tract and the surrounding reticular f o r m a t i o n 12-14,19. The nucleus of the solitary tract contains a n u m b e r of catecholaminergic fibers and nerve terminals 9,26,34,37. M o r e o v e r , catecholaminergic receptors 39'41, as well as catecholamine-synthesizing I and catabolic enzymes 31 are found at this level and a potassium-evoked, c a l c i u m - d e p e n d e n t release of catecholamines in the nucleus of the solitary tract has been d e m o n s t r a t e d in rat brain slices 35. Thus a transmitter role for catecholamines within this structure is highly p r o b a b l e . I n d e e d it was previously d e m o n -
Correspondence: A. Jean, Laboratoire de Neurobiologie Fonctionelle, Departement de Physiologie et Neurophysiologie (UA CNRS 205), Facult6 des Sciences et Techniques de Saint-J6r6me, F-13397 Marseille Cedex 13, France. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
70 strated that catecholaminergic mechanisms, within this region, are involved in breathing and blood pressure regulation 5A02232"33"42. Moreover, previous work suggests an action of catecholamines on the swallowing reflex 3'4. The present work was therefore designed in order to investigate the influence of catecholamines at the bulbar level, on laryngeal-initiated swallowing. Experiments were carried out by means of catecholamine microinjections performed within the lateral solitary complex (LSC), involving the tractus solitarius, the lateral part of the nucleus of the solitary tract and the surrounding reticular formation, which contains both the laryngeal afferent terminals and the swallowing interneurons. MATERIALS AND METHODS
into the muscles; (ii) the intrapharyngeal pressure was recorded through a catheter connected tc~ it pressure transducer (P 1008, Narco bio-Systemst. Both EMG activity and intrapharyngeal pressure were displayed on a polygraph (Mingograf 800, EIcma Sch6nander). During some experiments, the activity of the medullary swallowing neurons was recorded extracellulary through the central barrel (filled with 2 M NaCI) of a 7-barrelled glass micropipette, and visualized on an oscilloscope (Tektronix 565). Heart rate and body temperature were monitored continuously throughout the experiment. In some experiments, the respiratory rhythm was recorded by means of a thermistor placed in the tracheal cannula and the arterial blood pressure was monitored through a catheter inserted in the right carotid artery and connected to a pressure transducer.
Surgical procedure The experiments were performed on 56 adult Wistar rats, weighing from 250 to 300 g, anesthetized with ketamine (180 mg/kg) given i.p. A midline incision was made on the ventral side of the neck and a tracheal cannula was inserted. One or both superior laryngeal nerves (SLN) were dissected free from the surrounding tissues and were placed on bipolar electrodes. A catheter was inserted into the right jugular vein, in order to maintain anesthesia by a continuous i.v. perfusion of ketamine (10 mg/h). The animals were then fixed in a stereotaxic frame (David Kopf Instruments) in the position described by De Groot 8. After occipitoparietal craniotomy, the floor of the fourth ventricle was exposed by removing the overlying part of the cerebellum, and covered with warm liquid paraffin.
Stimulation and recordings In order to trigger swallowing, the SLNs were stimulated through copper wire bipolar electrodes. In the present study, SLN stimuli consisted of long trains of pulses (6-7 s duration) at 30 Hz (150-200 pA, 0.3 ms duration) which induce a succession of several swallows called rhythmic swallowing (cf. Fig. 1A and refs. 19, 20). In order to monitor the swallowing reflex: (i) the electromyographic (EMG) activity of suprahyoid muscles (digastric, mylohyoid and geniohyoid muscles) was recorded by means of bipolar copper wire electrodes, insulated except at the tip and inserted
Microinjections Pressure microinjections of drug solutions were performed through 7-barrelled glass micropipettes (10-15/~m o.d. at the tip) using an injection device (Neurophore BH2, Med. System). The following agents were used: noradrenaline hydrochloride (NA; Sigma), dopamine hydrochloride (DA; Sigma), apomorphine hydrochioride (Sigma), clonidine hydrochloride (Boehringer Ingelheim) and phentolamine hydrochloride (Ciba). NA, DA and clonidine were dissolved in a physiological solution (NaC1, 140 raM; KCI, 2.7 raM; CaC12, 1.7 mM; MgC12, 0.11 mM; NaHCO 3, 12 mM: Na2HPO 4, 0.35 mM; glucose, 5.6 raM) with ascorbic acid (3 mM). Phentolamine was dissolved in a NaC1 solution (154 mM) and apomorphine in an ascorbic acid solution (6 mM). The pH of the drug solutions was 7.4 except for the apomorphine solution (pH = 6). The procedure used consisted of repetitive injections of a small volume (5 nl) of the drug solution, each performed with long duration (4 s) pressure pulses in order to prevent the leakage of the injectate along the pipette track. Usually, the drug administration was carried out by injecting 10 pulses of the solution (corresponding to a total volume of 50 nl) over a 50-s period. Calibration of volumes injected through each barrel of the micropipette was carried out under microscopic observation, by measuring the diameter of an exuded droplet such as previously used (cf. ref. 20),
71
At the end of the experiment, Methyl blue (0.2%) contained in one barrel of the 7-barrelled micropipette, was applied into the injection site by either pressure or iontophoretic (15/~A, DC, for 3 min) ejection. The brainstem was removed after intracardiac perfusion of the animal with 10% formalin. Unstained frozen sections (thickness 50/~m) were cut and examined for histological localization of the injection site.
Following the positioning of the micropipette into the injection site, stimulation trains (6-7 s duration) were delivered to the ipsilateral SLN every 60 s. A control record, including at least 5 stimulation trains, was first obtained. The microinjection was then performed between two stimulation trains over a 50-s period, by applying 10 pressure pulses. Stimulation and recording were maintained until recovery. The effect of catecholaminergic agents on swallowing was quantified by considering the number of swallows elicited by each laryngeal stimulation train. A reference value was first established for each microinjection, by calculating the mean number of swallows elicited by the 5 stimulation trains preceding the injection. The mean number of swallows for the 5 stimulation trains following the injection was then calculated and this value was expressed as a % of the reference value. The data obtained were expressed as means + S.D. Statistical analysis was performed using the paired t-test. P values of 0.05 or less were considered to be significant.
Experimental procedure and statistical analysis
RESULTS
and according to the technique described by McCaman et al. 23. Pressure was adjusted between 80 and 150 kPa in order to obtain a 5-nl droplet when a 4-s duration pressure pulse was applied to the micropipette barrel. Such parameters were found to ensure an accurate calibration of volumes within the nanoliter range 38. Calibration was performed twice, at the beginning and at the end of each experiment. Only results obtained without change between the two calibrations were taken into account.
Histological controls
The effects of catecholaminergic agents were investigated on rhythmic swallowing elicited by long repetitive SLN stimulation (cf. ref. 20). Microinjections of catecholaminergic agents were performed stereotaxically into the LSC, which corresponds to a region between 0.2 and 0.6 mm rostral to the caudal tip of the area postrema (taken as the rostrocaudal and lateral zero), 0.7 mm to 1 mm laterally and 0.7 to 1 mm deep. This region contains both the swallowing afferent terminals and the swallowing interneurons involved in the programming of the reflex 15,19. In some cases the activity of the swallowing interneutons was recorded in order to confirm the accurate placement of the micropipette tip. Since it has been shown that the laryngeal afferents terminate mainly within the ipsilateral nucleus of the solitary tract 11'17'~s and that swallowing induced by unilateral SLN stimulation is triggered by the interneurons localized within the ipsilateral nucleus 12,13, microinjections were performed ipsilaterally to the SLN stimulation. Each animal received a maximum of two microinjections. These were performed into the same site, without removing the micropipette, when comparison of effects was required. In addition, some animals received local treatment with an antagonist.
Effects of noradrenergic agents on the ipsilaterally SLN-elicited swallowing Microinjections of NA (n = 57) were performed into the LSC in doses ranging between 0.1 and 5 nmol. These injections decreased the number of rhythmic swallows elicited by each laryngeal stimulation train (Fig. 1). The effect appeared as early as the first stimulation train after the injection and the maximal decrease was reached from 1 to 3 min after the beginning of the injection. The maximal effect, which could result in the total disappearance of swallows when high doses of NA (>2.5 nmol) were microinjected, remained for 5-10 rain. The return to the preinjection level was gradual, over a 20-40-min period. A total recovery was usually observed. Furthermore, the NA microinjections increased the latency and decreased the amplitude and the duration of the remaining swallows. It should be noted that neither the baseline of the EMG recording nor the resting level of the intrapharyngeal pressure were modified by the NA injections. Some NA microinjections (n = 9) were performed outside the LSC (i.e., 0.5 mm distant from the active site). These injections were without effect on swallowing. In addition, the
72 B
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...~.,
L
.~.
I,.
L
dose of N A (0.25 nmol in 50 nl), was followed after
100,
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forming two successive N A microinjections into the same site. The first one, which corresponded to a low recovery by a second microinjection corresponding to a higher dose of N A (2.5 nmol in 50 nl; Fig. 2A).
_ r,"
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.~.,.,
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NA Physiological 0.25n mol Solution
Fig. 1. Effects of NA microinjections on rhythmic swallowing. A: effects of a 2.5-nmol NA microinjection. EMG, electromyographic activity of the suprahyoi'd muscles; P, intrapharyngeal pressure; St, laryngeal stimulation', 1, control recording obtained before the injection. The ipsilateral SLN was stimulated every 60 s with long trains of pulses eliciting rhythmic swallowing; 2, recording obtained at the end of the injection. Note the marked decrease in the number and the amplitude of swallows; 3 and 4, recordings obtained 18 and 39 min after the end of the injection, respectively. B: effects of NAvs vehicle microinjections. Diagrams summarizing results obtained on the number of rhythmic swallows. Effects of NA (0.25 nmol, 50 nl) and vehicle solution (50 nl) are represented by the open and stippled columns, respectively. Results representing the mean value + S.D. of 6 experiments are expressed as % of control ( . . . . P < 0.001).
effect of microinjection of lower doses of N A (0.025 nmoi) were also tested during some experiments (n = 4). All these injections, performed within an active site, failed to significantly modify the swallowing reflex.
Comparison with vehicle microinjections. The effects of N A injections (n = 6) were compared to those induced by control microinjections of the vehicle (physiological saline solution). Each control microinjection (50 nl) was followed, 15 min later, by a N A microinjection (0.25 nmol, 50 nl) performed within the same site. All the N A microinjections significantly decreased the n u m b e r of rhythmic swallows (54 + 19%, P < 0.001) whereas control injections produced only a weak decrease (5 + 1 t % ) which was not significant (Fig. 1B). Dose-response relationship. The d o s e - r e s p o n s e relationship of the N A - i n d u c e d inhibition of swallowing was examined in some animals (n = 4), by per-
B
%
100"
4
st
A
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50.
i
50'
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NA 025nmol
***~
NA 25nmol
Clonidine 25nmol
C
% 100.
]_ ~0'
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NA 25 nmol
Phentolamine + NA 25 nmol
Phentolamine 04 nmol
Fig. 2. A: diagram showing the dose-effect relationship of NA injections to the number of rhythmic swallows. Results of 4 experiments (mean value + S.D.) are expressed as % of control. The effect of the lower dose of NA (0.25 nmol, 50 nl) significantly differs (P < 0.02) from that obtained with the higher dose (2.5 nmol, 50 nl). B: effects of clonidine microinjections (2.5 nmol, 50 nl) on the number of swallows. Results (mean value + S.D.) of 6 experiments are expressed as % of control. C: effect of phentolamine pretreatment on the inhibitory action of NA. Results of 4 experiments (mean value _+ S.D.) are expressed as % of control. Note that the inhibitory action of the NA microinjection (2.5 nmol in 50 nl) is weaker after the phentolamine pretreatment (stippled column) than before (left column). Effect of phentolamine alone is represented by the right column. *, P < 0.05; **, P < 0.02; ****, P < 0.001.
69
BRE 12098
Effect of Catecholamines on the Swallowing Reflex After Pressure Microinjections into the Lateral Solitary Complex of the Medulla Oblongata J.P. KESSLER and A. JEAN Laboratoire de Neurobiologie Fonctionnelle, Ddpartement de Physiologie et Neurophysiologie (UA CNRS 205), Facultd des Sciences et Techniques de Saint-Jdr6me, MarseiUe (France) (Accepted 25 March 1986) Key words: Nucleus of the solitary tract - - Swallowing - - Pressure microinjection - Noradrenaline - - Dopamine - - Medullary polysynaptic reflex - - Rat
The present study was carried out to elucidate the influence of catecholamines on swallowing, a polysynaptic reflex organized by an interneuronal network localized mainly within the lateral solitary complex (LSC) of the medulla oblongata. The effects of catecholaminergic agents were investigated in the rat, on rhythmic swallowing elicited by repetitive stimulation of the superior laryngeal nerve (SLN). Catecholaminergic agents were microinjected by pressure application, through multibarrelled glass micropipettes, into the LSC including the tractus solitarius, the swallowing region of the nucleus of the solitary tract and the adjacent reticular formation. Microinjections of noradrenaline (NA, 0.1-5 nmol, 50 nl) induced a significant decrease of the number and the amplitude of the rhythmic swallows elicited by stimulation of the ipsilateral SLN. This inhibitory effect was dose-related. Microinjections of clonidine (2.5 nmol, 50 nl), dopamine (0.25-2.5 nmol, 50 nl) and apomorphine (0.5 nmol, 50 nl), also inhibited swallowing. No significant modification of swallowing was induced by control injections of the vehicle (50 nl) within the active sites. Moreover the NA-induced inhibition of swallowing, was significantly antagonized by pretreatment with the a-adrenergic blocker phentolamine applied locally in the LSC. Furthermore neither blood pressure, nor respiratory rhythm were consistently modified by the catecholaminergic microinjections, indicating that the catecholamine-induced inhibition of swallowing was not a secondary side effect originating from alteration of these functions. It can therefore be concluded that the present results demonstrate the existence within the LSC of a catecholaminergic inhibition of the swallowing reflex. This inhibitory effect likely arises from activation of specific catecholaminergic receptors and affects the swallowing structures localized within the LSC, i.e., the laryngeal swallowing afferents running in the solitary tract and/or the swallowing interneurons within the nucleus of the solitary tract.
INTRODUCTION The nucleus of the solitary tract is a structure which receives a m a j o r input from visceral afferents running in the IX and X cranial nerves 7'18'29'36. Furt h e r m o r e , this nucleus was found to be involved in the organization and the regulation of m a n y vegetative functions such as breathing and b l o o d pressure regulation as well as digestive tract motility 6'27,3°. In this connection, it was d e m o n s t r a t e d that the swallowing reflex is organized within the nucleus of the solitary tract and the a d j a c e n t reticular formation 15'16'24. I n d e e d this reflex can be elicited by activation of laryngeal afferents, running in the X cranial nerve, which were shown to t e r m i n a t e within the nu-
cleus of the solitary tract u'17'18. In addition, the interneurons involved in the triggering and the p r o g r a m ming of swallowing are localized within the lateral portion of the nucleus of the solitary tract and the surrounding reticular f o r m a t i o n 12-14,19. The nucleus of the solitary tract contains a n u m b e r of catecholaminergic fibers and nerve terminals 9,26,34,37. M o r e o v e r , catecholaminergic receptors 39'41, as well as catecholamine-synthesizing I and catabolic enzymes 31 are found at this level and a potassium-evoked, c a l c i u m - d e p e n d e n t release of catecholamines in the nucleus of the solitary tract has been d e m o n s t r a t e d in rat brain slices 35. Thus a transmitter role for catecholamines within this structure is highly p r o b a b l e . I n d e e d it was previously d e m o n -
Correspondence: A. Jean, Laboratoire de Neurobiologie Fonctionelle, Departement de Physiologie et Neurophysiologie (UA CNRS 205), Facult6 des Sciences et Techniques de Saint-J6r6me, F-13397 Marseille Cedex 13, France. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
70 strated that catecholaminergic mechanisms, within this region, are involved in breathing and blood pressure regulation 5A02232"33"42. Moreover, previous work suggests an action of catecholamines on the swallowing reflex 3'4. The present work was therefore designed in order to investigate the influence of catecholamines at the bulbar level, on laryngeal-initiated swallowing. Experiments were carried out by means of catecholamine microinjections performed within the lateral solitary complex (LSC), involving the tractus solitarius, the lateral part of the nucleus of the solitary tract and the surrounding reticular formation, which contains both the laryngeal afferent terminals and the swallowing interneurons. MATERIALS AND METHODS
into the muscles; (ii) the intrapharyngeal pressure was recorded through a catheter connected tc~ it pressure transducer (P 1008, Narco bio-Systemst. Both EMG activity and intrapharyngeal pressure were displayed on a polygraph (Mingograf 800, EIcma Sch6nander). During some experiments, the activity of the medullary swallowing neurons was recorded extracellulary through the central barrel (filled with 2 M NaCI) of a 7-barrelled glass micropipette, and visualized on an oscilloscope (Tektronix 565). Heart rate and body temperature were monitored continuously throughout the experiment. In some experiments, the respiratory rhythm was recorded by means of a thermistor placed in the tracheal cannula and the arterial blood pressure was monitored through a catheter inserted in the right carotid artery and connected to a pressure transducer.
Surgical procedure The experiments were performed on 56 adult Wistar rats, weighing from 250 to 300 g, anesthetized with ketamine (180 mg/kg) given i.p. A midline incision was made on the ventral side of the neck and a tracheal cannula was inserted. One or both superior laryngeal nerves (SLN) were dissected free from the surrounding tissues and were placed on bipolar electrodes. A catheter was inserted into the right jugular vein, in order to maintain anesthesia by a continuous i.v. perfusion of ketamine (10 mg/h). The animals were then fixed in a stereotaxic frame (David Kopf Instruments) in the position described by De Groot 8. After occipitoparietal craniotomy, the floor of the fourth ventricle was exposed by removing the overlying part of the cerebellum, and covered with warm liquid paraffin.
Stimulation and recordings In order to trigger swallowing, the SLNs were stimulated through copper wire bipolar electrodes. In the present study, SLN stimuli consisted of long trains of pulses (6-7 s duration) at 30 Hz (150-200 pA, 0.3 ms duration) which induce a succession of several swallows called rhythmic swallowing (cf. Fig. 1A and refs. 19, 20). In order to monitor the swallowing reflex: (i) the electromyographic (EMG) activity of suprahyoid muscles (digastric, mylohyoid and geniohyoid muscles) was recorded by means of bipolar copper wire electrodes, insulated except at the tip and inserted
Microinjections Pressure microinjections of drug solutions were performed through 7-barrelled glass micropipettes (10-15/~m o.d. at the tip) using an injection device (Neurophore BH2, Med. System). The following agents were used: noradrenaline hydrochloride (NA; Sigma), dopamine hydrochloride (DA; Sigma), apomorphine hydrochioride (Sigma), clonidine hydrochloride (Boehringer Ingelheim) and phentolamine hydrochloride (Ciba). NA, DA and clonidine were dissolved in a physiological solution (NaC1, 140 raM; KCI, 2.7 raM; CaC12, 1.7 mM; MgC12, 0.11 mM; NaHCO 3, 12 mM: Na2HPO 4, 0.35 mM; glucose, 5.6 raM) with ascorbic acid (3 mM). Phentolamine was dissolved in a NaC1 solution (154 mM) and apomorphine in an ascorbic acid solution (6 mM). The pH of the drug solutions was 7.4 except for the apomorphine solution (pH = 6). The procedure used consisted of repetitive injections of a small volume (5 nl) of the drug solution, each performed with long duration (4 s) pressure pulses in order to prevent the leakage of the injectate along the pipette track. Usually, the drug administration was carried out by injecting 10 pulses of the solution (corresponding to a total volume of 50 nl) over a 50-s period. Calibration of volumes injected through each barrel of the micropipette was carried out under microscopic observation, by measuring the diameter of an exuded droplet such as previously used (cf. ref. 20),
71
At the end of the experiment, Methyl blue (0.2%) contained in one barrel of the 7-barrelled micropipette, was applied into the injection site by either pressure or iontophoretic (15/~A, DC, for 3 min) ejection. The brainstem was removed after intracardiac perfusion of the animal with 10% formalin. Unstained frozen sections (thickness 50/~m) were cut and examined for histological localization of the injection site.
Following the positioning of the micropipette into the injection site, stimulation trains (6-7 s duration) were delivered to the ipsilateral SLN every 60 s. A control record, including at least 5 stimulation trains, was first obtained. The microinjection was then performed between two stimulation trains over a 50-s period, by applying 10 pressure pulses. Stimulation and recording were maintained until recovery. The effect of catecholaminergic agents on swallowing was quantified by considering the number of swallows elicited by each laryngeal stimulation train. A reference value was first established for each microinjection, by calculating the mean number of swallows elicited by the 5 stimulation trains preceding the injection. The mean number of swallows for the 5 stimulation trains following the injection was then calculated and this value was expressed as a % of the reference value. The data obtained were expressed as means + S.D. Statistical analysis was performed using the paired t-test. P values of 0.05 or less were considered to be significant.
Experimental procedure and statistical analysis
RESULTS
and according to the technique described by McCaman et al. 23. Pressure was adjusted between 80 and 150 kPa in order to obtain a 5-nl droplet when a 4-s duration pressure pulse was applied to the micropipette barrel. Such parameters were found to ensure an accurate calibration of volumes within the nanoliter range 38. Calibration was performed twice, at the beginning and at the end of each experiment. Only results obtained without change between the two calibrations were taken into account.
Histological controls
The effects of catecholaminergic agents were investigated on rhythmic swallowing elicited by long repetitive SLN stimulation (cf. ref. 20). Microinjections of catecholaminergic agents were performed stereotaxically into the LSC, which corresponds to a region between 0.2 and 0.6 mm rostral to the caudal tip of the area postrema (taken as the rostrocaudal and lateral zero), 0.7 mm to 1 mm laterally and 0.7 to 1 mm deep. This region contains both the swallowing afferent terminals and the swallowing interneurons involved in the programming of the reflex 15,19. In some cases the activity of the swallowing interneutons was recorded in order to confirm the accurate placement of the micropipette tip. Since it has been shown that the laryngeal afferents terminate mainly within the ipsilateral nucleus of the solitary tract 11'17'~s and that swallowing induced by unilateral SLN stimulation is triggered by the interneurons localized within the ipsilateral nucleus 12,13, microinjections were performed ipsilaterally to the SLN stimulation. Each animal received a maximum of two microinjections. These were performed into the same site, without removing the micropipette, when comparison of effects was required. In addition, some animals received local treatment with an antagonist.
Effects of noradrenergic agents on the ipsilaterally SLN-elicited swallowing Microinjections of NA (n = 57) were performed into the LSC in doses ranging between 0.1 and 5 nmol. These injections decreased the number of rhythmic swallows elicited by each laryngeal stimulation train (Fig. 1). The effect appeared as early as the first stimulation train after the injection and the maximal decrease was reached from 1 to 3 min after the beginning of the injection. The maximal effect, which could result in the total disappearance of swallows when high doses of NA (>2.5 nmol) were microinjected, remained for 5-10 rain. The return to the preinjection level was gradual, over a 20-40-min period. A total recovery was usually observed. Furthermore, the NA microinjections increased the latency and decreased the amplitude and the duration of the remaining swallows. It should be noted that neither the baseline of the EMG recording nor the resting level of the intrapharyngeal pressure were modified by the NA injections. Some NA microinjections (n = 9) were performed outside the LSC (i.e., 0.5 mm distant from the active site). These injections were without effect on swallowing. In addition, the
72 B
A 1 EMB
% •
...~.,
L
.~.
I,.
L
dose of N A (0.25 nmol in 50 nl), was followed after
100,
.J,
forming two successive N A microinjections into the same site. The first one, which corresponded to a low recovery by a second microinjection corresponding to a higher dose of N A (2.5 nmol in 50 nl; Fig. 2A).
_ r,"
1
3 l
.~.,.,
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]_
NA Physiological 0.25n mol Solution
Fig. 1. Effects of NA microinjections on rhythmic swallowing. A: effects of a 2.5-nmol NA microinjection. EMG, electromyographic activity of the suprahyoi'd muscles; P, intrapharyngeal pressure; St, laryngeal stimulation', 1, control recording obtained before the injection. The ipsilateral SLN was stimulated every 60 s with long trains of pulses eliciting rhythmic swallowing; 2, recording obtained at the end of the injection. Note the marked decrease in the number and the amplitude of swallows; 3 and 4, recordings obtained 18 and 39 min after the end of the injection, respectively. B: effects of NAvs vehicle microinjections. Diagrams summarizing results obtained on the number of rhythmic swallows. Effects of NA (0.25 nmol, 50 nl) and vehicle solution (50 nl) are represented by the open and stippled columns, respectively. Results representing the mean value + S.D. of 6 experiments are expressed as % of control ( . . . . P < 0.001).
effect of microinjection of lower doses of N A (0.025 nmoi) were also tested during some experiments (n = 4). All these injections, performed within an active site, failed to significantly modify the swallowing reflex.
Comparison with vehicle microinjections. The effects of N A injections (n = 6) were compared to those induced by control microinjections of the vehicle (physiological saline solution). Each control microinjection (50 nl) was followed, 15 min later, by a N A microinjection (0.25 nmol, 50 nl) performed within the same site. All the N A microinjections significantly decreased the n u m b e r of rhythmic swallows (54 + 19%, P < 0.001) whereas control injections produced only a weak decrease (5 + 1 t % ) which was not significant (Fig. 1B). Dose-response relationship. The d o s e - r e s p o n s e relationship of the N A - i n d u c e d inhibition of swallowing was examined in some animals (n = 4), by per-
B
%
100"
4
st
A
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i
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***~
NA 25nmol
Clonidine 25nmol
C
% 100.
]_ ~0'
T
Ii
NA 25 nmol
Phentolamine + NA 25 nmol
Phentolamine 04 nmol
Fig. 2. A: diagram showing the dose-effect relationship of NA injections to the number of rhythmic swallows. Results of 4 experiments (mean value + S.D.) are expressed as % of control. The effect of the lower dose of NA (0.25 nmol, 50 nl) significantly differs (P < 0.02) from that obtained with the higher dose (2.5 nmol, 50 nl). B: effects of clonidine microinjections (2.5 nmol, 50 nl) on the number of swallows. Results (mean value + S.D.) of 6 experiments are expressed as % of control. C: effect of phentolamine pretreatment on the inhibitory action of NA. Results of 4 experiments (mean value _+ S.D.) are expressed as % of control. Note that the inhibitory action of the NA microinjection (2.5 nmol in 50 nl) is weaker after the phentolamine pretreatment (stippled column) than before (left column). Effect of phentolamine alone is represented by the right column. *, P < 0.05; **, P < 0.02; ****, P < 0.001.