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Laboratoire de Physiologie Neurovégétative (UMR CNRS 6153, UMR INRA 1147), Faculté des Sciences et Techniques Saint-Jérôme, Université Aix-Marseille 3, Cases postales 351–352, Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20, France
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(Received 2 September 2003;
accepted after revision 24 November 2003;
first published online 28 November 2003)
Corresponding author M. Bouvier: Laboratoire de Physiologie Neurovégétative (UMR CNRS 6153, UMR INRA 1147), Faculté des Sciences et Techniques Saint-Jérôme, Université Aix-Marseille 3, Cases postales 351–352, Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20, France. Email: michel.bouvier{at}univ.u-3mrs.fr
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Introduction |
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With a view to elucidating how Il-1ß is involved in the effects of leptin, it was proposed here to investigate the effects of this drug on leptin-sensitive intestinal vagal units to determine in particular whether its inhibitory effects on type 2 units may also involve Il-1ß. In order to establish whether each type of unit is specifically involved in the transmission of information about either inflammation or ingestion, and whether this information may vary depending on the chemical environment, we also investigated the interactions between leptin and CCK and the interactions between Il-1ß and CCK.
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Methods |
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Anaesthesia
Anaesthesia was induced by administering a gaseous mixture (2% halothane, 98% air; Bélamont, Neuilly sur-Seine, France). The trachea was cannulated and a catheter was inserted into a radial vein, through which the main anaesthetic (chloralose, 80 mg kg-1, I.V., Sigma, Saint Quentin-Fallavier, France) was administered intravenously. The depth of anaesthesia was monitored by checking that the palpebral reflex was absent and the pupils constricted. In addition, the blood pressure and heart rate were systematically monitored throughout the experiment to detect any changes in the level of anaesthesia. The appropriate depth of anaesthesia was characterized by constricted pupils, stable blood pressure (114.3 ± 8.2 mmHg, n= 34) and heart rate (119 ± 0.7 beats min-1, n= 34), and by the absence of the palpebral reflex. Whenever any of these parameters changed, showing that the anaesthesia was weakening, a flush of chloralose (8 mg kg-1, I.V.) was administered. To prevent the occurrence of any movement artefacts due to electrical stimulation of the nerves, the animals were paralysed by intravenously administering Flaxedil (gallamine triethiodide, 4 mg kg-1, I.V., Sigma, Saint Quentin-Fallavier, France) and ventilated with a respiratory pump. The cats were fixed in a Horsley-Clarke apparatus in the supine position, and their body temperature was maintained at 37 ± 1°C by means of a heating blanket.
In each experiment, the surgical procedure began when the values of the parameters characterizing the appropriate depth of anaesthesia were reached (after approximately 40–60 min). The stabilization time allowed to elapse between the end of the surgery and the start of the tests with the compounds was 2 h.
At the end of each experiment, the cats were killed by administering an overdose of barbiturates (sodium thiopentone, 70 mg kg-1, I.V., Sanofi, Libourne, France).
Electrophysiological recording techniques
The activity of the vagal interoceptors was routinely recorded at the level of the right nodose ganglion (Mei, 1978, 1983; Gaigéet al. 2002), which was reached via a short lateral incision in the neck. The ganglion was then placed on a special metallic plate, designed to prevent any artefacts from occurring as the result of the respiratory and carotid movements. The activity of the vagal afferent units was recorded by means of extracellular glass microelectrodes filled with 3 M KCl solution, as previously described (Mei, 1970). It has been reported that recordings obtained by means of glass electrodes filled with KCl (3 M) are similar to those obtained with glass electrodes filled with NaCl (2 M; Mei, 1968), or with metal electrodes (S. Gaigé, unpublished observations), which clearly indicates that using KCl (3 M) does not affect the discharge pattern of vagal afferent neurones under our experimental conditions. The electrodes were fixed to a micromanipulator (Mei, 1983) by means of a surgical microscope so that they could be implanted exactly into the gastro-intestinal region of the nodose ganglion, which is located in its caudal part (Mei, 1970; Zhuo et al. 1997). Electrodes were made using a vertical glass-stretching apparatus to obtain tips ranging between 1 and 3 µm in diameter with resistances from 2 to 5 M
. The discharge patterns of the vagal afferent units were displayed on an oscilloscope (TEKTRONIX, type R564B, Beaverton, OR, USA) and a thermosensitive paper recorder (Astromed, type DASH IV, 78190 Trappes, France). The activity of these units was also recorded on magnetic tape (DTR biologic, 38640 Claix, France) for further data processing. With the amplitude discrimination (WPI, model 121, New Haven, CT, USA) and shape recognition (Mac Laboratory, New Haven, CT, USA) procedures used, it was possible to select the electrical activity of a single afferent fibre, and thus to work under unitary recording conditions. Data were processed by computer in the form of frequency histograms showing the number of action potentials per second (AP s-1). To ensure that the full effects were displayed, the recordings were started 2 min before the injections were administered and lasted for 17 min.
Stimulation techniques
Electrical stimulation of the vagus nerve. The right cervical vagus nerve was dissected along 1 cm in the neck, 4–5 cm below the nodose ganglion, and was placed on stimulating electrodes consisting of two platinum wires placed in a Plexiglass gutter. Rectangular stimulation shocks (1 ms in duration and 15–30 V in amplitude) were delivered via a Grass stimulator connected in series to an isolation unit. This electrical stimulation made it possible to monitor all the responses via the recording electrode and to check whether the location of the electrode tip was accurate. This set-up was also used to record the conduction velocity (approximately 1 m s-1) of the unmyelinated vagal fibres.
Mechanical stimulation of the mechanoreceptors. A segment of small intestine including the duodenum and the proximal part of the jejunum was isolated between two cannulas. The more anterior of these, which was connected to a vertically adjustable reservoir containing physiological saline at 37°C, was inserted into the proximal jejunum so that various perfusion pressures could be obtained. The posterior cannula used to empty the intestinal loop was placed 20 cm lower down, and was connected to a pressure transducer (Telco, 94250 Gentilly Seine, France). The isolated intestinal loop was distended by moving the reservoir vertically. With the experimental protocol used here, it is possible to make pressure adjustments ranging between 0 and 100 cmH2O. With distensions of 20 cmH2O, it was possible to selectively activate the intestinal mechanoreceptors. Moderate digital pressure was also applied to locate these endings more exactly.
Chemical stimulation of the mechanoreceptors. Whenever an interoceptor was identified mechanically, intra-arterial chemical stimuli were applied. Pharmacological drugs were administered by performing close intra-arterial injections through a catheter introduced into the right femoral artery so that its tip was located at the level of the coeliac artery irrigating the upper part of the small intestine. A post mortem 1% methylene blue injection (Sigma, Saint Quentin-Fallavier, France) was carried out to check that the substances injected irrigated only the upper part of the small intestine. To prevent the occurrence of any tachyphylaxic events, the time elapsing between successive injections was set at 60 min. When testing whether CCK, SP and phenylbiguanide (PBG) modified the effects of Il-1ß and leptin, and to ensure that the discharge frequency had returned to its baseline value, we chose an interval of 20 min: in our experiments, the activatory effects of CCK on the discharge frequency of intestinal afferents lasted about 11 min and previous authors have indicated a similar duration of 12 min for these effects (Schwartz et al. 1991). In addition, we reported in a previous paper that the excitatory effects of Il-1ß on intestinal motility, which were probably mediated via the activation of vagal mechanoreceptors, were present 20 min after pretreatment by CCK (Gaigéet al. 2003). Likewise, when testing whether Il-1ra modified the effects of Il-1ß and leptin, this interval was reduced to 5 min, since this is the time this antagonist takes to block the interleukin-1 receptors (Gaigéet al. 2002, 2003).
Drugs
The following drugs were used: recombinant human interleukin-1ß (Il-1ß, AMGEN Thousand Oaks, USA), murine leptin lyophilized from a sterile filtered solution in phosphate-buffered saline (PBS; AMGEN Thousand Oaks, USA), recombinant human interleukin-1ß receptor antagonist (Il-1ra; AMGEN), sulphated cholecystokinin-8 (CCK; Sigma, Saint Quentin-Fallavier, France), substance P (SP; Sigma), phenylbiguanide (PBG; Sigma) and atropine sulphate salt (Sigma), CCK-A (lorglumide sodium salt) and CCK-B (CR 2945) receptor antagonists (Sigma).
All the substances were diluted in 1 ml of physiological saline and administered within 5 s. Each injection was followed by a flush with 1 ml of saline solution. Il-1ß (Bucinskaite et al. 1997; Kurosawa et al. 1997) and leptin were injected at the following doses: 0.1, 1 and 10 µg (99.50, 995.02, 9950.25 pmol and 6.25, 62.50, 625.00 pmol, respectively). CCK, SP and PBG were injected in a 10 µg dose (8.75, 7.42 and 56.43 nmol, respectively). All these doses were similar to those previously used by Gaigéet al. (2002) to activate vagal afferent fibres. Il-1ra and atropine were administered in a 250 µg dose (14.71 and 369.39 nmol, respectively; Bouvier & Gonella, 1981; Gaigéet al. 2002). CCK-A and CCK-B receptor antagonists were administered in a 250 µg dose (Kurosawa et al. 2000). The weight of the animals was not taken into account, since the drugs were injected by performing close intra-arterial injections into the intestine.
Statistical analysis
When the activity of several mechanoreceptors was recorded in the same animal, only the first unit recorded was included in the statistical analysis. At each injection, the mean discharge frequencies (AP s-1) of the afferent neurones were calculated during 30 s periods before (controls) and after the time of drug administration (effects). At each injection, we calculated the percentage difference between the mean discharge frequencies. Results are expressed as mean percentages ±S.E.M. The values given in parentheses are the mean discharge frequency before the injection ±S.E.M.versus the mean discharge frequency after the injection ±S.E.M. Statistical analysis was performed using Student's paired or unpaired t test to assess the significance of the results. An analysis of variance followed by a Scheffépost hoc test were also carried out to compare the effects of multiple drug injections. The significance level was taken to be P < 0.05.
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Results |
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Effects of leptin on the activity of intestinal mechanoreceptors
Leptin was injected intra-arterially (I.A.) at a dose of 10 µg to 34 mechanoreceptors. In 16 mechanoreceptors, leptin significantly increased the mean discharge frequency by 110 ± 21% with a latency of 9 ± 1 S and a duration of 308 ± 43 S (Table 1; Fig. 1). In 12 mechanoreceptors, leptin significantly decreased the mean discharge frequency by 20 ± 6% with a latency of 8 ± 1 S and a duration of 143 ± 21 S (Table 1; Fig. 1). In the six remaining mechanoreceptors, leptin had no effect on the mean discharge frequency. These results are similar to those obtained in our previous study (Gaigéet al. 2002). The mechanoreceptors are therefore again referred to here as type 1 units in the case of the 16 neurones which were activated and type 2 units in that of the 12 neurones which were inhibited.
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Il-1ß was injected intra-arterially (I.A.) at the following doses: 0.1, 1 and 10 µg (99.50, 995.02, 9950.25 pmol). The effects of Il-1ß on the three types of units defined in terms of their response to leptin were analysed. The effects of Il-1ß administered to the 34 mechanoreceptors were as follow: the 16 type 1 units were slightly activated (Fig. 2), the 12 type 2 units were more strongly activated (Fig. 3) and the six leptin-insensitive units showed no effect (not illustrated).
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). These effects had the following latencies: 5 ± 1, 6 ± 1 and 6 ± 1 s, respectively, and the following durations: 253 ± 38, 288 ± 31 and 308 ± 34 s, respectively. The responses of six type 2 units were also studied at three increasing doses (0.1, 1 and 10 µg) of Il-1ß. Il-1ß increased (P < 0.01, P < 0.001 and P < 0.001, respectively) the mean discharge frequency (80 ± 10, 158 ± 14 and 262 ± 26%, respectively; Table 1, Fig. 4B, 
). These effects had the following latencies: 6 ± 1, 6 ± 1 and 6 ± 2 s, respectively, and the following durations: 189 ± 26, 207 ± 24 and 230 ± 33 s, respectively. Significant differences between the responses to three doses (0.1, 1 and 10 µg) of Il-1ß were observed in the type 1 units and the type 2 units: the type 2 unit responses to Il-1ß were significantly (P < 0.05, P < 0.05 and P < 0.01, respectively) more intense and (P < 0.05 at all these doses) had shorter durations than the type 1 unit responses. It is worth noting that there was no significant difference between the spontaneous responses recorded from type 1 units (1.98 ± 0.41 AP s-1) and type 2 units (2.05 ± 0.38 AP s-1).
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The effects of the vehicle were investigated in the case of each unit and no significant changes in spontaneous activity were recorded after intra-arterial administration. In three type 1 units and three type 2 units, atropine (250 µg, I.A.), a muscarinic receptor blocking agent, administered before Il-1ß (1 µg) did not affect the excitatory responses (not illustrated). Sectioning the vagus nerve caudally to the nodose ganglion abolished the spontaneous activity of all three types of units (type 1, n= 3; type 2, n= 3; Il-1ß-insensitive, n= 4), and no effects of Il-1ß on the chemosensitive mechanoreceptors were observed (n= 6) (not illustrated). At all the doses tested, Il-1ß injection did not affect the intraluminal intestinal pressure, the arterial pressure (103.42 ± 7.59 versus 107.72 ± 9.51 mmHg) and the cardiac frequency (71.52 ± 0.84 versus 72.87 ± 0.58 beats min-1, n= 34).
The activity of several mechanoreceptors was recorded in six cats. In three of these cats, type 1 and type 2 units were identified; and in the others, all three types of units (type 1, type 2, and Il-1ß-insensitive) were detected (not illustrated).
Effects of CCK, SP and PBG on the activity of intestinal mechanoreceptors
Effects of CCK. CCK (10 µg, I.A.) increased the mean discharge frequency in all the units tested (n= 28), whether they were type 1 units (n= 16) or type 2 units (n= 12). The mean discharge frequency of the leptin- and Il-1ß-insensitive units was not affected by the injection of CCK (n= 6). In the type 1 units, a significant increase in the mean discharge frequency (Table 1, Fig. 1) was observed, amounting to 832 ± 87%, with a latency of 7 ± 1 S and a duration of 481 ± 76 s. The significant increase in the mean discharge frequency of 240 ± 40% observed with the type 2 units after CCK (Table 1, Fig. 1) had a latency of 7 ± 1 S and a duration of 244 ± 25 s. A significant difference (P < 0.01) was found to exist between the activatory effects of CCK on type 1 and type 2 units (Fig. 1).
Effects of SP. SP in a 10 µg dose (I.A.) had no effect on the type 2 units (n= 4; Table 1, Fig. 1); whereas SP significantly increased the mean discharge frequency by 137 ± 11% with a latency of 7 ± 2 S and a duration of 116 ± 21 S in the case of the type 1 units (n= 4; Table 1, Fig. 1).
Effects of PBG. PBG in a 10 µg dose (I.A.) had no effect on the type 2 units (n= 4; Table 1, Fig. 1), but significantly increased the mean discharge frequency by 857 ± 91% with a latency of 9 ± 3 S and a duration of 73 ± 29 s in the case of the type 1 units (n= 5; Table 1, Fig. 1).
Effects of leptin on the activity of intestinal mechanoreceptors after administration of CCK
In all the 16 type 1 units, leptin injected 20 min after CCK (10 µg, I.A.) induced a significant increase in mean discharge frequency of 308 ± 49% with a latency of 9 ± 1 S and a duration of 323 ± 51 s. CCK was found to enhance (P < 0.01) the effects of leptin alone (Table 1). In all the 12 type 2 units, CCK administered 20 min before leptin was found to inhibit (P < 0.05) the effects of leptin (Table 1).
Effects of Il-1ß on the activity of intestinal mechanoreceptors before and after administration of CCK, SP and PBG
Effects of Il-1ß before and after CCK.
In seven type 1 units, Il-1ß was administered in doses of 0.1, 1 and 10 µg 20 min after CCK injection (10 µg, I.A.). In all seven units tested, significant increases (P < 0.01, P < 0.001 and P < 0.001, respectively) of 82 ± 22%, 321 ± 32% and 342 ± 47%, respectively, were observed in the mean discharge frequency (Table 1; Fig. 4A, ). In all the cases studied, CCK was found to significantly enhance (P < 0.05, P < 0.01 and P < 0.01, respectively) the effects of Il-1ß described above (Figs 1 and 4A, •). On the other hand, the latencies (6 ± 2, 6 ± 1 and 6 ± 2 s, respectively) and durations (273 ± 33, 314 ± 47 and 310 ± 36 s, respectively) were not significantly different from those obtained with Il-1ß prior to CCK administration. Nor did the latencies of the effects of Il-1ß differ significantly from those of the effects of CCK.
In the 16 type 1 units, Il-1ß was administered at 1 µg 20 min after CCK injection (10 µg, I.A.). In all 16 units tested (n= 16 including the seven units presented in Fig. 4A, and •), a significant enhancement of the effects of Il-1ß (98 ± 17 versus 314 ± 29%) was observed (P < 0.01) (Table 1). An example of the effects of Il-1ß on a type 1 unit after CCK is given in Fig. 2.
In six type 2 units tested with the three doses (0.1, 1 and 10 µg) of Il-1ß, CCK was found to significantly inhibit (P < 0.01, P < 0.001 and P < 0.001, respectively) the effects of Il-1ß described above (Table 1; Fig. 4B, 
). In the 12 type 2 units, CCK injection performed 20 min before Il-1ß (1 µg) significantly (P < 0.001) inhibited the excitatory effects of Il-1ß described above (n= 12 including the 6 units presented in Fig. 4B, and ) (Table 1). An example of the effects of Il-1ß on a type 2 unit after CCK is illustrated in Fig. 3.
It is worth noting that the injection of vehicle performed 20 min after CCK affected neither type 1 units (n= 3) nor type 2 units (n= 3) (not illustrated). Two injections of 1 µg Il-1ß performed at an interval of 20 min both induced a similar increase in the discharge frequencies of the type 1 units (n= 3) and those of the type 2 units (n= 3) (not illustrated). In four type 1 units and three type 2 units, the responses of Il-1ß (1 µg) administered 20 min after CCK were not affected by atropine (250 µg, I.A.) pretreatment (not illustrated).
Effects of Il-1ß before and after SP. In the four type 1 units tested with Il-1ß and SP, injecting Il-1ß alone (1 µg) significantly increased the mean discharge frequency by 107 ± 24% with a latency of 6 ± 1 s and a duration of 307 ± 38 S (Table 1). Il-1ß administered to these four units 20 min after SP injection (10 µg, I.A.) also induced a significant increase of 109 ± 25% in the mean discharge frequency with a latency of 6 ± 1 S and a duration of 294 ± 37 s. Four type 2 units were tested with Il-1ß and SP. Il-1ß injected alone (1 µg) significantly increased the mean discharge frequency by 157 ± 19% with a latency of 6 ± 1 S and a duration of 185 ± 22 S (Table 1). Il-1ß administered 20 min after SP injection (10 µg, I.A.) also significantly inhibited the mean discharge frequency by 155 ± 16% with a latency of 6 ± 1 S and a duration of 196 ± 20 s. The excitatory effects of Il-1ß on type 1 and type 2 units observed before SP did not differ significantly from those observed after SP.
Effects of Il-1ß before and after PBG. In the five type 1 units tested with Il-1ß and PBG, injecting Il-1ß alone (1 µg) significantly increased the mean discharge frequency by 100 ± 28% with a latency of 5 ± 1 S and a duration of 294 ± 40 S (Table 1). Il-1ß administered in the same dose 20 min after PBG (10 µg, I.A.) induced a significant increase in the mean discharge frequency of 102 ± 21% with a latency of 6 ± 2 S and a duration of 298 ± 16 s. Four type 2 units were tested with Il-1ß and PBG. Il-1ß injected alone (1 µg) significantly increased the mean discharge frequency by 155 ± 12% with a latency of 7 ± 1 s and a duration of 185 ± 37 S (Table 1). Il-1ß administered in the same dose 20 min after PBG injection also significantly increased the mean discharge frequency by 157 ± 12% with a latency of 7 ± 0 s and a duration of 205 ± 46 s. Previously administering PBG did not significantly affect the responses of the units of either type to Il-1ß injection.
Effects of CCK-A and CCK-B receptor antagonist on the responses to Il-1ß
In three type 1 units and three type 2 units tested with CCK-A and CCK-B receptor antagonists, 1 µg Il-1ß injection alone significantly increased (P < 0.05 and P < 0.01, respectively) the mean discharge frequency by 99 ± 18 and 164 ± 21% with latencies of 5 ± 1 and 6 ± 1 s and durations of 297 ± 52 and 187 ± 38 s, respectively. Injecting CCK-A and CCK-B receptor antagonist (250 µg, I.A. each) 5 min before Il-1ß (1 µg) did not affect these two types of excitatory responses (Table 1).
In these type 1 and type 2 units, CCK injection (10 µg, I.A.) significantly increased the mean discharge frequency by 807 ± 92 and 239 ± 59% with latencies of 7 ± 2 and 7 ± 1 s and durations of 476 ± 61 and 236 ± 54 s, respectively. The two antagonists administered together in the same doses before CCK (10 µg, I.A.) completely blocked (P < 0.001 and P < 0.01, respectively) the CCK-excitatory responses in the type 1 and type 2 units (Table 1). In those cases where both antagonists were administered 5 min before CCK, Il-1ß (1 µg) injected 20 min after CCK (10 µg, I.A.) induced the same response as that obtained when Il-1ß was injected alone (n= 3, type 1 units; n= 3, type 2 units) (not illustrated). CCK-A and CCK-B receptor antagonist pretreatment applied before CCK therefore blocks the effects of CCK pretreatment on the effects of Il-1ß in the type 1 and type 2 units (not illustrated).
In addition, Il-1ß administered 20 min after CCK and 5 min after both antagonists induced the same response as that obtained when Il-1ß was injected 20 min after CCK (n= 3, type 1 units; n= 3, type 2 units) (not illustrated).
Effects of Il-1ra on the responses to Il-1ß and leptin
In the four type 1 units tested with Il-1ra, the endogenous antagonist of Il-1ß receptor, 1 µg Il-1ß injection alone significantly increased the mean discharge frequency by 108 ± 34% with a latency of 6 ± 1 s and a duration of 290 ± 46 s (Fig. 5Aa). Injecting Il-1ß at the same dose 5 min after Il-1ra (250 µg, I.A.) had no further significant effects (Table 1, Fig. 5Aa). The same result was obtained in the four type 1 units tested with Il-1ra and leptin. Injection of 10 µg (I.A.) leptin alone significantly increased the mean discharge frequency by 103 ± 25% with a latency of 8 ± 1 s and a duration of 304 ± 53 s (Fig. 5Ab). Leptin injected at the same dose 5 min after Il-1ra (250 µg, I.A.) had no further significant effects (Table 1, Fig. 5Ab).
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Effects of intestinal distension on the three types of mechanoreceptors depending on the effects of leptin and Il-1ß
The effects of a 20 cmH2O distension of the intestine on the three types of units defined in terms of their response to leptin and Il-1ß were analysed. The three types of units responded differently to mechanical stimulation. In the 13 type 1 units, distension significantly increased the mean discharge frequency by 143 ± 16% (Table 1). In the nine type 2 units, distension significantly increased the mean discharge frequency by 295 ± 55% (Table 1). In the six leptin- and Il-1ß-insensitive units, distension significantly (P < 0.01) increased the mean discharge frequency by 278.54 ± 69.74% (1.98 ± 0.24 versus 7.61 ± 0.87 AP s-1). There was no significant difference between the responses to intestinal distension observed in type 2 units and leptin- and Il-1ß-insensitive units, whereas these responses differed significantly (P < 0.01) from those of type 1 units.
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Discussion |
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In the present work, we show that the inhibitory effects of leptin on type 2 units were increased by Il-1ra. In addition, we demonstrate that Il-1ß induced excitatory responses in both types of units. After pretreatment with CCK, these excitatory effects were enhanced in type 1 units and blocked in type 2 units. Our results strongly suggest that leptin has direct effects on type 2 units, whereas on type 1 units, these effects are mediated through the release of Il-1ß.
Effects of Il-1ß on intestinal vagal afferents
As demonstrated for leptin in a previous paper (Gaigéet al. 2002), the effects of Il-1ß are definitely of peripheral origin and are not related to arterial distension associated with drug injection. Moreover, these effects are not due to motor activation, since Il-1ß injection did not affect intestinal intraluminal pressure (Gaigéet al. 2003) and atropine, generally used to inhibit motor activity, did not modify these effects of Il-1ß.
Our results are in agreement with electrophysiological data specifically indicating that Il-1ß enhances the spontaneous activity of hepatic (Niijima, 1996) and gastric (Kurosawa et al. 1997, 2000; Ek et al. 1998) vagal nerve fibres. Contrary to what was observed by these studies; in which excitatory effect latencies of approximately 30 min were recorded, in our experiments, for the first time, the latencies of these effects were found to be very short (about 5 s). In addition, the short latencies of the excitatory effects observed here suggest that this substance acts directly on sensory endings. It is worth noting that this short latency is similar to that recorded in the case of CCK, with which a direct effect can also occur (Mei, 1978; Mei et al. 1996). In our study, the excitatory effects of Il-1ß disappeared after the injection of Il-1ra, which suggests that Il-1ß acts on specific receptors. Furthermore, cell bodies and fibres of the vagal sensory neurones express the mRNA corresponding to the Il-1ß receptor (Ek et al. 1998). The excitatory effects of Il-1ß on intestinal vagal afferent fibres are consistent with numerous data showing that the peripheral Il-1ß message is transmitted via a vagal pathway to the brain, where it serves to depress food-motivated behaviour (Bret-Dibat et al. 1995), to induce fever (Watkins et al. 1995; Gordon, 2000) and hyperalgesia (Watkins et al. 1994), and to increase the extracellular noradrenaline concentration in the paraventricular nucleus in the hypothalamus (Fleshner et al. 1995; Hansen & Krueger, 1997; Ishizuka et al. 1997).
Interactions between Il-1ß and CCK
In the type 1 units, the excitatory effects of Il-1ß were enhanced by CCK, whereas in the type 2 units, they were inhibited by CCK. This cannot have been due to a tachyphylactic disorder, since two injections of Il-1ß given 20 min apart had identical effects on both types of intestinal vagal afferent fibres. In the type 1 units, the excitatory effects of Il-1ß after CCK were blocked by applying Il-1ra pretreatment, which shows that the enhancement of the effects was due to the binding of Il-1ß to specific receptors. In addition, the effects of Il-1ß alone on both types of units, and after CCK on the type 1 units, had the same latencies, which indicates that the same activation mechanisms were involved. In addition, the enhancement of the Il/1ß effects observed in the type 1 units was a peripheral process, since afferent neurone activation (latency about 5 s) always occurs prior to the motor effects (latency about 20–25 s) (Gaigéet al. 2003). This was confirmed by the fact that the effects of Il-1ß after CCK persisted under atropine. SP and PBG, which are routinely used to activate sensory visceral receptors (Paintal, 1973; Mei et al. 1996), activate type 1 units and have no effect on type 2 units. The fact that in both types of units, neither SP nor PBG alter the activatory effects of Il-1ß indicates that these Il–1ß–CCK interactions are actually specific. The enhancement of the Il-1ß effects observed after applying CCK pretreatment to type 1 units cannot have been due to the well known excitatory effect of CCK on the basal activity (see Gaigéet al. 2002). CCK is known to activate vagal afferent nerve fibres via CCK-A and CCK-B receptors (Schwartz et al. 1994; Wank, 1995). This was confirmed by our results showing that the excitatory effects of CCK on the intestinal afferent nerve fibres are blocked by CCK-A and CCK-B receptor antagonists. There exist data in the literature indicating that Il-1ß-induced excitation of vagal afferent nerve fibres is partly mediated by a release of CCK, which acts on specific receptors and thus directly activates mechanosensitive intestinal and gastric afferent fibres (Bucinskaite et al. 1997; Kurosawa et al. 1997, 2000). However, the results of the present study indicate that the excitatory effects of Il-1ß on intestinal vagal fibres are not mediated by CCK-A or CCK-B receptors, since the effects of Il-1ß on these two types of units are not blocked after pretreatment by CCK-A and CCK-B receptor antagonists. Moreover, the enhancement and blockade of the effects of Il-1ß induced by CCK on the type 1 and type 2 units, respectively, are abolished upon applying CCK-A and CCK-B receptor antagonists, which indicates that for CCK/Il–1ß interactions to occur, it is necessary for CCK to bind to specific receptors. This result also seems to indicate that Il-1ß is able to directly activate intestinal vagal afferent nerve fibres. In addition, this blockade of the responses to CCK suggests that independent transduction mechanisms may mediate the response to CCK and to Il-1ß. This idea is corroborated by studies showing that vagal sensory fibres express the mRNA corresponding to the Il-1ß receptors (Ek et al. 1998) and by the presence of CCK-A and CCK-B receptor binding sites in the vagus nerve (Corp et al. 1993). Furthermore, in type 2 units, CCK pretreatment blocks the Il-1ß excitatory effects, whereas CCK administration enhances spontaneous discharge patterns. These results show that a CCK binding step is required to modify the effects of Il-1ß on both types of units. The changes in the effects of Il-1ß induced by CCK pretreatment probably occur at the sensory ending level, since the process of CCK binding to specific receptors seems to modify the sensitivity of the Il-1ß receptors on the vagal sensory endings so as to mediate or control various pathological responses as well as food intake behaviour. Bucinskaite et al. (1997) have described a sensitization of the responses of gastric vagal afferent nerve fibres to CCK. Our results show the existence of a different mechanism whereby CCK sensitizes Il-1ß receptors on type 1 units. This is consistent with previous reports that CCK plays a role in the Il-1ß-induced excitation of gastric vagal afferents and with data showing that the responses of gastric vagal afferent nerves in CCK-A receptor-deficient rats were more sensitive to intravenous administration of Il-1ß than those of the control animals (Kurosawa et al. 1997, 2000). The present results on type 2 units are in agreement with the latter data, since CCK seems to be able to desensitize the Il-1ß receptors present on intestinal sensory endings. Our findings on the interactions between CCK and Il-1ß do not yield any information about the transductive mechanisms underlying the potentiation of the intestinal vagal mechanosensitive responses. However, interestingly, the fact that CCK is able to interfere with the effects of Il-1ß as well as with those of leptin 20 min after its application supports the hypothesis that CCK may affect membrane permeability: previous data obtained on the myenteric sensory neurones indicate that these changes in membrane permeability may have long-term effects (Clerc et al. 1999; Rugiero et al. 2003).
Interactions between Il-1ß and leptin
As established in our previous study (Gaigéet al. 2002) and confirmed by the present results, leptin induces an excitatory response which is enhanced by CCK in type 1 units, as well as an inhibitory response which is blocked by CCK in type 2 units. Il-1ra pretreatment blocks the excitatory effects of leptin in type 1 units, also providing evidence for the existence of functional links between leptin and Il-1ß. These findings are supported by numerous data showing that leptin is not only involved in the control of food intake and body weight, but also in regulating immune responses (Bik et al. 2001). Leptin receptors have been described in macrophages and granulocytes in the lamina propria of the rat jejunum (Lostao et al. 1998).
As regards the excitatory effects of leptin on type 1 units, it is possible that leptin may induce a release of Il-1ß, which in turn may activate the vagal sensory endings. Il-1ß and leptin have similar excitatory effects, which are potentiated by CCK and blocked by Il-1ra. The existence of functional links between leptin and Il-1ß has been corroborated by data showing that leptin induces Il-1ß transcript by binding to the leptin receptors present in glial cells in the brain (Hosoi et al. 2000), that the effects of leptin on food intake and body temperature are mediated by a release of Il-1ß (Luheshi et al. 1999) and that leptin up-regulates the production of pro-inflammatory cytokines both in vivo and in vitro (Loffreda et al. 1998).
As regards the inhibitory effects of leptin on type 2 units, our results show that these effects are not due to a release of Il-1ß, since the type 2 units are activated by Il-1ß. It is therefore possible that leptin may act directly on the vagal afferents, as suggested by several authors showing the existence of leptin receptors in vagal afferents (Buyse et al. 2001; Burdyga et al. 2002; Peiser et al. 2002). The fact that the inhibitory effects of leptin are enhanced in the presence of Il-1ß may be due to the fact that leptin is able to induce the secretion of Il-1ra, as found to occur in human monocytes by Gabay et al. (2001). The intensity of the inhibitory effects of leptin on type 2 units therefore varies depending on the Il-1ra levels.
As strongly suggested in a previous study (Gaigéet al. 2002), our present results confirm that at least two different types of intestinal vagal mechanoreceptors are involved in the effects of leptin and Il-1ß. It is worth noting that these units were both present in the same animal, which confirms that the various effects obtained were not due to the physiological condition of each animal. Although these two types of neurones cannot be differentiated in terms of their basal discharge frequency, morphology or conduction velocity (Harper & Lawson, 1985), they respond differently to mechanical stimulation and to various substances such as SP, PBG, CCK, leptin and Il-1ß (Gaigéet al. 2002). These stimuli may act either independently or together on neural endings. The responses to combined stimuli may differ from responses to single stimuli. The responses of these various units therefore depend on the chemical environment. Thanks to these characteristics, they are able to transmit a variety of information to the CNS, mainly about peripheral inflammatory states and ingestion.
Our findings confirm that single vagal afferent neurones possess a polymodal sensitivity, since they respond to mechanical stimulation and to endogenous administration of substances such as CCK, Il-1ß and leptin. This polymodal sensitivity is a general characteristic of the vagal mechanosensitive afferents (Schwartz et al. 1991). The results of the present study show that the information mediated by intestinal vagal afferent neurones is integrated first at peripheral level and secondly in the CNS, where it is used to produce the appropriate responses, such as variations in intestinal motility (Gaigéet al. 2003). Since type 1 and type 2 units are CCK-sensitive, they might be involved in the control of food intake. They are also Il-1ß-sensitive and might therefore also be involved in the transmission of information about peripheral inflammation to the CNS. The main difference between type 1 and type 2 units is that type 1 units require a release of Il-1ß to be responsive to leptin, whereas type 2 units do not. This difference may have some functional relevance. It is possible that type 2 units may generate acute satiety signals under physiological conditions whereas type 1 units may control food intake under pathological conditions (during inflammatory conditions, for instance).
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