Fig 1.
A. Schematic representation of the brainstem at the DVC level showing the central pattern generator of swallowing (SwCPG) and the solitary tract (ST). Coronal diagram of the medulla drawn from the atlas of Paxinos and Watson [27] corresponding to the level of glutamate and DON injection sites (between 0.2 and 0.5 mm in front of the caudal edge of the area postrema and 0.5 to 0.7 mm lateral) and to the level of stimulation of the solitary tract (ST). Although microinjections and ST stimulations were made on either side of the medulla, all injection sites have been reported to the left side and dotted in the hatched area corresponding to the region of the interstitial and intermediate solitary tract nucleus (NTS) subnuclei. Histological micrographs illustrating the stimulating ST site show the bipolar electrode tract (see *). Amb, ambiguous nucleus; AP, area postrema; OL, inferior olive; RVL, rostroventral reticular nucleus; SpT5, spinal trigeminal tract; ST, solitary tract; 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus; 12n, hypoglossal nerve. B. Parameters recorded in the rat during the experiment. Stim, stimulation of the superior laryngeal nerve (SLN), at 15 pulses/s during 5 s (pulse parameters: 2.5V and 0.06ms); EMG, sublingual muscle electromyogram (note rhythmic swallowing triggered by SLN stimulation); ECG, electrocardiological activity; Resp, respiration (note respiration blockade during SLN stimulation), ∫EMG, electromyogram envelope signal normalized, Sw Number, number of swallows triggered by SLN stimulation; ECG Frequency and Resp Frequency; “o” above the recording are event markers for ECG, Resp and EMG. Note that ST stimulation produced similar responses.
Fig 2.
Intravenously injected DON inhibited rhythmic swallowing triggered by SLN stimulation.
A. Typical sublingual muscle electromyogram (note the rhythmic swallowing induced by SLN stimulation). DON (3mg/kg bw) injection induced a rapid and powerful inhibition of the number of swallows. 3 minutes after DON application, only three swallow were triggered by SLN stimulation. B. Electrocardiogram and respiratory activity recorded before, during and after SLN stimulation. Note the blockade of respiration induced by swallowing during SLN stimulation in control condition and at recovery 74 min after DON injection.
Fig 3.
Dose-dependent DON inhibition of rhythmic swallowing triggered by SLN or ST stimulation.
Time course of the effect of various DON doses intravenously injected: 3mg/kg bw (16 trails; A), 1.5mg/kg bw (7 trials, B) and 0.3mg/kg bw (9 trials; C) on the number of swallows recorded over 5 minute periods and until recovery. On the right side of A and B, note a focus on the kinetics of DON effect over the first 5 min. Black bars represent the mean value of the control recorded before DON injection (C, 100%). Data are represented as means ± SEM normalized to the control value. D: Comparison of the inhibitory effects of the three doses of DON on rhythmic swallowing triggered by SLN or ST stimulation. Note that time course variations according to the doses showed a significant inhibitory effect for DON 3mg/kg bw, compared with the other doses, beginning 35 minutes after injection and lasting 40 minutes. *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
Table 1.
Effects of DON injection on the real number of swallows triggered by SLN (or ST) stimulation.
Fig 4.
Effects of DON intravenous administration on c-Fos immunoreactivity.
Representative coronal sections illustrating the c-Fos labeling observed in the brainstem (A, B, C: NTS, D: RVL, E: OL) of I) control rats intravenously treated with NaCl (0.9%) and sacrificed 2h post-treatment, II) rats intravenously treated with DON (3mg/kg bw) and sacrificed 2h post-treatment. III) Quantification of the number of c-Fos immunoreactive nuclei in the brainstem observed in rats treated either with DON (3mg/kg bw) or NaCl (0.9%). *P<0.05, **P<0.01 significantly different from NaCl-treated rats. Box explanation: upper horizontal line of box, 3rd quartile; lower horizontal line of box, 1st quartile; color separation within box, median; upper horizontal bar outside box, max; lower horizontal bar outside box, min. 4V, fourth ventricle; AP, area postrema; cc, central canal; NTS-C, caudal part of the NTS; NTS-R, rostral part of the NTS; NTS-P, postremal division of the NTS; OL, inferior olive; RVL, rostroventral reticular nucleus. Scale bar: 100 μm.
Fig 5.
DON (570ng/kg bw; 10 trials) microinjection within the central pattern generator of swallowing (SwCPG) inhibited rhythmic swallowing triggered by SLN stimulation.
A. Electromyogram from sublingual muscles (note the rhythmic swallowing induced by SLN stimulation). DON microinjection at 570ng/kg bw induced a rapid and powerful inhibition of the number of swallows. 30 seconds after DON application, no more swallow was triggered by SLN stimulation. This effect was transient since the swallows recovered after 45 minutes. B. Electrocardiogram and respiratory activity recorded during 30 seconds (before, during and after SLN stimulation). Note the blockade of respiration induced by swallowing during SLN stimulation in control condition and at recovery 45 min after DON injection.
Fig 6.
DON microinjection within the central pattern generator of swallowing (SwCPG) inhibited rhythmic swallowing triggered by SLN or ST stimulation in a dose depend manner.
Time course of the effects of various doses of DON: 570ng/kg bw (10 trials; A), 150ng/kg bw (10 trials; B), 60ng/kg bw (10 trials; C) on the number of swallows recorded over 5 minute periods (for 570ng/kg bw and 150ng/kg bw) or over 30 second periods (for 60ng/kg bw). On the right side of A and B, note a focus on the kinetics of DON effect over the first 5 min. Black bars represent the mean value of the control recorded before DON injection (C, 100%). Data are represented as means ± SEM normalized to the control value. Note time course variations according to the doses showing a significant inhibitory effect immediately after DON microinjection for 570ng/kg bw and 150ng/kg bw. At 60ng/kg bw, DON microinjection did not modify rhythmic swallowing triggered by SLN stimulation. D: Comparison of the effects of the three doses of DON during the first 25 minutes after injection. Note the powerful effect of 570ng/kg bw and 150ng/kg bw DON doses. *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.