Figure 5. TeNT activity in the brainstem after injection in the WP rapidly spreads to nuclei controlling vital functions, including respiration.

(A) TeNT was injected in the left WP (1 ng/kg in a final volume of 1 μL) that caused the appearance of cl-VAMP (red) at the level of different brainstem areas: by day 1 the paragigantocellular reticular nucleus (PGRN), involved in the regulation of respiratory and autonomic cardiovascular functions, and by day 3 trigeminal motor (TM), hypoglossal (HN), and ambiguus (NA) nuclei, controlling mastication, swallowing, and the upper airways (larynx and pharynx), respectively; scale bars, 500 μm. (B) Scheme illustrating the experimental setup used to measure the intraesophageal pressure in living mice, which provides an accurate air volume exchanged by the animal during the respiratory cycle: a buttoned needle connected to a pressure sensor is inserted in the mouse esophagus to measure the pressure; the signal is amplified and digitalized by computer. (C) Respirograms from naive (top trace) and TeNT-treated mice 1 day (central trace) and 3 days (bottom trace) after WP injection. Each trace deflection reports the pressure variations occurring during a single respiratory act, which highlight the progressive reduction in the air volume exchanged during CT; 1 day after TeNT, when VAMP cleavage is confined in the FN, few, if any, changes are present compared to naive respiration; at day 3 deflections at each respiratory act appeared markedly reduced, suggesting a deterioration in the ability of the mouse to breathe. (D) Quantification of the respiratory ability reported as “inferred ventilation index” (IVI), calculated as the overall volume of air exchanged by the animal over 20 seconds (see Methods); means ± SD; ****P < 0.0001 assessed by 1-way ANOVA with multiple comparisons and Bonferroni’s test. The analysis was done with 4 animals per time point.