Figure 1.
Effect of BoNT/A on longitudinal isometric muscle contraction from mouse ileal segments.
(A) Spontaneous ileal contraction frequency as a function of time under control conditions (black colums), and after BoNT/A (105 LD50/ml) injection into the lumen of ligated mouse ileal segments (white columns). BoNT/A (105 LD50/ml) was preincubated with GST-SV2C/L4 (20 µg/ml for 30 min at room temperature prior to being inoculated into ligated intestinal loop. Examples of spontaneous contractions before (B), and after 4 h of BoNT/A treatment (C). Note that BoNT/A significantly decreased, but not completely the frequency and amplitude of the spontaneous contractile activity. Data are from 3 independent experiments. (D). Time-dependent reduction of electrically-evoked longitudinal muscle contractions after BoNT/A injection into intestinal lumen. (E) Representative continuous control recording showing spontaneous contractions before and after single electrical field stimulation (30 Hz for 25 s, indicated by the trace below the evoked recording). Note the changes in spontaneous contractions following the evoked one. (F) In ileal segment treated for 4 h with BoNT/A, carbachol (20 µM) applied to the external medium (arrow) induced a sustained muscle contraction, indicating that ACh receptors were still functional. The line segment above the tension record indicates the time of carbachol application.
Figure 2.
Binding of HcA to frozen sections of the mouse upper small intestine.
(A) Sections were overlaid with Alexa488-HcA and were counterstained with TRITC-Phalloidin. HcA staining (green) was observed in intestinal crypts, small cells in intestinal villi beneath the enterocytes, as well as in filaments from the submucosa. (B) Counterstaining with TRITC-UEA1 (red) labeling of Paneth cells in intestinal crypts and small cells in intestinal villi. (C) Counterstaining with anti-neurofilament (NF) antibodies and co-labelling (arrowhead) of neurofilaments with HcA in the submucosa. (scale bars = 50 µm).
Figure 3.
Visualization of HcA in the mouse intestine following inoculation into the intestinal lumen.
(A) HcA-Cy3 (0.5 µg) was injected into ligated ileum, duodenum, or jejunum loop. After 30 min of incubation, the samples were fixed and the mucosal fluorescence was measured as the number of HcA-positive cells per crypt. The number of labeled cells was similar between the three intestinal segments, but significantly lower (*) in the competition assay with BoNT/A (2.5 µg) in an ileal loop (Student's t test). (B) Detection of HcA (green) in the intestinal crypts and in some submucosa areas. No or only weak labeling is observed in the intestinal villi. (C) Visualization of HcA in intestinal crypt lumen (arrow-head) and inside some intestinal crypt cells (arrow), and (D) on filaments in the submucosa (black arrow head) after 30 min incubation. (E) HcA labeling of filaments (corresponding to neurofilaments, see Figure 7) in the musculosa after 90 min incubation. (scale bars = 20 µm).
Figure 4.
Visualization of HcA in neuroendocrine cells from mouse intestinal crypts.
Fluorescent HcA (0.5 µg) was injected into the lumen of a mouse ileum (A, B) or duodenum (C) loop, and after 30 min incubation the intestinal loop was washed and prepared for immunostaining with chromogranin A or serotonin antibodies. (A) Two cells stained with chromogranine A antibodies in an intestinal crypt (dotted circle) were co-labeled with HcA (green). Magnification of one cell (square) shows a uniform punctuate distribution of chromogranin A staining, whereas HcA was preferentially localized at the basal pole. Phase contrast shows that the chromogranin A-immunoreactive cell contained no large granules, in contrast to Paneth cell (black cross). (B) Co-labeling of a cell from an intestinal crypt (dotted circle) with HcA (green) and serotonin antibodies (red). Magnification of the cell (square) shows a basal distribution of both HcA and serotonin. Note that a cell extension was also labeled with serotonin antibodies but not by HcA (arrowhead), and that the serotonin-immunoreactive cell contained no large granules as in Paneth cells (black cross) (scale bars = 10 µm). (C) Co-labeling of HcA (red) with serotonin (green) in a duodenum crypt cell. Neurofilament staining (blue) was observed at the crypt periphery.
Figure 5.
Transcytosis of BoNT/A through intestinal and neuroendocrine cell monolayers, and SV2C-dependent entry of HcA into cells.
(A) Transcytosis of BoNT/A trough the intestinal neuroendocrine cell line STC-1, m-ICcl2, and Caco-2 cells. Cells were grown on filters (Transwell) until confluence. Integrity of cell monolayers was confirmed by ZO-1 labeling and non-permeability to FITC-labeled dextran (4300 Da). BoNT/A was added to the upper chamber and 60 min after incubation at 37°C, the toxin was assayed in the lower chamber by mouse bioassay. The results were expressed as the mean percentage (n = 9) of transcytosed BoNT/A corresponding to the ratio of mouse lethal dose 50 between the lower and upper well (inoculation titer). BoNT/A transport was significantly higher (*) in m-ICcl2 and in cells treated with anti-proteases (Student's t test). (B) Immunoblotting of cell lysates separated by SDS-PAGE with anti-chromograninA, anti-SV2A, anti-SV2B, anti-SV2C, and anti SV2C-L4 antibodies. (C) m-ICcl2 and STC-1 grown on glass cover slips were exposed to HcA-Cy3, alone or in combination with a 10-fold more molar concentration of SV2C/L4-GST for 10 min at 37°C. SV2C/L4 impaired HcA uptake in both m-ICcl2 and STC-1 cells. (D) The number of HcA fluorescent patches per µm2 in m-ICcl2 and STC-1 cells were reduced by 97% and 94% respectively (p<0.0001) by incubation with SV2C/L4. Each column represents the mean ± SEM (n = 250).
Figure 6.
Neuronal cell types recognized by HcA in the intestinal submucosa after its inoculation into the intestinal lumen (30 min incubation).
(A) Cells and neuronal structures labeled with HcA (green) were immunoreactive to anti-neurofilament (NF, red), indicating that HcA reached neuronal structures in the submucosa. Note that only few neurons and neuronal extensions were labeled with HcA (B) Co-staining with ChAT shows that some but not all ChAT-immunoreactive neurons were labeled with HcA. (C) Co-staining with anti-VIP antibodies showing no colocalisation with HcA. (D) Co-staining with anti-glutamate. Note that only a few neurons stained with anti-glutamate antibodies colocalized with HcA (scale bar = 20 µm). (E) Co-staining with anti-serotonin antibodies. Only few neurons stained with anti-serotonin antibodies colocalized with HcA. Note in the phase contrast in (E) that serotonin labeling was observed in some crypt intestinal cells and a few nerve endings in the submucosa. (F) Anti-SV2C and anti-neurofilament antibodies stained neurons and thin neuronal extensions in the submucosa. HcA labeled only certain nerve endings (arrows). Note that some SV2C-immunoreactive large cells with thicker extensions were neither stained with anti-neurofilament antibodies nor with HcA. (G) Quantification of colocalization between HcA and neurotransmitter markers in mouse intestinal submucosa. Results are expressed as colocalization index for which 1 represent 100% of colocalization between HcA and a neuronal marker. Colocalization index represents the ratio of the number of HcA positive terminal endings colocalized with one neurotransmitter marker to the total number of terminal endings labeled with HcA. Columns represent means ± SD accounting for at least 50 cells in three different experiments.
Figure 7.
HcA labeled cholinergic neurons in the musculosa after 90 min exposure in the intestinal lumen.
Co-staining with anti-ChAT and/or anti-neurofilament antibodies. Note that HcA preferentially stained neuronal cell extensions and to a lower extent cell bodies. (scale bar = 20 µm).