Preferential Entry of Botulinum Neurotoxin A Hc Domain through Intestinal Crypt Cells and Targeting to Cholinergic Neurons of the Mouse Intestine
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.