Volume 128, Issue 5, Pages 1317-1326 (May 2005) Serotonin Excites Neurons in the Human Submucous Plexus via 5-HT3 Receptors  Klaus Michel, Florian Zeller, Rupert Langer, Hjalmar Nekarda, Dagmar Kruger, Terri J. Dover, Catherine A. Brady, Nicholas M. Barnes, Michael Schemann  Gastroenterology  Volume 128, Issue 5, Pages 1317-1326 (May 2005) DOI: 10.1053/j.gastro.2005.02.005 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 5-HT had an excitatory effect on a subset of human submucous neurons. (A) Fluorescent image of a submucous ganglion taken with the CCD camera from the NeuroCCD system at a resolution of 70 × 70 pixels. Individual neurons of the ganglion can be distinguished by the strong staining of the outer membrane; all cell bodies are numbered from 1 to 5. (B) Cell counts were verified by staining with the pan-neuronal HuC/D, which labeled 5 neuronal cell bodies. The change in fluorescence intensity is shown in D for 3 of these neurons. (C) Position of the CCD detectors in 2 × 2 binned mode, which was used for the measurement. In this graph the signals of the CCD detectors are shown in an extremely condensed mode. The outline of the ganglion shown in A can be seen. Because with this magnification many detectors record the activity of 1 nerve cell the data from the detectors that monitor the activity of individual neurons were binned to give the traces shown in D. (D) The expanded traces from CCD signals show the responses of neurons 1, 4, and 5 to pressure applications of 600 ms 5-HT (black bar). Two neurons showed a discharge of action potentials, whereas neuron 5 showed no effect. (E) Responses of the same neuron to single-pulse electrical stimulation of interganglionic fiber tracts, 5-HT, and 2-methyl-5-HT. Electrical stimulation evoked 2 responses: the first signal is a compound action potential arising from nerve fibers and the second response is a fast excitatory postsynaptic potential—triggered action potential. A pressure application of 5-HT (800 ms) or 2-methyl-5-HT (600 ms) onto the same neuron revealed similar responses (action potential discharge) that lasted throughout the recording period of 1.26 s. (F) The transient rapidly desensitizing 5-HT3—mediated response. The first application of 2-methyl-5-HT evoked a response that lasted for 288 ms discharging 7 action potentials. Partial recovery occurred 210 s after the first application with a response duration of 187 ms discharging 3 action potentials. Recovery of the response 470 s after the first application with a response duration of 313 ms discharging 5 action potentials. Gastroenterology 2005 128, 1317-1326DOI: (10.1053/j.gastro.2005.02.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 5-HT3 receptor activation did not evoke an increase in Isc (chloride secretion) in human mucosa/submucosa preparations (Ussing chamber experiment). (A) Serosal application of 10 μmol/L 2-methyl-5-HT (▴) had no effect on Isc in a human colon preparation whereas electrical stimulation of the preparation (arrows) before and after 2-methyl-5-HT evoked a transient increase in Isc, showing the viability of the preparation. (B) In contrast, serosal application of 10 μmol/L 2-methyl-5-HT (▴) onto guinea pig proximal colon evoked a prominent increase in Isc. The tissue also was stimulated electrically before and after the application of 2-methyl-5-HT (arrows). Note the different scale bars for Isc in A and B. (C) A human colon preparation did not respond to 2-methyl-5-HT (▴) but showed a prominent increase in Isc after addition of the nicotinic agonist DMPP (▵). Gastroenterology 2005 128, 1317-1326DOI: (10.1053/j.gastro.2005.02.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Immunohistochemical demonstration of the 5-HT3A and 5-HT3B receptor subunits. (A) Immunolabeling of a ganglion in the human submucous plexus with an antibody against the 5-HT3A subunit. (B) The same ganglion as in A stained with an antibody against the human neuronal protein HuC/HuD, which labels all neurons. All anti-HuC/HuD—positive neurons are immunoreactive for the 5-HT3A antibody. (C) Immunolabeling of another human submucous ganglion with an antibody against the 5-HT3B subunit. (D) The same ganglion as in C stained with anti-HuC/HuD. All anti-HuC/HuD–positive neurons are immunoreactive for the 5-HT3B antibody. Scale bar in A applies also for B–D. (E) Ability of antibody MSD-3A (1:500) and AP86/3 (1:500) to recognize selectively the h5-HT3A and h5-HT3B subunit, respectively, in HEK293 cells stably expressing the h5-HT3A subunit (HEK-5-HT3A) or both the h5-HT3A and h5-HT3B subunits (HEK-5-HT3A/3B) or HEK293 cells transiently transfected with the h5-HT3B subunit (HEK-h5HT3B) or untransfected HEK293 cells (HEK293). All pictures for a given primary antibody result from the same exposure. Typical fields are displayed from a single experiment that was repeated 3 times. Scale bar applies to all panels. (F) Detection of h5-HT3B–like immunoreactivity by SDS polyacrylamide gel electrophoresis/Western blot. Lane 1, HEK293 cells stably expressing with h5-HT3A subunit (5.0 μg protein); lane 2, HEK293 cells stably expressing both the h5-HT3A and h5-HT3B subunits (5.0 μg protein); lane 3, large intestine (13.0 μg protein); lane 4, small intestine (13.8 μg protein). Data shown are from a single representative experiment that was repeated 3 times. Gastroenterology 2005 128, 1317-1326DOI: (10.1053/j.gastro.2005.02.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Detection of h5-HT3A and h5-HT3B mRNA by RT-PCR. RT-PCR products resolved on 1% agarose gels using primers specific for h5-HT3A subunit (top) and h5-HT3B subunit (bottom) transcripts. Lane 1, muscle layers/myenteric plexus; lane 2, mucosa; lane 3, submucosa/submucous plexus; lane 4, HEK293 cells stably expressing the h5-HT3A subunit (top) or HEK293 cells stably expressing the h5-HT3A and h5-HT3B subunits (bottom); lane 5, no template. Results shown for each reaction are from a single representative experiment repeated at least 3 times (predicted size of RT-PCR products; h5-HT3A 555 bp and h5-HT3B 445 bp are consistent with the sizes resulting from HyperLadder I (Bioline, London, UK); far left lane for each gel). Gastroenterology 2005 128, 1317-1326DOI: (10.1053/j.gastro.2005.02.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions