Glial Cell–Derived Neurotrophic Factor Enhances Synaptic Communication and 5- Hydroxytryptamine 3a Receptor Expression in Enteric Neurons  Fanning Zeng, Robert P. Watson, Mark S. Nash  Gastroenterology  Volume 138, Issue 4, Pages 1491-1501 (April 2010) DOI: 10.1053/j.gastro.2009.11.048 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Colonic inflammation was induced in mice by supplementing drinking water with dextran sulfate sodium. After the indicated periods, colonic tissue was removed and messenger RNA was extracted. Gdnf expression levels were determined by quantitative PCR. Each data point contained 4–6 animals. There were significant increases in (A) interleukin (IL)-1β and (B) Gdnf expression levels in the inflamed tissues. *P < .05, **P < .01. Gastroenterology 2010 138, 1491-1501DOI: (10.1053/j.gastro.2009.11.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 (A) After 10 days in vitro, cultures were stained with anti–neurofilament 200 (green) and anti-synaptophysin (red) antibodies. GDNF cultures displayed a marked increase in neurite formation, as well as much denser staining for synapses. Scale bar, 50 μm. (B) Neurite outgrowth was visualized by anti-NF200 staining, and quantified with ImageJ/NeuriteTracer. Each group includes 20 images. ***P < .001. (C) Myenteric neurons were loaded with Fluo-4 and challenged with PMA. Upon PKC activation, [Ca2+]i oscillations in GDNF-treated cultures were robust and synchronized, whereas only small and nonsynchronous responses were detected in control cultures. Inset shows a magnified view of the boxed region. (D) Synchronous [Ca2+]i oscillations were blocked by 1 mmol/L hexamethonium and 1 μmol/L tetrodotoxin. Gastroenterology 2010 138, 1491-1501DOI: (10.1053/j.gastro.2009.11.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 (A) Up-regulation of Htr3a was shown by quantitative PCR. In GDNF-treated cultures, Htr3a expression levels increased approximately 4-fold (P < .05), whereas changes in Htr3b and Htr4 transcripts were not significant. Data were pooled from 3 independent experiments. (B) Htr3a transcript levels were measured in the colon tissues after the induction of inflammation, and were up-regulated significantly by day 7. (C) In situ hybridization of Htr3a in tissues from naive and inflamed colonic tissues, where Htr3a expression was observed only in myenteric or submucosal ganglia. Arrow indicates a submucosal neuron, arrowheads indicate myenteric ganglia. Data shown were generated using the 499–698 probe but identical data were obtained with the 241–555 probe. Sense probes for both showed no obvious staining. (D) Neurons were loaded with Fluo-4, followed by challenge with 100 μmol/L carbachol (cch nachr agonist), 50 μmol/L 5-HT, 50 μmol/L γ-aminobutyric acid, and 100 μmol/L ATP (P2X-receptor agonist). Traces represented the mean response (± standard error of the mean) of all the neurons in the field, identified by high K+ solution. Up-regulated responses to 5-HT were evident in neurons cultured with GDNF (n = 98–103). (E) 5-HT responses in GDNF neurons were blocked by 1 μmol/L ondansetron. (F) Bar chart shows the percentage of neurons that responded to 5-HT. Tetrodotoxin (ttx) indicates that the experiments were performed with 1 μmol/L TTX in bath solution. Data were pooled from 3 independent cultures each. *P < .05, **P < .01, ***P < .001. Gastroenterology 2010 138, 1491-1501DOI: (10.1053/j.gastro.2009.11.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 (A) LY294002 reversed GDNF effects on Ca2+ oscillations. Cultured neurons were challenged with PMA (as in Figure 2C). Synchronized [Ca2+]i oscillations were counted within 5 minutes of PMA application (data from 5 experiments). ***P < .001. (B) Cells were cultured with GDNF ± LY294002 for 7 days. Responses to different agonists were measured by Ca2+ imaging as in Figure 3D. 5-HT response remained unchanged in LY294002-treated neurons. Similar results were obtained with NVP-BEZ235, a dual PI3K and mammalian Target Of Rapamycin (mTOR) inhibitor. The data are summarized in panel C. (D) Application of 1 μmol/L ondansetron has no effects on PMA-induced [Ca2+]i oscillations. Gastroenterology 2010 138, 1491-1501DOI: (10.1053/j.gastro.2009.11.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 (A) No difference in A-type K+ currents were observed between control and GDNF-treated neurons (P > .05). Traces represent the mean value of IA from 11–13 neurons. The insert shows the protocol used to elicit A-type K+ currents. (B) IK was suppressed in GDNF-treated neurons (P < .01). Traces represent the mean value of IK, and the standard errors were expressed as the shaded area. (C) Representative traces from control (top) and GDNF-treated (bottom) neurons. Control neurons fired more APs upon a train of current injections (n = 13–14). The data are summarized in panel D. (E) Superimposed APs from both groups. APs in GDNF (red) were significantly wider than those in control (black). Data (width at half maximum) are summarized in panel F. ***P < .001. Gastroenterology 2010 138, 1491-1501DOI: (10.1053/j.gastro.2009.11.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 (A) LY294002 did not rescue IK in GDNF-treated neurons. Currents were measured after 8–9 days in culture. P value was calculated against the control (n = 11–13 each). (B) IK was measured in control (black) and GDNF-treated (gray) neurons on day 1 and day 7. IK was inhibited nearly 50% in GDNF cultures by day 7, whereas IK in control neurons remained the same (P > .05). (C) Cells were loaded with Fura 2, and responses to 5-HT application were measured by Ca2+ imaging. GDNF treatment increased responses to 5-HT over the culturing period, whereas no change was observed in controls. *P < .05, ***P < .001. Gastroenterology 2010 138, 1491-1501DOI: (10.1053/j.gastro.2009.11.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 (A) TEA blocks IK. Traces show the mean value of IK before (black) and after (blue) 5 mmol/L TEA application. (B) Bar chart shows the number of APs generated upon current injection (protocol as in Figure 4C and D). (C) Superimposed APs before (black) and after (blue) TEA application. (D) Cells were cultured under the conditions indicated. Responses to 5-HT were measured as in Figure 6C. TEA increased 5-HT in both control and GDNF-treated cultures, however, the BKCa channel opener attenuated responses to 5-HT. (E) Cells were cultured with 5 mmol/L TEA for 7 days and Htr3a transcript levels were measured. Compared with controls, a moderate but significant increase of Htr3a expression was observed (P < .05; n = 4). *P < .05, ***P < .001. Gastroenterology 2010 138, 1491-1501DOI: (10.1053/j.gastro.2009.11.048) Copyright © 2010 AGA Institute Terms and Conditions