## 3 Results
### Distribution of Serotonergic Neurons in the Myenteric Ganglia of Controls
The insulin treatment reduced the proportion of 5-HT-IR neurons that are similar to the control level. Data are expressed as mean \(\pm\) SEM. **_p_ < 0.01 and ****_p_ < 0.0001 (between controls and diabetics); \({}^{\circ}\)_p_ < 0.05 and \({}^{\circ\circ\circ\circ}\)_p_ < 0.0001 (between diabetics and insulin-treated diabetics). C—controls; D—diabetes; and ID—insulin-treated diabetics. Figure 5: Representative fluorescent micrographs from the duodenum (**a**), ileum (**b**), and colon (**c**) of insulin-treated diabetics after 5-HT-HuCD immunohistochemistry. On the myenteric plexus whole-mount preparations, asterisks indicate neurons that are labelled for HuCD and arrowheads point to myenteric 5-HT-IR varicosities. Scale bars are 20 \(\upmu\)m.  
Proportion of Myenteric ganglia Containing 5-HT-IR Neurons and the Amount of the Serotonergic Neurons in These Ganglia  
In controls, the total number of 5-HT-IR neurons per ganglia is extremely low and varied between 0.31 \(\pm\) 0.06 and 0.46 \(\pm\) 0.1 in the three investigated gut segments (Figure 6). However, only 25-30% of the myenteric ganglia contain serotonergic neurons (Figure 7), and the number of 5-HT-IR neurons is 1-2 in these ganglia (Figure 8). Figure 6: Number of 5-HT-IR neurons of the duodenum, ileum, and colon of controls, diabetics, and insulin-treated diabetics. The total number of 5-HT-IR myenteric neurons was elevated in each gut segment of diabetics. In insulin-treated diabetic rats, the total number of 5-HT-IR neurons remained close to the control level. Data are expressed as mean \(\pm\) SEM. **\(p\) < 0.01 (between controls and diabetics); \({}^{\circ\circ}\)\(p\) < 0.01 (between diabetics and insulin-treated diabetics). C—controls; D—diabetics; and ID—insulin-treated diabetics. Figure 7: Percentage of myenteric ganglia containing 5-HT-IR neurons. In controls and insulin-treated diabetics, 20–30% of the myenteric ganglia contain 5-HT-IR neurons. In diabetic rats, a gut segment-specific increase was observed with the most pronounced elevation in the colon where 50% of the myenteric ganglia contain 5-HT-IR neurons. C—controls; D—diabetics; and ID—insulin-treated diabetics. In diabetics, the total number of 5-HT-IR neurons per ganglia increased in each gut segment; however, the increase was significant only in the colon (_p_ < 0.01) where the number of 5-HT-IR neurons was doubled relative to the controls (1.05 \(\pm\) 0.14 vs. 0.46 \(\pm\) 0.1; Figure 6). In the diabetic colon, half of the ganglia contained serotonergic neurons (Figure 7) where an average of two cells were found (Figure 8). A significant elevation of the total number of 5-HT-IR neurons per ganglia containing serotonergic neurons was observed in the duodenum compared to the controls (2.13 \(\pm\) 0.21 vs. 1.25 \(\pm\) 0.1; Figure 8). The immediate insulin treatment resulted in low levels of 5-HT-IR hyperenteric neurons similar to the controls in each intestinal segment in all investigated aspects such as the total number of serotonergic neurons per ganglia (Figure 6), the percentage of hyperenteric ganglia containing serotonergic neurons (Figure 7), and the total number of 5-HT-IR neurons per serotonergic ganglia (Figure 8).