Author:

Abstract:

GI functions, such as meal digestion, nutrient absorption and enzyme secretion, highly depend on the action of the enteric nervous system (ENS). The ENS influences and is influenced by appetite-related molecules, microbiota and possibly harmful agents, all of which is crucial to controlappetite and to maintain health. A well-functioning ENS is of key importance to a healthy life, and chronic impairment of ENS activity as observed in metabolic and inflammatory disorders, severely affects quality oflife and might even lead to central neurodegeneration. Thus, it is highly important to better understand ENS physiology in basal conditions as well as after dietary interventions. Therefore, the aim of the present doctoral project was to study the activity of enteric nerves during fasting (as mimicked by CORT incubation) and after a fructose diet; and to develop an approach to measure nutrient sensing in the ENS using life imaging techniques. We found that fasting results in elevated plasma levels of the hormone CORT, and increased expression of the CORT-converting enzyme 11β-HSD-1. In addition, we demonstrated that 11β-HSD-1 was expressed in myenteric neurons of mouse ileum. However, till now we were unable to identify whether or not a specific subclass of neuronsexpressed 11β-HSD-1. Interestingly, we showed that CORT decreased neuronal activity in the myenteric plexus of mouse ileum, as measured byCa2+-imaging. More specifically, incubation with CORT reduced EFS-induced Ca2+ amplitudes and response durations in myenteric neurons of mouse ileum; and induced the expression of modulators (synaptobrevin, CB1) important in the fine tuning of neurotransmission. Subsequently, in order to investigate how these results were related to functional changes, we studied small intestinal motility after CORT-incubation and showed that CORT reduced mixing movements in the ileum. In view of the increased addition of free fructose in soft drinks and industrialized foods, and the observed relation between fructose consumption and cognitive impairments in rodents, we studied the effects of a six-week fructose diet onneuronal activity in the murine ENS. In order to investigate if the fructose effects were reversible, we also included a ‘recovery’ group. We found that, caloric intake, body weight and blood glucose levels did not change between groups; however, mRNA expression levels of the specific fructose transporter GLUT5 were significantly decreased in the fructose-fed animals. Next, we showed that high K+-induced depolarization of duodenal submucous plexus neurons induced significantly lower Ca2+ amplitudesafter fructose consumption. This fructose-induced decrease in neuronal activity was also observed after 5-HT stimulation; moreover, the amount of 5-HT responders was significantly decreased in the fructose-fed mice.At the level of gene expression, no changes could be found in synaptobrevin, CaV2.2 and 5-HT3 receptor levels; while CaV2.1 levels were increased in the recovery group and SERT mRNA was reduced after fructose consumption. The rise in CaV2.1 mRNA expression after switching back to normalwater, suggests rebound compensation during the recovery period. Next, in view of the reported fructose-induced impairments in intestinal permeability, gene expression of tight junction proteins was measured, and Ussing chamber experiments were performed. Although fructose consumption reduced ZO-1 and occludin mRNA levels, permeability was not altered. Using a novel transgenic mouse that expresses the Ca2+ indicator GCaMP3 exclusively in neural crest derivatives, we developed a new technique to study nutrient sensing in the ENS. First, we showed that itwas possible to focus on the neuronal layers throughout the newly developed intact intestinal tissue preparations; and that the spontaneous activity of the neurons in these preparations was limited. Next, we found that, upon EFS-induced depolarization of the epithelial layer, neuronal responses were induced in the submucous plexus. By discriminating betweenthe intestinal regions, we showed that epithelial stimulation in the duodenum and the jejunum caused more submucous neurons to respond comparedto the ileum; whereas no responders could be found in the colon after EFS-induced stimulation of the mucosa. In addition, using luminal nutrient perfusion, we showed that only a fraction of the responders, induced by EFS, were activated by glucose or fructose; and only in the duodenum, overlap between the glucose and fructose responders could be found. Preliminary results showed that the SCFA acetate induced activity in 18.5 % of jejunal myenteric neurons, of which the majority had large cell bodies, reminiscent of Dogiel type II morphology. Finally, using a setup to inject 5-HT into one single villus, we demonstrated that 21.3 % of the studied myenteric neurons responded, and that the responding neurons were mainly the ones with a large diameter. In conclusion, the data obtained in this doctoral thesis provide new insights, which may be used toimprove dietary prevention strategies and therapeutical approaches in the context of food intake disorders and Western diet-related diseases. Furthermore, the new experimental techniques will allow us to dissect different circuitry (if any) that is activated upon addition of different nutrients, which will further improve our understanding of ENS physiology.