Alterations in mouse enteric nervous system activity and primary cilia localization across reproductive states

The enteric nervous system, an essential division of the peripheral nervous system, expands from the esophagus to the anus, containing an estimated 500 million neurons in humans and 500,000 in mice. Neurons and glia are organized in two ganglionated plexus: the myenteric plexus (formerly Auerbach’s plexus) situated between the longitudinal and the circular muscle layers, and the submucosal plexus that lies beneath the mucosa layer. The ENS is involved in multiple functions such as gastrointestinal secretion and absorption, digestion, muscle contraction activity, intestinal motility, regulation of blood flow, interaction with immune and endocrine system. Apart from these relatively independent tasks it also communicates with the central nervous system, a connection that is referred to as the gut-brain axis. Dysfunction of the ENS or the gut-brain axis can lead to gastrointestinal, neurological, and sometimes even psychiatric disorders. During pregnancy, the female physiology goes through numerous changes to adapt to the needs of both the mother and the developing fetus. Currently, it is not known to what extent the gut, and more specifically the ENS, is remodeled to accommodate this demanding situation. Pregnancy hormones, such as progesterone and estrogen, can affect the number and morphology of neuronal cells. Additionally, alterations in neurotransmitter levels can induce changes in intestinal motility, secretion, and absorption, in response to the new requirements of pregnancy. Therefore, pregnancy and lactation may well affect intestinal functions with an important impact on the overall well-being of the mother. Understanding how changes in the reproductive cycle affect ENS function is crucial to contribute to the broader knowledge of gastrointestinal diseases during pregnancy and to improve the treatment efficiency. To achieve this, we utilize virgin, pregnant and lactating Wnt1- GCaMP3 mice in which all enteric neurons and glia express a genetically encoded Ca2+ indicator (GCaMP3). This mouse strain enables us to conduct Ca!" imaging studies and activate enteric nervous cells with different stimuli. Additionally, we perform immunohistochemical analyses to study the localization, morphology and quantity of responding neurons and glia cells, as well as video recordings to evaluate gut motility. Given that the primary cilium, a small sensory cellular antenna of approximately 2-4 μm, is present on many progenitor cells capable of accommodating tissue changes, we aim to investigate its expression during the reproduction. Primary cilia are present in most mammalian cells including those in the ENS. They are non-motile structures composed of a microtubule-based cytoskeletal core known as axoneme, a basal body, and a membrane. Although their actual role remains unclear, primary cilia are likely involved in cellular signaling pathways, sensory function, and neurogenesis. Our goal is to better map the distribution of primary cilia within the gut and localize their presence in neurons and glial cells using immunostaining techniques.