Citation

Maternal obesity is a rapidly growing problem in the United States as only 2 out of 5 pregnant individuals enter gestation at a normal weight, with over 30% reporting obesity pre-pregnancy. In children born to obese mothers, we see metabolic dysfunction in high rates of childhood obesity, adulthood obesity, and cardiovascular disease. Additionally, there is evidence of neurological dysfunction in these offspring, including increased rates of anxiety and depression, autism spectrum disorder, and ADHD. Turning to animal studies, where investigators have exposed the offspring to a high fat diet during the perinatal period in the absence of obesity, we see a similar phenotype emerge; weight gain, fat deposition, anxiety-like behavior, depressive-like behavior, and stress sensitivity. This combination of metabolic and neurologic pathologies led us to investigate vagal neurocircuits, as they display plasticity to environmental changes, develop and refine during the perinatal period, play roles in mediating feeding behavior, and induce gastric pathologies that have significant comorbidities with anxiety and mood disorders. Vagal control of the gastrointestinal tract is involved in modulating gastric motility, including gastric emptying rates. The adaptability of these vagal neurocircuits to modulate gastric motility after environmental changes is important in maintaining proper digestion and avoiding the consequences of gastroparesis such as nausea, bloating, and abdominal pain. To investigate how vagal neurocircuits, and their subsequent control of gastric motility, may be altered following perinatal high fat diet (pHFD) exposure, pregnant rat dams were put on either a control (14% kcal from fat) or high fat (60% kcal from fat) diet starting on embryonic day 13, when vagal neurocircuits begin to develop. The mothers remained on the respective diets while nursing, and the pups were aged until at least postnatal day 28 (P28) before use in subsequent experiments. A combination of in vitro electrophysiology, in vivo gastric motility recordings, gastric emptying assays, and neuronal tracing experiments were performed to investigate two major aims; how pHFD exposure causes intrinsic changes to DMV neurons (aim 1) and extrinsic innervation (aim 2) to vagal neurocircuits, altering neural activity that leads to loss of gastric control. For the first aim, I investigated how pHFD exposure led to changes in neuronal activity within the dorsal motor nucleus of the vagus (DMV), the nucleus containing vagal motor neurons that project to the stomach and modulate motility. These neurons are under tonic GABAergic control from the NTS to regulate the rate these neurons fire. GABA is inhibitory at this synapse as it acts through the GABAA receptor, allowing negatively charged chloride (Cl-) ions to enter the cell, leading to a hyperpolarization and decrease in firing activity. This is made possible because the Cl- gradient is set by K+-Cl- cotransporter (KCC2), a co-transporter that keeps extracellular Cl- levels high. Loss of this transporter would make GABA less inhibitory and reduce its ability to modulate DMV neuronal activity. Using cell-attached and perforated patch clamp electrophysiology, I determined that GABA was indeed less inhibitory on DMV neurons from pHFD-exposed rats, and pharmacological blockade of the KCC2 transporter reveled a loss of KCC2 activity. To rescue this neurologic and gastric phenotype and directly test causality, I used viral transfection techniques to insert KCC2 directly into DMV neurons of pHFD rats. This restored GABA’s ability to inhibit these neurons and accelerated their gastric emptying rates. Lastly, developmentally promoting activation of TrkB receptors via treatment with the agonist elevated KCC2 expression, restored GABAergic inhibition, and accelerated gastric motility. These results would suggest that pHFD exposure leads to a downregulation of KCC2 levels, potentially through reductions in BDNF-TrkB signaling, impairing the ability to modulate these vagal neurocircuits and leading to gastroparesis. For my second aim, I investigated how pHFD exposure altered projections from the paraventricular nucleus of the hypothalamus (PVN) to the DMV. The PVN contains both oxytocin (OXT) and corticotropin releasing factor (CRF) neurons, both innervating the DMV and playing important roles in modulating gastric motility in response to stress. I performed neuronal tracing experiments, which uncovered a loss of PVNOXT neurons projecting to the DMV in pHFD rats. Using in vivo gastric motility recordings, in which I fitted a miniature strain gauge onto the corpus muscle of the ventral stomach of an anesthetized rat to measure contractile activity, I found that injection of OXT into the DMV led to a contraction in the stomach of pHFD rats, the opposite of what we see in controls. Further investigation, using in vitro whole cell patch clamp electrophysiology, revealed that there is tonic release of CRF onto pHFD-exposed DMV neurons that when blocked, normalized the cellular response to OXT. Similarly, when I blocked CRF receptors in the DMV and then injected OXT during motility recordings, I was able to induce a gastric relaxation. Taken together, these results suggest that pHFD exposure leads to a loss of PVNOXT innervation to the DMV and subsequent changes to gastric motility driven by tonic PVNCRF input. These studies have allowed us to uncover the mechanistic basis for gastric dysfunction observed in response to pHFD exposure. I combined established experimental techniques such as perforated and cell-attached patch clamp electrophysiology, in vivo gastric motility recordings, and gastric emptying assays to answer these novel experimental questions, from the level of the single neuron to behavioral outcomes in the freely moving animal. The combined results of these studies demonstrate neurodevelopmental changes in pHFD-exposed animals that may be leading to the pathology we see in human populations, including gastric motility disturbances, metabolic dysfunction, and high rates of anxiety and mood disorders. Additionally, loss of this vagal control of gastric functions is likely to exacerbate these neurological and metabolic changes, positioning itself to be a potential therapeutic target. Lastly, we uncovered these neurodevelopmental changes because of pHFD exposure in the absence of maternal obesity, making these results applicable to all future mothers.