Date Published: February 22, 2019
Publisher: Public Library of Science
Author(s): Michela Bistoletti, Valentina Caputi, Nicolò Baranzini, Nicoletta Marchesi, Viviana Filpa, Ilaria Marsilio, Silvia Cerantola, Genciana Terova, Andreina Baj, Annalisa Grimaldi, Alessia Pascale, Gianmario Frigo, Francesca Crema, Maria Cecilia Giron, Cristina Giaroni, Kenji Hashimoto.
Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity.
Numerous studies have established that the naturally occurring commensal microbiota, which in the human gut is composed of about 3.8X 1013 bacterial cells belonging to approximately 2000 species, represents an essential organ for the host homeostasis by contributing to the metabolism of nutrients, development of the immune host defence and maturation of the gastrointestinal (GI) tract . The complex array of cellular elements constituting the enteric microenvironment (enterocytes, enteroendocrine cells, immunocytes, smooth muscle cells, interstitial Cajal cells, enteric neurons and glial cells) responds to microbial factors, mainly via pattern recognition receptors (e.g. Toll-like receptors, TLRs) [2,3], neurotransmitter, neuropeptides and neurohormone receptors [4–6]. The commensal gut microbial flora influences the development and function of the enteric nervous system (ENS), which consists of two complex interconnected neuroglial networks, constituting the submucosal and myenteric plexus . Microbiota-induced neuronal plasticity is, however, not limited to the ENS but can potentially activate responses in the central nervous system (CNS), via activation of neuroendocrine and metabolic pathways, along the microbiota-gut-brain axis . Several preclinical studies, carried out in animals fed with specific dietary regimens and in transgenic animals or germ-free (GF) rodents, have shown that dysfunction of the gut microbiota during early life (infancy, childhood and adolescence) has important consequences, not only on the normal gut functions, but also on the brain and behaviour, including pain perception, stress response and anxiety . During adolescence neurons undergo crucial structural, neurochemical and molecular changes in response to genetic and environmental signals both in the CNS and in the ENS [10,11]. During this stage of life, the symbiotic microbial flora experiences dynamic processes, such as changes in the composition and relative abundance of various microbial constituents, which may influence neuronal development [11,12]. Environmental insults, such as the use of antibiotics, stress, harmful events and poor diet, may result in dysbiosis-induced neuronal vulnerability both in the ENS and CNS. This neuronal susceptibility to microbiota alterations contributes to the onset of chronic functional GI disorders, such as irritable bowel syndrome (IBS), which is associated with psychological distress and psychiatric comorbidity, including depression [9, 13]. From this perspective, it is important to evaluate whether changes in the symbiotic microbial flora during adolescence may influence the expression of some factors involved in neuronal development and plasticity both in the ENS and CNS. In this study, we have focused on the role of brain-derived neurotrophic factor (BDNF) and its high affinity receptor tropomyosin-related kinase B (TrkB). BDNF plays a central role in promoting neuronal survival and growth, synaptic plasticity and reinforcement of synaptic communication [14,15]. In the CNS, the neurotrophin is a key molecule influencing mood, behaviour and cognitive functions, such as learning and memory, and any alteration of its levels are related to development of psychiatric disorders, such as anxiety and depression [16,17]. The normal gut microbiota influences the expression of BDNF in brain regions crucially involved in the development of correct behavioral patterns, such as the hippocampus and cortex [18,19]. A relationship between development of behavioral disorders, such as anxiety and cognitive deficits, and altered BDNF levels in different CNS regions was demonstrated both in GF mice and in adolescent mice after antibiotic treatment-induced dysbiosis [20–22]. In the GI tract, BDNF is released from different cell types, including enterocytes, enteric glial cells and neurons and plays a fundamental role in modulating both sensory and motor functions [23–26]. BDNF represents a neurotransmitter/neuromodulator in enteric neuronal circuitries, favouring the local release of enteroendocrine molecules and neurotransmitters from sensory and motor neurons, enhancing peristalsis and gut motility both in laboratory animals and in humans [23,24,27,28]. The neurotrophin plays also a modulatory role on visceral pain perception, as demonstrated in studies carried out on rat models of IBS showing that BDNF may favour the development of visceral hypersensitivity by activating both intrinsic and extrinsic primary afferent neurons [25,29]. In addition, BDNF released from dorsal root ganglia and spinal cord contributed to the development of exaggerated visceromotor responses to colorectal distension in a rat model of colitis . In spite, of the central role played by BDNF in the microbiota-gut-brain axis, there are no reports, at least up to our knowledge, concerning the occurrence of BDNF and TrkB expression changes in the ENS, caused by alterations in the gut commensal microflora during adolescence. The demonstration of such relationship would be, even more, interesting since, in the ENS of juvenile mice, important rearrangements of both excitatory and inhibitory neurotransmitter pathways regulating intestinal motility occur after antibiotic-induced dysbiosis . Thus, the aim of this study was to evaluate the expression of BDNF and TrkB in the ENS of juvenile mice after a massive chronic antibiotic treatment, by means of morphological and molecular biology approaches. In view of the ability of the gut microbiota to influence neurotrophin expression in the CNS, we also investigated BDNF and TrkB levels in the hippocampus and prefrontal cortex.
In this study, we provide evidence that a 2-week antibiotic treatment carried out in juvenile mice induces changes in the expression of BDNF and of its high affinity receptor, TrkB, in both the ENS and CNS. However, such alterations are different along the gut-brain axis, since BDNF and TrkB levels are up-regulated in the ENS, downregulated in the hippocampus and unmodified in the prefrontal cortex, suggesting that dysbiosis may predispose to regionally differentiated effects on BDNF signaling. The observed changes are closely superimposable to BDNF alterations observed in animal models of IBS and IBS patients alike, suggesting that an antibiotic treatment in adolescence may predispose to functional gut disorders later in life [13,39]. A two-week course of high‐dose, broad-spectrum, poorly-absorbable antibiotics is a cost‐effective model to characterize the many effects of gut dysbiosis on ENS and CNS structure and function, as shown previously by us and others [3,31,40,41]. However, we have recently reported that ABX treatment in juvenile mice determines a marked reduction in bacterial loads in mouse feces  and, consistent with previous observations, the faded commensal stimulation impacts gut neuromuscular function, brain anxiety and cognitive behaviors as well as key neuroimmune modulators of gut-brain dialogue in a manner comparable to that described in germ-free mice .
In summary, the present data suggest that a dysbiosis induced by a massive antibiotic treatment during adolescence may alter BDNF and TrkB expression both in the ENS and CNS, although with different outcomes in the two nervous systems, later in life. We cannot exclude that these molecular changes may contribute to alter specific neurotransmitter pathways, involved in the development of functional gut diseases, such as IBS. These results further strengthen the concept that appropriate manipulation of the gut microbiome during adolescence, may reduce the probability of developing severe disorders that could affect the gut and the CNS later. This is all the more interesting in view of the worldwide increasing incidence of functional gastrointestinal disorders affecting the adolescent and young population [64,65].