Neonatal Antibiotics Disrupt Motility and Enteric Neural Circuits in Mouse Colon.

Early life antibiotics and microbiota alterations are linked to increased susceptibility to gastrointestinal (GI) diseases commonly associated with enteric neuropathy and dysmotility.1, 2 The GI tract houses a complex microbiota ecosystem that interacts with the enteric nervous system (ENS). The early postnatal period is a critical window for functional coupling between the developing ENS and microbiota,2, 3 but little is known about the bidirectional signaling or the impact of early life antibiotics on gut pathophysiology. Although there are reports of microbiota regulating the ENS,4, 5, 6 there are no systematic studies of the effects of antibiotics on the neonatal ENS in a more clinically relevant, non–germ-free state.

Antibiotics, including vancomycin, often are administered to preterm infants to prevent GI diseases, such as necrotizing enterocolitis. To investigate the impact of early life antibiotics on gut microbiota and ENS development, we administered vancomycin orally to neonatal mice to reduce systemic effects and assess its impact on the ENS and gut motility. Vancomycin or water was given to pups in each litter, from birth to postnatal day (P)10. By P10/P11, microbiota composition and diversity were altered significantly in the small and large intestines. This included significant increases in the relative abundance of Firmicutes at the expense of Bacteroidetes and Proteobacteria (Figure 1A), and perturbations of gram-positive and gram-negative families of bacteria (Supplementary Figure 1G). In vitro spatiotemporal mapping methods showed increased frequency and anal propagation speed of colonic contractions of vancomycin-treated pups compared with control littermates (Figure 1B–D). Increased colonic motility in vancomycin-fed pups involved significant changes in the composition and function of colonic myenteric neurons (Figure 2). The density of colonic myenteric cells expressing the pan-neuronal marker, Hu, was notably reduced after vancomycin treatment, whereas S100β+ glial density was unaffected. Moreover, proportions of neuronal nitric oxide synthase (nNOS) neurons were reduced, but proportions of calbindin neurons, a subtype of cholinergic neurons, were increased. In contrast, no neurochemical or motility differences were found in the duodenum (Supplementary Figure 2A–C). The unaffected duodenal ENS suggests that acute drug neurotoxicity is not the major effector.

Figure 1

Neonatal vancomycin alters microbiota composition and disrupts colonic motility in P10/P11 mice. (A) Microbiota composition at the phylum taxonomic level. (B) Spatiotemporal maps of colonic motility from water- (control [Con]) and vancomycin-fed (Vanco) pups, propagating contractions indicated by arrowheads. (C) Frequency and (D) propagation speed represented as means ± SD. (A and C) *Mann–Whitney test, (D) *2-tailed unpaired t test.

Supplementary Figure 1

5-HTP supplementation prevented some vancomycin-induced effects on the ENS and microbiota. Graphs indicate the (A) frequency and (B) speed of colonic contractions, (C) colonic neuronal (Hu+) density, and amplitude of [Ca2+]i transients (ΔFi/F0) from all cells stimulated with a (D) 20 pulse train and a (E) single pulse stimulus, and (F) α diversity. All data were examined from P10/P11 of water-, vancomycin-, and vancomycin + 5-HTP–fed pups. All graphs represent means ± SD. (A and F) Treatment effects were assessed with a Mann–Whitney or a 2-tailed unpaired t test. (G) Summary of microbiota composition at the family taxonomic level and (H) principal coordinate analysis of colonic mucosa samples from P10/P11 of water-, vancomycin-, and vancomycin + 5-HTP–fed pups. (G) Vancomycin treatment reduced the abundance of specific gram-positive families, including peptostreptococaceae and clostridiaceae 1. However, in accordance with other studies,11, 12 a more general dysbiosis was observed, in which some gram-negative families also were affected by vancomycin. OTU, operational taxonomic unit.

Figure 2

Neonatal vancomycin (Vanco) disrupts the structure and function of enteric neurons in P10/P11 mouse colon. (A) Myenteric plexus immunostained for pan-neuronal Hu (red). (B) Hu+ density quantification. (C) Myenteric plexus immunostained for Hu (red), nNOS (blue), and calbindin (green). (D) Proportions of nNOS+ and calbindin+ neurons relative to Hu+ neurons. (E) Fluorescence micrographs of 20 pulse train (20 Hz)-evoked [Ca2+]i responses in myenteric neurons (GCaMP3 signal at rest [t = 0]; and after 20 pulse train stimulation [t = 4.2 s]). (F) Eight representative responding neurons in each condition are marked numerically (panel E), and their color-coded traces show 20 pulse-evoked [Ca2+]i responses. (G) Amplitude of [Ca2+]i transients (ΔFi/F0) from all cells stimulated with a 20 pulse train vs a single pulse stimulus. Graphs represented as means ± SD. *,#Two-tailed unpaired t test. Con, control.

Supplementary Figure 2

Neonatal exposure to vancomycin has no effect on S100β+ myenteric glia density, duodenal ENS, and motility, but significantly reduced body weight. (A) Frequency and speed of duodenal contractions. (B) Neuronal (Hu+) and glial (S100β+) density measured from the myenteric plexus of the colon and duodenum of control (Hu+, n = 9; S100β+, n = 11) and vancomycin-fed (Hu+, n = 8; S100β+, n = 11–14) mice. S100β+ glia from the mucosa, and not enteric plexi, previously were reported to be microbiota-dependent. Our observations of the ineffectiveness of vancomycin treatment on S100β+ immunostaining in the myenteric plexus is in accordance with this, but we cannot exclude the possibility that the antibiotic treatment may have affected mucosal glia, which were not examined in our study. (C) Proportion (relative to Hu+ neurons) of nNOS+ and calbindin+ myenteric neurons in the duodenum of control and vancomycin-fed P10/P11 mouse pups. (D) Body weight of P10/P11 control and vancomycin-fed mice. All graphs represent means ± SD. Treatment effects were assessed with a 2-tailed unpaired t test. Calb, calbindin; Con, control; Vanco, vancomycin.

Ca2+-imaging performed on colons from mice expressing the Ca2+-indicator GCaMP3 in all enteric neurons and glia showed that more neurons responded to electrical stimulation of interganglionic fiber tracts after vancomycin treatment (Supplementary Table 1). This increase in transmission to myenteric neurons was mediated by a specific slow transmission 20 pulse train-evoked [Ca2+]i response, whereas single-pulse evoked responses were unaffected (Figure 2E–G). Post hoc immunofluorescence located these larger train-evoked [Ca2+]i transients to neurons lacking nNOS (Supplementary Table 1). Thus, reduced nNOS+ neurons, including inhibitory motor neurons, and the increased proportion and synaptic excitation of calbindin+ and other excitatory neurons in the myenteric circuitry, may contribute to the antibiotic-induced boost in colonic motility.

Supplementary Table 1

Electrically Evoked [Ca2+]i Transients of Neurochemically Identified Neurons in P10/P11-Treated Pups

Electrical stimulation	Neurochemistry	Control, ΔFi/F0	Vancomycin, ΔFi/F0	Respondersa
1 pulse	Calbindin+	0.1 ± 0.04 (6)	0.1 ± 0.02 (40)	1.4b
	nNOS+	0.1 ± 0.02 (23)	0.1 ± 0.01 (58)
	Calbindin-/nNOS-	0.1 ± 0.01 (64)	0.1 ± 0.01 (122)
20 pulse	Calbindin+	0.4 ± 0.07 (21)	0.7 ± 0.04 (113)b	1.9c
	nNOS+	0.4 ± 0.03 (55)	0.5 ± 0.02 (154)
	Calbindin-/nNOS-	0.4 ± 0.02 (162)	0.6 ± 0.02 (366)c
			Vancomycin + 5-HTP (ΔFi/F0)
1 pulse	Calbindin+	0.11 ± 0.01 (60)	0.13 ± 0.01 (70)	1.6c
	nNOS+	0.09 ± 0.00 (68)	0.11 ± 0.01 (86)c
	Calbindin-/nNOS-	0.11 ± 0.01 (123)	0.13 ± 0.01 (141)b
20 pulse	Calbindin+	0.35 ± 0.01 (137)	0.53 ± 0.03 (129)c	1.1d
	nNOS+	0.35 ± 0.01 (163)	0.46 ± 0.02 (145)c
	Calbindin-/nNOS-	0.36 ± 0.01 (255)	0.51 ± 0.02 (208)c

Responders refers to the proportion of neurons responding in vancomycin or vancomycin + 5-HTP normalized to their controls, chi-square test.

P < .01, 2-tailed unpaired t test, number of neurons examined written in parentheses.

P < .0001, 2-tailed unpaired t test, number of neurons examined written in parentheses.

P < .05, 2-tailed unpaired t test, number of neurons examined written in parentheses.

In adult mouse colon, antibiotics modulate serotonin (5-hydroxytryptamine [5-HT]) levels and gut motility via microbiota signals that promote expression of tryptophan hydroxylase 1; the rate-limiting enzyme in enteric mucosal 5-HT biosynthesis.6, 8 At P10/P11, vancomycin-fed pups had significantly fewer colonic 5-HT+ mucosal cells, which we confirmed by quantitative mass spectrometry of 5-HT levels and its biosynthetic intermediates (Supplementary Figure 3A–C). Decreased 5-HT can accelerate gut motility. Indeed, colonic migrating complexes in tryptophan hydroxylase 1 knockout mice are abnormal and propagate faster compared with wild-type mice, consistent with our observations of faster colonic contractions in vancomycin-fed pups. The 5-HT transporter Serotonin-selective reuptake transporter (SERT) (Slc6a4) gene expression was higher in vancomycin-treated pups, but expression of enterochromaffin cell lineage (chromogranin A) and tryptophan hydroxylase 1 was unaltered (Supplementary Figure 3D). Thus, we assessed the role for 5-HT signaling in vancomycin-induced colonic motility by co-treating pups with vancomycin and 5-hydroxy-L-tryptophan (5-HTP) from birth to P10/P11. 5-HTP supplementation prevented the antibiotic-induced loss of the 5-HT metabolite, 5-hydroxyindole acetic acid, loss of myenteric neuronal density, and an increase in colonic contraction frequency (Supplementary Figures 3C and 1A and C). However, synaptic activity in the myenteric plexus remained enhanced, as was the speed of colonic contractions in vancomycin + 5-HTP–treated animals, showing that more than one pathway mediates vancomycin effects in neonatal animals (Supplementary Figure 1B, D, and E, and Supplementary Table 1). Similarly, although 5-HTP supplementation prevented the effects of the antibiotic on microbial α-diversity, the microbiota composition differed significantly from controls (Supplementary Figure 1F–H). 5-HTP co-treatment also reduced SERT expression (Supplementary Figure 3D), which may account for protective effects on neuron density, because enteric neurogenesis is related inversely to SERT.

Supplementary Figure 3

Vancomycin-induced effects on the gut are linked to alterations in 5-HT availability. (A) Confocal micrograph of the midcolon cross-section of a vancomycin (Vanco)- and a water (control [Con])-fed pup immunostained for 5-HT (5-HT+ cells indicated by arrowheads, enlarged example in inset). (B) Number of 5-HT+ cells in the colonic mucosa of water- and vancomycin-fed pups. (C) Levels of tryptophan, 5-HT, and 5-hydroxyindole acetic acid (5-HIAA); and (D) transcripts encoding Slc6a4, chromogranin A (CgA), and tryptophan hydroxylase 1 (Tph1) were quantified in the colonic mucosa of control-, vancomycin-, and vancomycin + 5-HTP–fed mouse pups. Concentrations of transcripts are expressed as copies/μL of amplified polymerase chain reaction mixture normalized to the concentration of Hprt. All data were examined at P10/P11. All graphs represent means ± SD. (B and C) Treatment effects were assessed with a Mann–Whitney or a 2-tailed unpaired t test.

Previous studies have identified microbial involvement in the regulation of ENS and gut motility by using germ-free mice or prolonged exposure to high doses of broad-spectrum antibiotic mixtures to abolish microbiota.4, 5, 6 Our studies advance these key findings by providing mechanistic insight into acute antibiotic treatment-induced enteric neuropathy and dysmotility at a critical point in development, the period immediately after birth. Future studies should identify the mediators of antibiotic-induced developmental effects in the neonate, elucidate modulatory mechanisms of 5-HTP supplementation, and determine the consequences for adults of these disturbances during the immediate post-natal period.