Early life adversity in piglets induces long-term upregulation of the enteric cholinergic nervous system and heightened, sex-specific secretomotor neuron responses

Abstract

Background: Early life adversity (ELA) is a risk factor for the later-life onset of gastrointestinal (GI) diseases such as irritable bowel syndrome (IBS); however, the mechanisms are poorly understood. Here, we utilized a porcine model of ELA, early weaning stress (EWS), to investigate the influence of ELA on the development and function of the enteric nervous system (ENS).

Methods: Female and castrated male (Male-C) piglets were weaned from their sow either at 15 days of age (EWS) or 28 days of age (late weaning control, LWC). At 60 and 170 days of age, ileal mucosa-submucosa preparations were mounted in Ussing chambers and veratridine- and corticotropin releasing factor (CRF)-releasing factor-evoked short circuit current (Isc ) responses were recorded as indices of secretomotor neuron function. Enteric neuron numbers and the expression of select neurotransmitters and their receptors were also measured.

Key results: Compared with LWC pigs, female, but not Male-C EWS, pigs exhibited heightened veratridine-induced Isc responses at 60 and 170 days of age that were inhibited with tetrodotoxin and atropine. Ileum from EWS pigs had higher numbers of enteric neurons that were choline acetyltransferase positive. Markers of increased cholinergic signaling (increased acetylcholinesterase) and downregulated mucosal muscarinic receptor 3 gene expression were also observed in EWS pigs.

Conclusions & inferences: This study demonstrated that EWS in pigs induces lasting and sex-specific hypersensitivity of secretomotor neuron function and upregulation of the cholinergic ENS. These findings may represent a mechanistic link between ELA and lifelong susceptibility to GI diseases such as IBS.

Keywords: cholinergic neurons; early life adversity; enteric nervous system development; irritable bowel syndrome; porcine model.

Figure 1. Veratridine-evoked I_sc in the porcine ileum

Porcine ileum (mucosa and submucosa) was mounted on Ussing chambers. After a 30 minute equilibration period, the serosal side of the tissue was exposed to veratridine (30 µM), in the presence or absence of a pre-treatment with tetrodotoxin (TTX), on the serosal side of tissues. I_sc was recorded at 1 minute intervals over a 50-minute period. Data is presented as the absolute peak change in I_sc in response to veratridine (A) or as a representative line tracing of treatment responses (B). Data were analyzed using a 1-Way ANOVA. *P<0.05 compared with other treatments.

Figure 2. Influence of early weaning stress (EWS) on veratridine-evoked I_sc responses in juvenile and adult pigs

Porcine ileum was obtained from late weaned control (LWC) pigs and EWS pigs at 60 d (A) and 170 d (B) of age and exposed to dose concentrations of veratridine. I_sc responses were recorded and data were presented as the absolute peak change in I_sc (ΔI_sc) in response to veratridine. Values represent means (±SE) for n=12 pigs/treatment. *P<0.05 LWC vs. EWS within each veratridine dose; Data were analyzed with a Student’s T-test.

Figure 3. Influence of early weaning stress (EWS) on CRF-evoked I_sc responses in juvenile pigs

Porcine ileum was obtained from late weaned control (LWC) pigs and EWS pigs at 60 d of age and exposed to increasing doses of CRF (0 µM, 0.05 µM, and 0.1 µM. I_sc responses were recorded and data were presented as the absolute peak change in I_sc (ΔI_sc) in response to CRF. Values represent means (±SE) for n=6 pigs/treatment. ^#P<0.05 vs. 0 µM CRF dose within weaning age group (LWC and EWS). Values represent means (±SE) for n=6 pigs/treatment. *P<0.05 LWC vs. EWS within each CRF concentration; Data were analyzed with a Student’s T-test.

Figure 4. Influence of sex and early weaning stress (EWS) on veratridine-evoked I_sc responses in juvenile and adult pigs

Porcine ileum was obtained from late weaned control (LWC) pigs (n=6 female; n=6 Male-C) and EWS pigs (n=6 Female; n=6 Male-C) at 60 d (A) and 170 d (B) of age and exposed to veratridine (30 µM). I_sc responses were recorded and data were presented as the absolute peak change in I_sc (ΔI_sc) in response to veratridine. Data were analyzed using a 2-way ANOVA (Factors: Sex and weaning age group). *P<0.05 compared with other treatments.

Figure 5. Effects of muscarinic receptor blockade on veratridine-evoked I_sc responses in late weaned control (LWC) and early weaning stress (EWS) pig ileum

Porcine ileum was obtained from female LWC pigs and EWS pigs at 60 d (A) and 170 d (B) of age and exposed to veratridine (100 µM), in the presence or absence of a pre-treatment with the muscarinic cholinergic receptor antagonist atropine (1 µM). I_sc responses were recorded and data were presented as the absolute peak change in I_sc (ΔI_sc) in response to veratridine. Data were analyzed using a 2-way ANOVA (Factors: Sex and weaning age group). Different letters (a,b,c) differ by P<0.05.

Figure 6. Influence of early weaning stress (EWS) on enteric neuron numbers and phenotype

The numbers of enteric neurons were quantified counting the number of PGP9.5-positive cells and expressing them as neurons/mm². The proportion of ChAT-, 5HT- and DBH-positive neurons were measured by immunohistochemistry in the submucosal plexus of ileum of LWC and EWS pigs at 60 d and 170 d of age and were expressed as a percentage relative to PGP9.5-positive neurons. (A) Number of PGP9.5-positive enteric neurons. (B) percentage of ChAT-positive/PGP-positive neurons. (C) percentage of 5HT-positive/PGP-positive neurons. (D) percentage of DβH -positive/PGP-positive neurons. (E and F), representative example of PGP9.5-ChAT double positive in LWC (E) and EWS (F) porcine ileum at 170 d of age. White arrows indicate double-positive cells. Values represent means (±SE) for n=12pigs/treatment. *P<0.05 LWC vs. EWS within each age time point; Data were analyzed with a Student’s T-test.

Figure 7. Impact of Early weaning stress (EWS) on enteric cholinergic markers in juvenile and adult pigs

Mucosal acetycholinesterase (AcHE) activity in LWC and EWS pigs presented as combined Male-C + female data at 60 d (A) and 170 d (D) of age or as individual Male-C and female data at 60 d (B) and 170 d (E) of age. Quantitative PCR was performed to measure the gene expression for muscarinic cholinergic receptor subtypes 1(CHRM1), 2 (CHRM2) and 3 (CHRM3) in the mucosa from 60 d (C) and 170 d (F) old pigs. Student’s T-tests were performed for each variable to detect differences between LWC and EWS pig groups. *P<0.05.