Functional programming of the autonomic nervous system by early life immune exposure: implications for anxiety

Abstract

Neonatal exposure of rodents to an immune challenge alters a variety of behavioural and physiological parameters in adulthood. In particular, neonatal lipopolysaccharide (LPS; 0.05 mg/kg, i.p.) exposure produces robust increases in anxiety-like behaviour, accompanied by persistent changes in hypothalamic-pituitary-adrenal (HPA) axis functioning. Altered autonomic nervous system (ANS) activity is an important physiological contributor to the generation of anxiety. Here we examined the long term effects of neonatal LPS exposure on ANS function and the associated changes in neuroendocrine and behavioural indices. ANS function in Wistar rats, neonatally treated with LPS, was assessed via analysis of tyrosine hydroxylase (TH) in the adrenal glands on postnatal days (PNDs) 50 and 85, and via plethysmographic assessment of adult respiratory rate in response to mild stress (acoustic and light stimuli). Expression of genes implicated in regulation of autonomic and endocrine activity in the relevant brain areas was also examined. Neonatal LPS exposure produced an increase in TH phosphorylation and activity at both PNDs 50 and 85. In adulthood, LPS-treated rats responded with increased respiratory rates to the lower intensities of stimuli, indicative of increased autonomic arousal. These changes were associated with increases in anxiety-like behaviours and HPA axis activity, alongside altered expression of the GABA-A receptor α2 subunit, CRH receptor type 1, CRH binding protein, and glucocorticoid receptor mRNA levels in the prefrontal cortex, hippocampus and hypothalamus. The current findings suggest that in addition to the commonly reported alterations in HPA axis functioning, neonatal LPS challenge is associated with a persistent change in ANS activity, associated with, and potentially contributing to, the anxiety-like phenotype. The findings of this study reflect the importance of changes in the perinatal microbial environment on the ontogeny of physiological processes.

Figure 1. Experimental design.

(A) A schematic timeline of the study design and experimental protocols. (B) A schematic representation of the respiratory testing procedure.

Figure 2. Effect of neonatal LPS exposure on plasma corticosterone levels.

Corticosterone levels in neonatal period (PNDs 5-7) (A), in adolescence (PND 50) (B), and in adulthood (PND 85) (C). (D) Demonstrates changes in circulating corticosterone following the respiratory testing in adult rats (PND 85). Filled bars represent neonatally-treated LPS rats, hollow bars represent neonatally-treated saline controls. Values are mean±SEM. *p<.05.

Figure 3. Effect of neonatal LPS exposure on anxiety-like behaviours in adulthood.

Effect of neonatal LPS exposure on a percentage of time spent in the closed arms of the EPM (A), on a mean number of exploratory head dips (B) and risk assessment behaviours (C) in the holeboard apparatus. Filled bars represent neonatally-treated LPS rats, hollow bars represent neonatally-treated saline controls. Values are mean±SEM. *p<.05.

Figure 4. Gene expression in the PFC, the hypothalamus and the hippocampus, presented as a fold change relative to the saline control.

(A–C) Represent changes in CRHR1, CRHBP and GABA-Arα2 mRNA levels in the PFC. (D–E) Represent changes in CRHR1 and CRHBP mRNA levels in the hypothalamus. (F–H) Represent changes in CRHR1, CRHBP and GR mRNA levels in the hippocampus. Values are mean±SEM. *p<.05, ***p<.001.

Figure 5. Effect of neonatal LPS exposure on TH protein, phosphorylation at Ser19, Ser31, Ser40 and TH activity levels.

Changes in TH are expressed as a fold change relative to the saline control in the adrenal glands in adolescence (PND 50) (A) and adulthood (B). Representative immunoblots demonstrate the effect of LPS and saline treatments. Filled bars represent neonatally-treated LPS rats, hollow bars represent neonatally-treated saline controls. Values are mean±SEM. *p<.05, **p<.01, ***p<.001.

Figure 6. Effect of neonatal LPS exposure on respiratory rate in adulthood.

(A) Representative raw data records of respiratory signal (mV), respiratory rate (cycles per minute (CPM)) and movement signal (mV) prior and following a sensory stimulus (broken vertical line). (B) Represents Δ changes from baseline (±SEM) in respiratory rate in response to acoustic stimuli as generated by the Change Detection model. (C) Represents changes (CPM±SEM) in respiratory rate prior and in response to light stimulus. Filled bars represent neonatally-treated LPS rats, hollow bars represent neonatally-treated saline controls. *p<.05.