Dietary supplementation with n-3 fatty acids from weaning limits brain biochemistry and behavioural changes elicited by prenatal exposure to maternal inflammation in the mouse model

Abstract

Prenatal exposure to maternal immune activation (MIA) increases the risk of schizophrenia and autism in the offspring. The MIA rodent model provides a valuable tool to directly test the postnatal consequences of exposure to an early inflammatory insult; and examine novel preventative strategies. Here we tested the hypotheses that behavioural differences in the MIA mouse model are accompanied by in vivo and ex vivo alterations in brain biochemistry; and that these can be prevented by a post-weaning diet enriched with n-3 polyunsaturated fatty acid (PUFA). The viral analogue PolyI:C (POL) or saline (SAL) was administered to pregnant mice on gestation day 9. Half the resulting male offspring (POL=21; SAL=17) were weaned onto a conventional lab diet (n-6 PUFA); half were weaned onto n-3 PUFA-enriched diet. In vivo magnetic resonance spectroscopy measures were acquired prior to behavioural tests; glutamic acid decarboxylase 67 (GAD67) and tyrosine hydroxylase protein levels were measured ex vivo. The main findings were: (i) Adult MIA-exposed mice fed a standard diet had greater N-acetylaspartate/creatine (Cr) and lower myo-inositol/Cr levels in the cingulate cortex in vivo. (ii) The extent of these metabolite differences was correlated with impairment in prepulse inhibition. (iii) MIA-exposed mice on the control diet also had higher levels of anxiety and altered levels of GAD67 ex vivo. (iv) An n-3 PUFA diet prevented all the in vivo and ex vivo effects of MIA observed. Thus, n-3 PUFA dietary enrichment from early life may offer a relatively safe and non-toxic approach to limit the otherwise persistent behavioural and biochemical consequences of prenatal exposure to inflammation. This result may have translational importance.

Figure 1

Experimental design. AMPH, amphetamine; EPM, elevated plus maze; GD, gestation day; MIA, maternal immune activation; OF, open field; PND, postnatal day; POL, PolyI:C; PPI, prepulse inhibition; PUFA, polyunsaturated fatty acids; SAL, saline. Groups: n3-POL, prenatal PolyI:C-exposed offspring treated with n-3 PUFA; n6-POL, prenatal PolyI:C-exposed offspring treated with n-6 PUFA; n3-SAL, prenatal saline-exposed offspring treated with n-3 PUFA; n6-SAL, prenatal saline-exposed offspring treated with n6-polyunsaturated fatty acids (n-6 PUFA) control diet.

Figure 2

Representative MRS voxel-of-interest (VOI) and MRS metabolite levels in adult offspring exposed to prenatal inflammation or saline, with or without n-3 PUFA intervention in peri-adolescence. Representative MRS voxel-of-interest (VOI) and spectra acquired in adult offspring: (a) Voxel-of-interest with the size of 1.2 × 2.6 × 2.5 mm³ was positioned in the anterior cingulate of each mouse. Mouse atlas reference of VOI is from Allen Institute: (b) Representative in vivo ¹H spectra with quantum estimation (QUEST) fitting from the anterior cingulate in each group. MRS raw spectrum is shown in black; estimate fitting is shown in red. MRS metabolite levels: (c) Greater N-acetylaspartate (NAA)/creatine (Cr) in n-6 PolyI:C-exposured group compared with all other groups (*P<0.05, **P⩽0.01); n-3 PUFA prevents the rise in NAA/Cr caused by prenatal PolyI:C exposure. (d) Lower myo-inositol (mIns)/Cr in PolyI:C-exposed group compared with Saline group. Groups: n3-POL, prenatal PolyI:C-exposed offspring treated with n-3 PUFA; n6-POL, prenatal PolyI:C-exposed offspring treated with n-6 PUFA; n3-SAL, prenatal saline-exposed offspring treated with n-3 PUFA; n6-SAL, prenatal saline-exposed offspring treated with n6-polyunsaturated fatty acids (n-6 PUFA) control diet. MRS, magnetic resonance spectroscopy; POL, PolyI:C; PUFA, polyunsaturated fatty acids; SAL, saline.

Figure 3

Mean percentage prepulse inhibition (%PPI). The bar plot represents mean %PPI across all prepulse and pulse stimuli. All values are mean±s.e.m. *P<0.05, **P<0.01. Groups: n3-SAL, prenatal saline-exposed offspring treated with n-3 PUFA; n6-SAL, prenatal saline-exposed offspring treated with n6-polyunsaturated fatty acids (n-6 PUFA) control diet.

Figure 4

GAD67 protein levels. (a) GAD67 protein level in nucleus accumbens (NAc), (b) caudate putamen (CP) and (c) medial prefrontal cortex (mPFC). β-actin is shown as a control for comparison. All values are mean±s.e.m. *P<0.05, **P<0.01. Histology panels represent coronal mouse atlas reference from Allen Institute, indicating regions-of-interest dissected for analyses (A=NAc, B=CP, C=mPFC). Groups: n6-POL, prenatal PolyI:C-exposed offspring treated with n-6 PUFA; n3-SAL, prenatal saline-exposed offspring treated with n-3 PUFA; n6-SAL, prenatal saline-exposed offspring treated with n6-polyunsaturated fatty acids (n-6 PUFA) control diet.

Figure 5

Metabolite and behaviour correlation. Mean %PPI and NAA/Cr correlation (a); Mean %PPI and mIns/Cr correlation (b). □,○,△,+ refer to n6-SAL, n6-POL, n3-SAL and n3-POL, respectively. Groups: n6-POL, prenatal PolyI:C-exposed offspring treated with n-6 PUFA; n3-SAL, prenatal saline-exposed offspring treated with n-3 PUFA; n6-SAL, prenatal saline-exposed offspring treated with n6-polyunsaturated fatty acids (n-6 PUFA) control diet.