Iron and n-3 fatty acid depletion, alone and in combination, during early development provoke neurochemical changes, anhedonia, anxiety and social dysfunction in rats

Figures & data

Figure 1. Flow diagram of the experimental design; n indicates the numbers in each group; * indicates an equal number of male and female rats. GD: Gestational day; ID: Iron deficient; n-3 FAD: n-3 fatty acid deficient; ID + n3 FAD: iron and n-3 fatty acid deficient.

Table 1. Litter sizes, body weights and weight gain in offspring.

	Control	ID	n-3 FAD	ID + n-3 FAD	P value^a
					ID	n-3 FAD	ID × n-3 FAD
Litter sizes at birth, n	12.3 ± 0.4	11.9 ± 0.4	11.9 ± 0.6	12.3 ± 0.5	1.000	1.000	0.412
Body weight PND 0, g	5.8 ± 0.3^a,b	5.7 ± 0.1^a,b	6.1 ± 0.2^a	5.5 ± 0.1^b	<0.001	0.923	0.036
Body weight PND 21, g	56 ± 2.5^a,b	59 ± 2.4^a,b	63 ± 2.2^a	53 ± 2.0^b	0.089	0.853	0.005
Total weight gain, g/21d	50.3 ± 2.4^a,b	53.0 ± 2.3^a,b	56.8 ± 2.2^a	47.2 ± 2.0^b	0.119	0.854	0.006
Body weight PND 45, g	193 ± 8.0^a	175 ± 5.8^a,b	191 ± 7.1^a	159 ± 4.5^b	<0.001	0.049	0.134
Total body weight gain, g/24d	137 ± 7.0^a	116. ± 4.5^b,c	128 ± 5.9^a,b	106 ± 3.6^c	<0.001	0.010	0.923
Males	164 ± 7.4^a	135 ± 3.3^b.c	152 ± 5.9^a,b	115 ± 4.7^c	<0.001	0.006	0.464
Females	111 ± 4.8	98 ± 3.4	104 ± 3.1	98 ± 4.2	0.012	0.398	0.395

Notes: Values are means ± SEM, n = 24 per group (n = 12 females, n = 12 males). ID, iron deficient; n-3 FAD, n-3 fatty acid deficient; ID + n-3 FAD, iron deficient and n-3 fatty acid deficient.

^a Two-way ANCOVA was used to test effects of dietary iron (deficient vs. sufficient) and dietary n-3 FA (deficient vs. sufficient), and ID × n-3 FA interactions, adjusted for sex, P < 0.05. Between-group differences were determined using one-way ANCOVA adjusted for sex, followed by Bonferroni’s post hoc test. Means in a row with superscripts without a common letter differ significantly, P < 0.05.

Table 2. Haemoglobin, and tissue iron concentrations and fatty acid composition of the different brain regions in offspring at PND 45.

	Control	ID	n-3 FAD	ID + n-3 FAD	P value^a
					ID	n-3 FAD	ID × n-3 FAD
Haemoglobin, g/dl	13.53 ± 0.34^a	9.45 ± 0.33^b	13.02 ± 0.47^a	8.63 ± 0.28^b	<0.001	0.057	0.626
Brain iron, ug/g tissue
Hip	17.12 ± 0.67^a	14.96 ± 0.56^a,b	17.01 ± 0.85^a	14.23 ± 0.70^b	0.001	0.548	0.659
Str	17.73 ± 0.88^a	13.48 ± 0.41^b	16.93 ± 0.73^a	13.01 ± 0.44^b	<0.001	0.323	0.800
FC	17.09 ± 0.84^a	14.52 ± 0.79^a,b	16.85 ± 0.88^a,b	13.80 ± 0.85^b	0.001	0.568	0.776
Tissue fatty acids, % of total FA
18:3 n-3 (ALA)
Hip	0.006 ± 0.001^a	0.007 ± 0.001^a	0.003 ± 0.000^b	0.002 ± 0.000^b	0.917	<0.001	0.069
Str	0.006 ± 0.000^a	0.006 ± 0.000^a	0.002 ± 0.000^b	0.002 ± 0.000^b	0.945	<0.001	0.890
FC	0.005 ± 0.000^a	0.005 ± 0.001^a	0.004 ± 0.001^a,b	0.002 ± 0.000^b	0.065	<0.001	0.067
20:5 n-3 (EPA)
Hip	0.02 ± 0.00^a	0.02 ± 0.00^a	0.01 ± 0.00^b	0.01 ± 0.00^b	0.454	<0.001	0.333
Str	0.02 ± 0.00^a	0.02 ± 0.00^a,b	0.01 ± 0.00^b,c	0.01 ± 0.00^c	0.308	<0.001	0.458
FC	0.01 ± 0.00^a	0.01 ± 0.00^a	0.01 ± 0.01^a	0.01 ± 0.00^a	0.384	0.180	0.061
22:6 n-3 (DHA)
Hip	14.18 ± 0.19^a	13.88 ± 0.13^a	4.25 ± 0.14^b	4.72 ± 0.26^b	0.683	<0.001	0.042
Str	12.42 ± 0.21^a	12.44 ± 0.26^a	3.91 ± 0.23^b	4.24 ± 0.17^b	0.414	<0.001	0.469
FC	17.55 ± 0.28^a	16.71 ± 0.33^a	4.53 ± 0.19^b	4.67 ± 0.23^b	0.170	<0.001	0.058
Total n-3 FAs
Hip	14.53 ± 0.19^a	14.23 ± 0.13^a	4.34 ± 0.14^b	4.85 ± 0.28^b	0.585	<0.001	0.039
Str	12.72 ± 0.21^a	12.75 ± 0.26^a	4.00 ± 0.23^b	4.35 ± 0.17^b	0.368	<0.001	0.466
FC	17.87 ± 0.28^a	17.09 ± 0.32^a	4.65 ± 0.19^b	4.80 ± 0.23^b	0.220	<0.001	0.068
18:2 n-6 (LA)
Hip	0.84 ± 0.02^b	0.96 ± 0.20^a	0.56 ± 0.01^d	0.69 ± 0.02^c	<0.001	<0.001	0.711
Str	1.02 ± 0.02^b	1.10 ± 0.01^a	0.68 ± 0.01^d	0.84 ± 0.02^c	<0.001	<0.001	0.017
FC	1.18 ± 0.02^b	1.28 ± 0.020^a	0.80 ± 0.03^d	0.91 ± 0.02^c	<0.001	<0.001	0.752
20:4 n-6 (AA)
Hip	14.02 ± 0.11^a,b	13.96 ± 0.11^b	14.36 ± 0.10^a	14.26 ± 0.10^a,b	0.439	0.003	0.823
Str	11.37 ± 0.17^b	11.50 ± 0.09^b	12.16 ± 0.20^a	12.31 ± 0.12^a	0.366	<0.001	0.927
FC	12.96 ± 0.05^b	13.03 ± 0.12^a,b	13.44 ± 0.08^a	13.19 ± 0.18^a,b	0.442	0.009	0.173
Total n-6 FAs
Hip	19.97 ± 0.13^b	20.32 ± 0.13^b	31.35 ± 0.20^a	30.87 ± 0.30^a	0.769	<0.001	0.049
Str	16.92 ± 0.22^b	17.28 ± 0.14^b	27.93 ± 0.33^a	28.33 ± 0.29^a	0.135	<0.001	0.979
FC	19.17 ± 0.10^b	19.86 ± 0.17^b	33.37 ± 0.23^a	32.79 ± 0.40^a	0.816	<0.001	0.012
Total n-6:n-3 ratio
Hip	1.38 ± 0.02^b	1.43 ± 0.02^b	7.42 ± 0.26^a	6.80 ± 0.37^a	0.232	<0.001	0.150
Str	1.33 ± 0.02^b	1.35 ± 0.02^b	7.04 ± 0.29^a	6.77 ± 0.30^a	0.516	<0.001	0.461
FC	1.08 ± 0.02^b	1.15 ± 0.01^b	7.39 ± 0.31^a	7.19 ± 0.38^a	0.828	<0.001	0.532

Notes: Values are means ± SEM, n = 20–24 per group (n = 12 females, n = 12 males). ID, iron deficient; n-3 FAD, n-3 fatty acid deficient; ID + n-3 FAD, iron deficient and n-3 fatty acid deficient; Hb, haemoglobin; Hip, hippocampus; Str, striatum; FC, frontal cortex; ALA, alpha-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; LA, linoleic acid; AA, arachidonic acid.

^a Two-way ANCOVA was used to test effects of dietary iron (deficient vs. sufficient) and dietary n-3 FA (deficient vs. sufficient), and ID × n-3 FA interactions, adjusted for sex, P < 0.05. Outcome variables that were not normally distributed were log transformed to perform ANCOVA. Between-group differences were determined using one-way ANCOVA adjusted for sex, followed by Bonferroni’s post hoc test. Means in a row with superscripts without a common letter differ significantly, P < 0.05.

Figure 2. Sucrose preference test (SPT), elevated plus maze test (EPM) and novel object recognition test (NORT) results. (A) Percentage of sucrose consumed in the SPT by male and female rats (ID P = 0.001). (B) Percentage of time spent in the open (ID P = 0.020) and closed arms (ID P = 0.008) of the EPM by male and female rats. (C) Percentage time spent exploring familiar (n-3 FAD P = 0.034) and novel objects during the NORT. Two-way ANCOVA was used to test effects of ID, n-3 FAD, and ID x n-3 FAD interactions, adjusted for sex. The results that were not normal distributed were log transformed to perform ANCOVA. Between-group differences were determined using one-way ANCOVA followed by Bonferroni’s post-hoc test (adjusted for sex). For the EPM, independent t-test were performed to determine significant difference between time spent in open vs closed arms. Values are means ± SEM, n = 22–24. Labelled means without a common letter differ significantly, ID, iron deficient; n-3 FAD; n-3 fatty acid deficient.

Figure 3. Social interaction test result. (A) The percentage of time female and male rats spent approaching one another (ID P < 0.001; n-3 FAD P = 0.001), (B) together (ID P < 0.001; n-3 FAD P < 0.001) and (C) moving away from each other (ID P < 0.001; n-3 FAD P = 0.001) during the SIT. (D) The percentage of time female and male rats spent anogenital sniffing (ID P = 0.033; n-3 FAD P = 0.011; IDxn-3FAD P < 0.001) during the SIT. Panels E and F show the amount of time the rats spent in self-directed behaviour: (E) Rearing (n-3 FAD P = 0.018) and (F) self-grooming (ID P = 0.011; IDxn-3 FAD P = 0.022). Two-way ANCOVA was used to test effects of ID, n-3 FAD, and ID x n-3 FAD interactions, adjusted for sex. The results that were not normal distributed were log transformed to perform ANCOVA. Between-group differences were determined using one-way ANCOVA followed by Bonferroni’s post-hoc test (adjusted for sex). Values are means ± SEM, n = 7–12, where split for sex (A, B, C, D) and n = 22–24 where results for male and female rats were combined (E, F). Labelled means without a common letter differ significantly. ID: iron deficient; n-3 FAD: n-3 fatty acid deficient.

Figure 4. Monoamine results for the striatum (NE, ID P = 0.012; DA, ID P = 0.006; DOPAC, ID P = 0.001). Two-way ANCOVA was used to test effects of ID, n-3 FAD, and ID x n-3 FAD interactions, adjusted for sex. The values were log transformed to perform ANCOVA. Between-group differences were determined using one-way ANCOVA followed by Bonferroni’s post-hoc test (adjusted for sex). Means with superscripts without a common letter differ (P < 0.05). Values are means ± SEM, n = 16–24 per group (n = 12 females, n = 12 males). ID: iron deficient; n-3 FAD: n-3 fatty acid deficient.

Figure 5. Monoamine results for the frontal cortex (NE, IDxn-3FAD P < 0.001; DA, ID P = 0.018; IDxn-3FAD P = 0.033; 5HT, ID P = 0.001; IDxn-3FAD P = 0.027). Two-way ANCOVA was used to test effects of ID, n-3 FAD, and ID x n-3 FAD interactions, adjusted for sex. The values were log transformed to perform ANCOVA. Between-group differences were determined using one-way ANCOVA followed by Bonferroni’s post-hoc test (adjusted for sex). Means with superscripts without a common letter differ (P < 0.05). Values are means ± SEM, n = 16–24 per group (n = 12 females, n = 12 males). ID: iron deficient; n-3 FAD: n-3 fatty acid deficient.

Figure 6. Serotonin results for the frontal cortex split by sex (female, ID P = 0.001; IDxn-3FAD P = 0.027; male, n-3FAD P = 0.005). Two-way ANCOVA was used to test effects of ID, n-3 FAD, and ID × n-3 FAD interactions, adjusted for sex. The values were log transformed to perform ANCOVA. Between-group differences were determined using one-way ANCOVA followed by Bonferroni’s post-hoc test (adjusted for sex). Means with superscripts without a common letter differ (P < 0.05). Values are means ± SEM, n = 16–24 per group (n = 12 females, n = 12 males). ID: iron deficient; n-3 FAD: n-3 fatty acid deficient.

Data availability statement

The authors declare that all data associated with the current study are available from the corresponding author upon reasonable request.