Impact of dietary n-3 polyunsaturated fatty acids on cognition, motor skills and hippocampal neurogenesis in developing C57BL/6J mice  Carola I.F. Janssen, Valerio Zerbi, Martina P.C. Mutsaers, Bas S.W. de Jong, Maximilian Wiesmann, Ilse A.C. Arnoldussen, Bram Geenen, Arend Heerschap, Frits A.J. Muskiet, Zeina E. Jouni, Eric A.F. van Tol, Gabriele Gross, Judith R. Homberg, Brian M. Berg, Amanda J. Kiliaan  Journal of Nutritional Biochemistry  Volume 26, Issue 1, Pages 24-35 (January 2015) DOI: 10.1016/j.jnutbio.2014.08.002 Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 1 Schematic overview of the study design. C57BL/6J dams were randomly assigned to n-3 def or n-3 adq at GD 0. Offspring was maintained on the corresponding diet throughout the whole study. Male littermates were randomly assigned to one out of three parallel groups. One group was tested for behavioral and cognitive parameters on PND 30 and subsequently sacrificed for brain biochemistry. A parallel group was tested for behavioral and cognitive parameters on PND 60 and subsequently underwent 31P MRS and was also sacrificed for brain biochemistry. A third group was used for brain histology at PND 60. One male pup from each dam was represented in each parallel group for testing and histology. Due to some technical problems during the experiments, not all mice could be included for statistical analysis for each parameter. 31P MRS, 31phosphorus magnetic resonance spectroscopy; GD, gestational day; n-3 adq, n-3 PUFA adequate; n-3 def, n-3 PUFA deficient; PND, postnatal day. Journal of Nutritional Biochemistry 2015 26, 24-35DOI: (10.1016/j.jnutbio.2014.08.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 2 Time line of the experiment. On GD 0, dams were randomly assigned to n-3 PUFA adequate or n-3 PUFA deficient diet for the remainder of the experiment. Offspring are fed the diets throughout life. Behavioral and cognitive tests on the male offspring were performed starting at PND 30 (pubertal age) and PND 60 (young adult age) and they were sacrificed for brain biochemistry. Additionally, mice that performed behavior and cognition also underwent 31P MRS before being sacrificed for brain biochemical purposes. In a parallel group, brain histology was performed at PND 60. 31P MRS, 31phosphorus magnetic resonance spectroscopy; GD, gestational day; PND, postnatal day. Journal of Nutritional Biochemistry 2015 26, 24-35DOI: (10.1016/j.jnutbio.2014.08.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 3 Locomotion in the open field. Activity (total walking distance) was measured over a 30-min period. Values represent mean±S.E.M., n=8–10 per diet, *P≤.05. n-3 adq, n-3 PUFA adequate diet; n-3 def, n-3 PUFA deficient diet; PND, postnatal day. Journal of Nutritional Biochemistry 2015 26, 24-35DOI: (10.1016/j.jnutbio.2014.08.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 4 Exploration in the open field. Different open field parameters were measured in a 30-min period. Values represent mean±S.E.M., n=8–10 per diet, *P≤.05. (a) n-3 adq diet increased time spent walking on PND 60 compared to PND 30. Furthermore, n-3 adq increased time spent walking compared to n-3 def on PND 60. (b) n-3 adq diet decreased time spent grooming on PND 60 compared to PND 30. Additionally, n-3 adq decreased time spent grooming compared to n-3 def on PND 60. (c) n-3 adq diet increased time spent leaning on PND 60 compared to PND 30. n-3 adq, n-3 PUFA adequate diet; n-3 def, n-3 PUFA deficient diet; PND, postnatal day. Journal of Nutritional Biochemistry 2015 26, 24-35DOI: (10.1016/j.jnutbio.2014.08.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 5 Latency to fall in the rotarod. Values represent mean±S.E.M., n=9–13 per diet, *P≤.05. On PND 30, n-3 adq diet increased latency to fall at acceleration compared to n-3 def. On PND 60, n-3 adq diet displayed a decreased latency to fall compared to PND 30. n-3 adq, n-3 PUFA adequate diet; n-3 def, n-3 PUFA deficient diet; PND, postnatal day. Journal of Nutritional Biochemistry 2015 26, 24-35DOI: (10.1016/j.jnutbio.2014.08.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 6 Startle response in the prepulse inhibition test. Mice were exposed to acoustic pulses, and the startle amplitude at 120dB of the mice was measured at the start and at the end of the experiment. Values represent mean±S.E.M., n=10 per diet, *P≤.05 and **P≤.01. (a) The mice displayed an overall habituation effect on PND 30. (b) On PND 60, there was also a habituation and additionally a diet effect. n-3 adq, n-3 PUFA adequate diet; n-3 def, n-3 PUFA deficient diet; PND, postnatal day. Journal of Nutritional Biochemistry 2015 26, 24-35DOI: (10.1016/j.jnutbio.2014.08.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 7 MWM learning and memory in young adult mice (PND 60). Spatial learning was measured in a 4-day acquisition phase, by determining the latency to find a hidden platform in the NE target quadrant. Spatial memory was tested in a probe phase in which the percentage of time spent in the target quadrant and the total number of platform crossings (of the previous location of the platform) were measured. Values represent mean±S.E.M., n=8–9 per diet, *P≤.05. (a) MWM acquisition: both diets show a learning curve. (b) MWM probe target quadrant: n-3 adq diet increased time spent in the NE quadrant. The dotted line represents the 25% chance level of finding the target quadrant. The n-3 def mice performed below 25% chance level while n-3 adq performed at 25% chance level. (c) MWM probe platform crossings: MWM, Morris water maze; n-3 adq increased platform crossings. n-3 adq, n-3 PUFA adequate diet; n-3 def, n-3 PUFA deficient diet; NE, northeast; NW, northwest; PND, postnatal day; SE, southeast; SW, southwest. Journal of Nutritional Biochemistry 2015 26, 24-35DOI: (10.1016/j.jnutbio.2014.08.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 8 DCX immunohistochemical staining performed on brain sections of young adult mice (PND 60). (a) Representative image of the DCX-positive cells in the hippocampus. Image taken using a 2.5× objective. The scale bar represents 500μm. Values represent mean±S.E.M., n=7–9 per diet, *P≤.05. (b) Representative image of the DCX-positive cells in the subgranular zone of the hippocampus. Image taken using a 20× objective. The scale bar represents 100μm. (c) Amount of DCX-positive cells in the subgranular zone of the hippocampus. DCX, doublecortin; n-3 adq, n-3 PUFA adequate diet; n-3 def, n-3 PUFA deficient diet; PND, postnatal day. Journal of Nutritional Biochemistry 2015 26, 24-35DOI: (10.1016/j.jnutbio.2014.08.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 9 Synaptophysin mRNA expression measured with qRT-PCR in the cortical region of the brain in pubertal (PND 30) and young adult mice (PND 60). Values represent mean±S.E.M., n=9–10 per diet, *P≤.05 and .05<#P<.075. On PND 30, n-3 adq diet increased expression of synaptophysin compared to n-3 def. On PND 60, n-3 adq decreased synaptophysin expression compared to PND 30. n-3 adq, n-3 PUFA adequate diet; n-3 def, n-3 PUFA deficient diet; PND, postnatal day. Journal of Nutritional Biochemistry 2015 26, 24-35DOI: (10.1016/j.jnutbio.2014.08.002) Copyright © 2015 Elsevier Inc. Terms and Conditions