The aim of this study was to evaluate the impact of oral administration of KO on depression, cognitive function and expression of genes linked to memory and changes in neuronal connectivity. The KO dose given to study rats is relevant for human consumption as the EPA and DHA amount given (1.25% KO of diet) corresponds to a human daily intake of 0.8 g EPA and DHA in an 8.4 MJ/day diet. A separate group that received the tricyclic antidepressant IMIP as a comparative positive control was included. The results showed that 7 weeks of KO supplementation significantly improved learning and working memory in the ALSAT and displayed significant antidepressant-like effects in the UALST and FST. In addition, KO enhanced expression of Bdnf mRNA, which is a gene implicated in neuronal growth and differentiation.
Cognition and depression in the Conditioned Light Extinction Test (CLET)
In general the same effects were seen in both genders with the exceptions pointed out. The CLET is an ethological model used to score learning ability using the ALSAT procedure [31, 32] and antidepressant-like effects using the UALST in rat .
The administration of KO did not result in any sign of sedation (similar number of total lever presses as in control group), a common problem with classic antidepressants and also seen in this study with IMIP. IMIP was previously shown to have a sedative effect on motor activity in rats . KO administration increased the ability of the rats to discriminate between the active and the inactive lever, which can be interpreted as a sign of improved learning and memory function. A significantly higher number of presses were placed on the active rewarding lever compared to both control and IMIP receiving rats. Whereas the control group showed signs of resignation when the active lever was made inactive on test day 3, both IMIP and KO contributed to an antidepressant-like effect and no reduction in the number of lever presses between day 2 and day 3 was observed despite the absence of positive reinforcement.
Wistar Unilever rats are very sensitive to resignation/depression . That is why these rats were chosen in this study to assess the antidepressant-like effects of KO, the primary objective of the study. With other laboratory rat strains, i.e. Wistar Han or Sprague–Dawley, generally rats learn to discriminate the active lever from the inactive one at the end of the first test session. In our study, after two-day training in the CLET, the KO and IMIP groups showed a higher number of active relative to inactive lever presses. However, the KO group displayed more active lever presses as early as day 1, at a time when the results with the control and IMIP groups did not show any discrimination. Although based on a single experiment, these results indicate that KO may accelerate learning processes. The light environment represents a stress for rats and is a disruptive factor in the acquisition of operant conditioning. Astaxanthin has anxiolytic-like effects  and is known to improve cognitive functions such as reaction time, attention and working memory [29, 36]. The exact role of the astaxanthin in KO remains to be determined and if the rather low amounts given to KO-treated rats help to exploit without stress the environmental parameters of the learning situation in the CLET and to display better cognitive performances remains to be proven.
During the UALST session, when the active lever is deactivated, only KO rats displayed lever pressing activity relatively high compared with that of control and IMIP rats. Thus, KO rats did not resign themselves to suffer passively the unavoidable aversive light stimulus by continuing to press more on the levers and to discriminate the active lever from the inactive one. This effect could be mainly due to the n-3 PUFAs, astaxanthin having no known effect on depression .
Depression in the FST
The antidepressant-like effect of KO was further studied using the FST. Dietary supplementation of KO resulted in reduced immobility time in water. This is consistent with studies demonstrating that alpha-linolenic acid (ALA) administration (ranging from 30 days to 15 weeks) and EPA and DHA administration (12 weeks) reduced immobility and climbing in the FST [14–18]. It is known that short-term administration of PUFAs fail to induce the antidepressant effects in FST [14, 37].
Improved reference and spatial memory was recently reported in the radial maze test for rats that had received a diet supplemented with isolated phospholipids from KO . Moreover, in these rats, DHA levels were shown to be significantly increased in the cerebral cortex, as well as in the hippocampus . The KO used in the present study was previously shown to similarly increase brain DHA levels in Zucker rats . This study was in comparison to FO, which did not lead to an increase in brain DHA levels. Underlying reasons explaining the difference between KO and FO administration might be related to different molecular forms of EPA and DHA (phospholipids versus triglycerides), which has recently been reviewed in more detail .
Taken together, these studies indicate that KO, which is rich in n-3 containing phospholipids, can increase DHA levels in the brain and may protect from depression-like behaviour, as measured by the UALST and FST. In addition, the study suggests that KO can improve learning acquisition and working memory as evaluated in the ALSAT.
Very recently, it was shown that dietary supplementation of KO attenuates inflammation and oxidative stress in ulcerative colitis in rats . Especially, the role of inflammatory processes on emotion is indicated by findings of a link between depression and elevated systemic inflammatory markers . It was demonstrated that antidepressant agents are able to prevent or suppress inflammation and depressive symptoms [42, 43] induced by systemic injection of cytokines [44, 45]. C-reactive protein (CRP), as a biomarker of inflammation , is involved in stroke and cognitive impairments , and KO phospholipids demonstrated anti-inflammatory responses, lowering CRP levels in human subjects [48, 49]. Thus, KO might be used to influence mood and cognition in subjects with an inflammatory condition.
Up-regulation of BDNF signalling and downstream gene expression is implicated in the action of several well-known antidepressant drugs [19, 23, 50]. N-3 PUFAs have also been shown to modulate BDNF expression and signaling in a brain region- and treatment-specific manner [18, 51, 52].
The present study shows that long-term KO administration to rats results in gender and brain region-specific changes in mRNA expression. Recent data also suggests that n-3 PUFAs can induce antidepressant effects associated with increased serotonergic neurotransmission and neurotrophin expression in the hippocampus [18, 51, 53].
Abnormalities in the PUFA content in the brain might affect mood control through effects on neurotrophin expression and signalling and deficiencies have been shown to reduce neurotrophin levels in the prefrontal cortex as well as in the hippocampus [54, 55]. 6 and 15 weeks of ALA deprivation results in significantly decreased levels of BDNF expression, CREB transcription factor activity, and p38 mitogen-activated protein kinase (MAPK) activity in the frontal cortex .
Although IMIP and KO treatments both have antidepressant-like behavioural effects, these treatments do not have identical effects on gene expression. IMIP and KO both induce Bdnf, but only IMIP enhanced the expression of genes previously associated with BDNF signaling and long-term synaptic plasticity [19, 56]. Hence, the antidepressant-like effects of IMIP and KO are correlated with distinct neurobiological mechanisms at the level of gene expression. However, it remains possible that KO regulates BDNF-responsive target genes on a different time course than does IMIP. The observed increase of Bdnf mRNA level observed in the present study might be a result of increased transcription or an effect on RNA turnover. Another study with n-3 PUFA supplementation (0.5% EPA and 1% DHA for 12 weeks) showed antidepressant effects, a change in CREB protein levels, but no increase in hippocampal BDNF protein levels . This might be explained by differences in types, forms and amounts of n-3 PUFAs, and the fact that real-time PCR is a more sensitive method then Western blot. On the other hand, inclusion of 1.2% DHA in the diet was able to increase BDNF protein levels in rat hippocampal tissue after brain trauma .
Arc has key regulatory roles in protein synthesis-dependent synaptic plasticity, memory formation, and postnatal cortical development [57, 58]. Interestingly, KO treatment induced Arc in the male prefrontal cortex, although Bdnf levels were not altered. On the other hand IMIP induced the expression of Bdnf and several BDNF-regulated genes, but did not increase Arc expression under these conditions. Another possible mechanism of Arc induction is through activation of muscarinic cholinergic receptors (mAchR). It has previously been shown in vitro and in vivo that activation of mAchR induces Arc expression [59–61].
In KO, the major phospholipid species is phosphatidylcholine, which may provide a source for acetylcholine biosynthesis . Learning ability has previously been associated with increased cerebral levels of acetylcholine after the intake of a DHA supplemented diet in a rat model and different sources of DHA intake increase the release of acetylcholine from the rat hippocampus . Future studies need to investigate possible changes in acetylcholine levels after KO administration.