Our study demonstrates that DHA supplementation significantly promotes neurite growth, synaptogenesis, and increases the levels of pre- and post-synaptic proteins involved in synaptic transmission and LTP, improving synaptic function. It is generally accepted that more synaptic connections can be made with longer neurites and a higher number of dendritic branches (Jan and Jan 2001
). It has been also shown that synaptic activity in developing neurons further promotes dendritic arbor elaboration and stabilizes dendritic structure, which is critical for synaptic remodeling during memory (Cline 2001
). As the synaptic activity was not altered by acute applications of DHA (data not shown), availability of DHA during development for neurite growth, synaptogenesis and synaptic protein expression may be an important aspect of ultimately enhancing hippocampus-related learning and memory function.
In the hippocampus, DHA is the major polyunsaturated fatty acid found while its precursor LNA is not detected (Tables S2 and S3). LNA at 1 μM which is far exceeding physiological concentrations of LNA expected as the free fatty acid did not affect the hippocampal neurite growth or synaptogenesis (data not shown). We observed that DHA at a concentration greater than 5 μM was toxic while the concentration below 0.5 μM did not exert measurable effects in our hippocampal neuronal cultures (data not shown). The free DHA concentration in the brain has been reported to be ~1.3 μM (Contreras et al. 2000
), and therefore, 1 μM DHA employed in our study was in the physiological concentration range.
The enhanced synaptic activity in DHA-treated neurons suggested a unique role of DHA in promoting active synapse formation and/or pre-synaptic neurotransmitter release, which was supported by the DHA-induced increase of synaptogenesis and synapsin protein expression (). Moreover, the primarily glutamatergic nature of the enhanced synaptic activity was consistent with the observation that DHA increased expression of glutamate receptors ( and ) without affecting GABA receptor levels. It has been demonstrated that GABAergic synapses are established before glutamatergic synapses during development (Tyzio et al. 1999
). Consistently, the baseline synaptic activity observed in control embryonic hippocampal neurons in 10 days in vitro
culture was mostly GABAergic (). The synergistic activation of GABA and NMDA receptors has been suggested to contribute to the maturation of glutamatergic synapses (Tyzio et al. 1999
). In this regard, the observed DHA-induced increase in NR expression may have played a significant role in the maturation of glutamatergic synapses, enhancing excitatory synaptic activity.
The observed increases in synaptic proteins may involve activation of transcriptional factors during development. DHA has been shown to be an endogenous ligand for RXR (de Urquiza et al. 2000
). RXR forms heterodimers with nuclear receptors such as retinoic acid receptor (RAR), peroxisome proliferator-activated receptors, liver X receptor, constitutive androstane receptor, farnesoid X receptor, pregnane X receptor, thyroid hormone receptor, vitamin D receptor, or Nurr1 for transcriptional regulation of target genes (Aarnisalo et al. 2002
; Lane and Bailey 2005
). It is well established that retinoic acid signaling plays an important role in neurodevelopment by regulating genes involved in the control of synaptic plasticity, cytoskeleton, and membrane assembly, as well as signal transduction and ion channel formation (Maden 2002
; Lane and Bailey 2005
). Although RAR plays the dominant role in the RAR–RXR dimer function (Kurokawa et al. 1994
), it is possible that DHA binding to RXR within the functional dimer enhances its transcriptional activity, as has been reported earlier for retinoid-dependent gene expression (Minucci et al. 1997
). It is also possible that Nurr1–RXR signaling played a role in preventing the loss of synaptic proteins as DHA binding to RXR in Nurr1–RXR heterodimers in embryonic CNS has been shown to support neuronal survival, particularly dopaminergic neurons (Wallen-Mackenzie et al. 2003
Under neurodegenerative conditions, such as in aging, reduction of GluR expression (Dyall et al. 2007
) and impairment of LTP in the DG has been observed (McGahon et al. 1999
), which was reversed by n
-3 fatty acid supplementation. Long-term dietary n
-3 fatty acid depletion in aging brains has been shown to decrease NMDA receptor subunits in the cortex and hippocampus, which were further decreased in transgenic mice with the human Alzheimer's disease gene APPswe
(Calon et al. 2005
). Our data indicated that DHA-depletion during development also leads to decreased expression of GluR and NR glutamate receptor subunits and retarded LTP in the young mouse hippocampus, suggesting that developmental DHA inadequacy can be similarly deleterious in young brains as in aged brains with neurodegenerative conditions.
It is clear from our data that the maternal intake of DHA at an early stage of development greatly influences the synaptic plasticity in the CA1 region of the offspring brain. Significant influence of prenatal availability of DHA on neuro-development in an early stage was apparent as hippocampal neuronal cultures obtained from DHA-deficient E18 embryos develop shorter neurites, less branches and reduced number of synapsin puncta in comparison to DHA-adequate neurons (). Our data also indicated that the developmental deficit, at least for neurite growth and synaptogenesis, because of prenatal DHA-deficiency can be reversed by DHA supplementation at an early stage (), supporting importance of postnatal DHA provision in neurodevelopment. DHA-dependent expression of synapsins and subunits of glutamate receptors, NR and GluR, was also evident from DHA-supplemented in vitro
embryonic cultures () as well as DHA-depleted hippocampal tissues at P-18 (). Although expression of synapsins and NR2A decreased in all regions of DHA-depleted hippocampus, it is interesting to note that the decrease of NR2A was particularly prominent in the CA3 region (). It has been reported that NMDA receptors in the CA3 region play a crucial role in fast learning of one-time experience (Nakazawa et al. 2003
) and associative memory recall (Nakazawa et al. 2002
), implying learning and memory deficit as a consequence of developmental DHA-deficiency.
Underlying mechanisms facilitating synaptic plasticity include the increased release of neurotransmitters and increased synaptic expression of glutamate receptors. Although several forms of LTP exist in the brain, the most prevalent form found in the hippocampal CA1 region involves enhanced sensitivity of post-synaptic neurons to glutamate (Nicoll and Malenka 1995
; Kerchner and Nicoll 2008
). This enhancement can be achieved by the increased activity of existing receptors and/or by increasing the number of receptors at the functional synapses. As LTP in Schaffer collateral-CA1 synapses is known to be mediated by two types of glutamate receptors, NMDA and AMPA receptors (Hayashi et al. 2000
; Grosshans et al. 2002
), the decreased expression of these receptors in DHA-depleted mouse hippocampi potentially contributed to the inhibited LTP. In addition, reduced synapsin1 expression may have played a role as synapsins have been shown to be involved in the formation of a readily releasable pool of vesicles for neurotransmitter release during action potential (Hvalby et al. 2006
; Baldelli et al. 2007
). The implication of the retarded LTP observed with reduced synapsin1 expression under the DHA-deficient condition is particularly noteworthy in light of the previous finding that lack of the synapsin1 gene is associated with human phenotypes displaying learning difficulties (Garcia et al. 2004
Phospholipase activity leading to ARA release has been implicated in neurite growth, particularly in retinoic acid-mediated signaling (Smalheiser et al. 1996
; Farooqui et al. 2004
). In addition, involvement of ARA metabolism by cyclooxygenase-2 in hippocampal long-term synaptic plasticity has been demonstrated (Chen et al. 2002
). The fact that hippocampal development does occur even under DHA-depleted conditions clearly indicates that other mechanisms are also in operation to support the basic neurite growth and synaptogenesis. Unlike the DHA supplementation which significantly increased the DHA level in hippocampal neuronal culture, ARA supplementation increased only its elongation product 22:4n
-6, suggesting that ARA levels are fairly well regulated (Table S1). Similarly, the ARA level was maintained upon in vivo
depletion of DHA, and the reduction of DHA was compensated by the increases of 22:4n
-6 and DPAn
-6, elongation products of ARA (Tables S2 and S3), suggesting a need to maintain ARA at relatively constant levels. Although our study addressed the impact of rather unexplored n
-3 fatty acids on hippocampal development and function, the important role of ARA or its metabolites in neurodevelopment and LTP may not be overlooked. Unlike ARAs involvement in LTP where cyclooxygenase-2-generated prostaglandin E2 regulates membrane excitability and long-term synaptic plasticity (Chen et al. 2002
), the synaptic activity enhanced by DHA supplementation most probably resulted from the DHA-promoted neurite growth and synaptogenesis, as acute application of DHA to the target neurons did not affect synaptic activity.
Our study demonstrates that DHA can promote neurite development, synaptogenesis and expression of synapsins and glutamate receptors, improving glutamatergic synaptic transmission. Inadequate expression of synapsins because of DHA-deficiency during development by maternal n
-3 fatty acid deprivation may hamper synaptogenesis and reduce the pre-synaptic pool of synaptic vesicles for neurotransmitter release during the action potential, similar to the case demonstrated for the hippocampal neurons lacking synapsin 1 (Baldelli et al. 2007
). Concomitantly, reduced expression of glutamate receptors may cause inadequate support for proper glutamatergic synaptic transmission and synaptic plasticity, contributing to impaired learning and memory. In conclusion, our data provide evidence that developmental DHA availability influences hippocampal neurite growth, synaptogenesis and synaptic protein expression critically involved in glutamatergic synaptic transmission and LTP. Compromised DHA-dependent neurodevelopment and synaptic function may be an important mechanism for the learning disability and memory deficit associated with dietary deficiencies of n
-3 fatty acids.