ARTICLE
Auteur(s) : Sylvie Vancassel, Sabah
Aïd, Isabelle Denis, Philippe Guesnet, Monique Lavialle
Unité de Nutrition et Régulation Lipidiques des Fonctions
Cérébrales, NuRéLiCe, INRA, domaine de Vilvert, 78352 Jouy-en-Josas
cedex, France
Introduction
The mammalian brain is particularly rich in polyunsaturated fatty
acids (PUFA), mainly arachidonic acid (AA, 20:4n-6) and
docosahexaenoic acid (DHA, 22:5n-6) for the n-6 and the n-3 series,
respectively. The brain contents of these PUFA are greatly affected
by feeding of oils containing their respective precursor, linoleic
acid (18:2n-6) and alpha-linolenic acid (18:3n-3). Moreover, DHA
could be obtained directly from aquatic sources.
The accumulation of DHA in brain membranes is particularly high
during the perinatal period, coinciding with the formation of
synapses [1, 2]. Thus, Cunnane et al. [3] have shown that during
the first 6 months of life of a human infant, the brain accumulates
around 5 mg of DHA every day, which represents half of the daily
accumulation in the entire body. Then, at the adulthood, DHA can
reach half of the quantity of PUFA inserted into the phospholipids
that build the structure of neuronal membranes.
In addition to this structural function, DHA plays an important
role in cell signaling, as a precursor of molecules such as
docosanoids [4, 5]. It also regulates the expression of many genes,
some of which are involved in the process of synaptic transmission
[6]. Thus, studies using DNA microarrays suggested that n-3 PUFA
regulate the expression of genes involved in cerebral energetic
metabolim (cytochrome c oxydase), synaptic plasticity
(α-synuclein), signal transduction (calmodulin I), neurogenesis or
yet cytoskeleton formation and neurotransmission process
[7-11].
n-3 PUFA and behaviour
For the last decades, many studies have revealed the role of n-3
PUFA, and more particularly DHA, in behaviour and learning. Thus,
rodents subjected to a diet deficient in DHA, or in its precursor,
showed reduced attention, modifications of habituation, anxiety and
locomotor response to novelty [12-16]. They also exhibited an
aggressive behaviour and increased depression symptoms in the
forced swim test [17]. The rodents’ learning ability was also
impaired, and their performance in spatial memory and
discrimination tasks was lower [18-20]. In addition, n-3
PUFA-deficient animals were more resistant to extinction,
suggesting a reduced behavioural flexibility [21]. However, most of
these effects were reversed by a dietary supplementation with long
chain n-3 PUFA [22-24].
These different kinds of behaviours are underlain by different
neuronal pathways that exert a reciprocal presynaptic control in
specific cerebral areas.
Neurotransmission is a fundamental function
In the brain, neurotransmission is a fundamental function that
allows a chemical communication between the different types of
neural cells, through the release of neurotransmitters by
exocytosis process. This process requires the fusion of the
vesicular and the neuronal membranes. Therefore, it strongly
involves phospholipids and fatty acids. It may not be a coincidence
that biological membranes that are naturally enriched in DHA, such
as neurons, rod outer segments and sperms, are predisposed to
undergo vesicle formation and fusion. In addition, exocytosis
involves a number of specialized proteins involved in targeting
vesicles to the active zone and in the mediation of vesicle fusion
with the presynaptic membrane, and some of them have been described
to be located in lipid rafts [25, 26]. Therefore, the lipid
environment, and more particularly the DHA levels in membranes, is
likely to influence the efficiency of the exocytosis.
In this context, we stated the hypothesis that the dietary
modulation of the DHA content of the brain could change the
neurotransmission function and then underlie the impairments of
cognitive processes. In particular, we focussed on the release of
dopamine in basal ganglia, for its key role in locomotion, reward
and motivation, of serotonin in the frontal cortex, involved in
anxiety and of acetylcholine in the septo-hippocampal pathway, for
its crucial role in learning and memory.
Effects of dietary n-3 PUFA deficiency on brain phospholipid
membrane composition
The most widely used method to study the function of DHA in the
nervous tissue is to induce its deficiency in the brain from the
lack of α-linolenic acid in the diet, during the gestation and
lactation periods, throughout one or several generations of
offspring.
We provided F2 rats with a diet free of rapeseed oil, compared
to rats fed a control diet containing rapeseed and peanut oils.
This leads to a severe decrease in DHA levels, notably in the
phosphatidylethanolamine (PE) of the frontal cortex, with a
well-established compensatory substitution by n-6 PUFA (mainly
22:5n-6, DPA), which are normally absent from brain membranes and
thus signs the status of deficiency in the rats [27]. However, we
showed that the range of variations of DPA and DHA levels following
an α-linolenic acid deprivation were not identical in every sub
brain regions. Thus, the most dramatic reduction of the DHA level
takes place in the frontal cortex, as compared to striatum,
hippocampus and septum. Very recently, Levant et al. [28] showed a
depletion of brain DHA content in female undergoing pregnancy and
lactation; this depletion was the greatest in the frontal cortex
compared to seven other cerebral structures. Inversely, Carrié and
collaborators [22] have shown that supplementation of deficient
mice with phospholipids enriched in DHA restored normal fatty acid
composition in all brain regions, except for frontal cortex. These
data illustrated the differences in avidity for DHA between the
cerebral areas previously described [29]. Therefore it seems that
all of the cerebral areas do not respond identically to the lack of
DHA precursor; and this may be in relation with functional
specificity.
Effects of diet-induced decrease in brain DHA on
neurotransmission function
Dysfunction of the dopaminergic mesocorticolimbic loop
We studied the dopaminergic system by measuring the levels of
dopamine and their metabolites (DOPAC and HVA) in the frontal
cortex. The results showed an important reduction of the
neurotransmitter levels in deficient rats (figure 1), whereas the
turnover of DOPAC and HVA increased (figures 2A and 2B)
[30, 31]. Moreover, we subsequently showed that the cortical
release of dopamine after inhibition of the noradrenaline and
dopamine uptakes (by inhibition of the transporters) was unchanged
in deficient rats, whereas, as expected, it increased in the
synaptic cleft of control rats (figure 3).
These data suggested that the process of presynaptic dopamine
storage could be altered in deficient animals. To support this
hypothesis, we looked at the vesicular stage, using dual-probe
microdialysis to monitor the release of dopamine from the vesicular
pool by tyramine stimulation in the frontal cortex and in the
nucleus accumbens, both structures being involved in reward and
learning process. The results showed that the response to tyramine
was significantly reduced in deficient rats, by 70% in the frontal
cortex (figure
4A) and by 90% in the nucleus accumbens (figure 4B) [32-34].
However, this effect was abolished by a resepine pretreatment,
which depletes the dopamine vesicular store, showing a reduced
dopamine reserve in the presynaptic vesicles. These results have
been confirmed by the study of the density of dopamine vesicles,
using immunolabeling with a dopamine antibody and in situ
hybridization of vesicular transporters [31, 33, 34].
All this led to the conclusion that the dopamine vesicle
compartment was reduced by 30% in the frontal cortex of deficient
rats, resulting in a reduced cortical inhibition on the ventral
parts, particularly on the nucleus accumbens. Moreover, the mRNA
expression of D2 receptors was 30% lower in the frontal cortex and
20% higher in the nucleus accumbens of deficient rats [31, 33].
Moreover, Kuperstein et al. [11] has studied in detail the
consequences of DHA deprivation in rats during the early
development on the expression of a battery of genes incoding
neurotransmitter receptors. In particular, they showed a remarkable
elevation of the dopamine D1 and D2 receptors genes, in discrete
regions of the mesolimbic and mesocortical pathways, notably the
nucleus accumbens, the prefrontal cortex and the hippocampus, or
yet in the ventral tegmental area. The authors attributed this
over-expression to a compensatory mechanism resulting from the
possible impairment of the dopamine synthesis, storage or
processing, in order to enable the targeted synapses to act even
with low levels of DHA.
To sum up, all these data suggest that an inadequate intake of
DHA results in a dysfunction of the dopamine mesocorticolimbic
loop, leading to an hypodopaminergia in the cortical areas,
responsible for inattention, and to an hyperdopaminergia in the
nucleus accumbens, responsible for hyperactivity and for an
inefficient reward process. The reduction of dopamine reserves can
then be related to the inappropriate behavioural response
previously observed in deficient animals, since they may now not be
sufficient to achieve a high release during stimulated cognitive
processes.
Alteration of dopamine-related behaviour
Interestingly, a lower dopaminergic activity in the frontal cortex
associated to a higher one in the nucleus accumbens, as we observed
in the deficient rats, is considered to be a biological substrate
of sensation-seeking that could induce locomotor hyperactivity [35,
36]. To explore this relationship, we measured the lomotor activity
in response to novelty in a population of rats placed in a cage of
activity for 1 hour. Despite all these rats fed the same standard
lab diet, we observed an inverse relationship between the DHA
levels in PE of the frontal cortex and the general motor activity:
hyperactive individuals having less DHA than hypoactive ones (figure 5) [37]. When
we compared rats fed a balanced diet and rats fed an α-linolenic
acid deficient diet from conception, we saw that the last exhibited
a severe hyperactivity in a circular corridor during the three
consecutive days of test (figure 6) (unpublished
results). Thus, the response to novelty was negatively linked to
the DHA content of phospholipid membranes, whereas no association
was found with n-6 PUFA.
Dysfunction of the hippocampal serotoninergic system (figure 7)
Using microdialysis, Kodas et al. [38] showed a dramatic increase
in the basal synaptic release of serotonin in the hippocampus of
adult awake rats fed an α-linolenic acid deficient diet, as
compared to controls. Inversely, the release was reduced under
pharmacological stimulation. The authors also studied the recovery
of the serotonin release after the deficient diet was switched to
an n-3 PUFA-adequate diet, at different stages of the
neurodevelopment (at birth, 7 or 14 days postnatal, or at weaning).
The results showed that, when given during the lactation period,
the adequate diet restored both the fatty acid composition of the
brain and the serotonin release. Whereas after weaning, the
adequate diet did not allow any recovery for 5-HT, despite a
normalization of DHA levels in the hippocampal membranes.
Dysfunction of the septo-hippocampal cholinergic system
The cholinergic system was investigated in accordance with its
critical role in the processes underlying arousal, attention,
learning and memory [39-41]. In order to check the proposal of an
alteration of the cholinergic system under DHA-deficient
conditions, we looked at the release of acetylcholine in
hippocampal and cortical synapses of rats fed with diets containing
different amounts of DHA (0 to 300 mg DHA/100 g diet)
supplied by egg-phospholipids.
We showed that first, at rest, the synaptic release of
acetylcholine was increased by 70% in the hippocampus of
α-linolenic acid-deficient animals as compared to controls
receiving adequate levels of n-3 and n-6 PUFA. But, the maximum
KCl-stimulated release was reduced by 30%, associated to a 70% loss
of DHA in phospholipid membranes (figure 8) [27]. Secondly,
we showed that a supply of 200 mg of DHA/100 g diet is
needed to ensure a release of acetylcholine and an incorporation of
DHA in the membranes equivalent to those of the control rats (figure 9) [42]. These
modifications were not related to changes in the catabolic
acetylcholinesterase and choline uptake activities, nor in the
density of the vesicular acetylcholine transporter.
Conclusion
These data show that the variation of the DHA contents in brain
phospholipid membranes is associated with the modification of
several neurotransmission systems, and more particularly with
changes in the release of neurotransmitters. Specific changes in
dopamine release in the frontal cortex and in the nucleus accumbens
seem to be linked, probably through anatomo-functional interactions
between the 2 areas. This can be connected to the effect of a DHA
deficit on the impairment of attention and of the reward process,
that contribute to the damaging of learning and to the slowing of
extinction. Moreover, the basal and the stimulated releases were
inversely affected for acetylcholine and 5-HT in the hippocampus;
this could be involved in reduced spatial learning and increased
anxiety in deficient animals.
It has been shown that these neurochemical changes could
potentially be reversed by an adequate diet, depending on when the
intervention occurs. In particular, weaning seems to be a pivotal
period after that all recovery was impossible.
However, it must be kept in mind that these different neuronal
pathways exert reciprocal presynaptic control in specific cerebral
areas to regulate the behavioural response.
We have now to understand what is the mechanism involved in the
modulation of the neurotransmission function depending on the
presence or not of DHA in brain phospholipids.
The regulation of the synaptic transmission is the result of a
complex metabolic cooperation between three intimate partners: the
endothelial cell for the energy supply, the astrocyte network for
the regulation of functional coordination between cells and
plasticity, and the presynaptic neuron for the release of
neurotransmitters.
We have shown that modifications of DHA levels in the
phospholipid membranes of the three cell types led to changes in
glucose transport [43] but also in gap junction coupling [44]. The
hypothesis can be made that the proportions of DHA in membranes may
have an impact on the morphological plasticity and on the different
astrocyte functions involved in the regulation of synaptic
transmission, and more particularly in the release of
neurotransmitter in the synaptic cleft.
These data will help to understand the relationship between the
n-3 PUFA metabolism and some neuropsychiatric disorders, such as
depression or schizophrenia, in which altered neurotransmission
systems are well known today. Thus, a PUFA imbalance could
contribute to the predisposition to central nervous system
pathologies by acting on the regulation of the neurotransmission
function. This highlights the importance of optimal nutritional n-3
PUFA supply to prevent or at least buffer impairments of brain
functioning.
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