Maternal nutrition plays a crucial role in the offspring’s brain development during pregnancy and during a critical period after birth. Initial studies on perinatal programming have shown that malnutrition during pregnancy and/or lactation induces metabolic disorders such as hyperphagia, adiposity, hyperlipidemia and glucose intolerance in offspring, even into adulthood [1
]. Moreover, growing evidence has revealed a modulation of the offspring behavior related to depression, anxiety-like behaviors and addiction (preference of hypercaloric foods and drugs) due to the consumption of a hypercaloric diet in the perinatal period, being associated with long-term structural and functional alterations in the neurodevelopment of the prefrontal cortex (PFC) [5
Behavioral disorders related to chronic stress/insults are associated with changes in glutamatergic and GABAergic neurotransmission in the hippocampus and prefrontal cortex [9
]. The adequate modulation of these neurotransmitters is essential for the adaptive behavioral response. During stressful experiences, including nutritional insult, there is neuroplasticity of glutamatergic signaling in the brain areas involved in stress response. An excessive glutamate activity and/or lack of gamma-aminobutyric acid (GABA) can generate negative associative memories, being associated with depression and anxiety [12
A fatty diet can be considered a stressor affecting central neurotransmission, especially in areas directly related to metabolic control, such as the hypothalamus or the hippocampus, but also in areas that control eating behavior, such as the prefrontal cortex [15
]. A high-fat diet during the perinatal period affects the cognitive functions of the offspring at different stages of age, and may be attributed to neurochemical changes in the hippocampus and PFC [16
There are a set of homeostatic processes that control the release of neurotransmitters after neuronal activation; the most highly characterized is the endocannabinoid system (ECS), comprising the endogenous lipids anandamide (AEA, arachidonoylethanolamide) and 2-arachidonoylglycerol (2-AG), their target cannabinoid (CB) receptors CB1 and CB2, and their synthetic and catabolic enzymes. ECS is a retrograde neuromodulatory system of the major excitatory (glutamatergic) and inhibitory (GABAergic) synapses in the brain. Endogenous cannabinoids are released postsynaptically, and bind to presynaptic cannabinoid receptors, thus inhibiting the release of glutamate or GABA [17
]. It is known that in the face of acute stress, the levels of the endocannabinoid 2-arachidonyl glycerol (2-AG) increase in involved brain areas such as the hippocampus and PFC [19
ECS develops during prenatal and early postnatal life, in humans and animals, in the PFC. ECS is highly concentrated at interneuron synapses, playing a critical role in the developmental trajectory of GABAergic interneurons [20
]. In addition, several studies have demonstrated that the ECS is altered in different brain areas of the offspring born from mothers with a caloric restriction or a hypercaloric diet during the perinatal period [3
Since anxiety disorders have been related to regional dysregulation of excitatory/inhibitory neurotransmission pathways, we investigated the possible implication of glutamatergic and GABAergic signaling in the anxiogenic action of the perinatal and/or postnatal palatable diets, in the PFC of the male and female offspring in adulthood. Given the modulatory role of the ECS in glutamatergic and GABAergic neurotransmission during development, we hypothesized that the expression of the main elements of this homeostatic system are also altered in the adult offspring’s PFC by the perinatal and/or postnatal exposure to a palatable diet.
With this aim, we analyze the expression of the receptors and the enzymes of synthesis and degradation of the endocannabinoids, as well as the expression of the main glutamatergic and GABAergic receptors in the PFC of the offspring in the adult state, after a perinatal and/or postnatal hypercaloric diet.
The present study indicates that hypercaloric diet consumption during the perinatal period induces anxiety-related behavioral changes in adult offspring that kept consuming the palatable diet after weaning (PP animals). Furthermore, increased anxiety in PP animals is associated with specific changes in the prefrontal cortex’s (PFC) mRNA expression of ionotropic and metabotropic glutamate receptors, such as Grin1, Gria1 and Grm5, and endocannabinoid signaling elements, such as the Cnr1 receptor or the endocannabinoid-synthesis/degrading enzymes Diacylglycerol lipase-beta (Daglβ) and N-acyl phosphatidylethanolamine phospholipase D (Nape-pld). The alterations suggest an enhanced glutamatergic activity in the PFC, associated with a reduced inhibitory input into the glutamate synapses through the cannabinoid CB1 receptor, whose expression was decreased. These changes are sex-dependent, being more marked in males.
Traditionally, maternal nutrition studies have focused on offspring metabolic diseases, such as glucose tolerance, insulin resistance, dyslipidaemia, hypertension and obesity, in humans and rodents [4
]. However, it is known that continued stress, such as malnutrition with an excess or deficiency in most nutrients, impacting fetal neurodevelopment, can cause neuropsychiatric disorders, including depression, anxiety and attention-deficit/hyperactivity disorder in offspring, even though to adulthood [32
]. Thus, both exposure to a high-fat diet and calorie restriction during the perinatal period can lead to eating disorders in offspring, such as hyperphagia and the preference for hypercaloric food [33
]. In addition to eating disorders, perinatal exposure to a high-fat diet has been shown to increase anxiety-related behaviors in offspring, this effect being independent of the type of diet consumed during postnatal life [36
Our results confirm that a free-choice palatable diet (P) during the perinatal period exerts an effect by itself on the offspring’s time in, and number of entries into, the central area of the open field test. However, we also observed an interaction between the maternal diet and the postnatal diets, affecting the same parameters mentioned, as well as the time in and number of entries into the open arms of the elevated plus maze test. Thus, the male PP group showed the most anxiety-like behaviors, associated with a decrease in these four behavioral indicators despite normal locomotion. These results also indicate a marked sexual dimorphism in long-term hypercaloric diet-induced anxiety behavior.Earlier studies reported the implications of a hypercaloric diet for brain structures and the neurotransmitter systems important in anxiety. For example, obese animals are susceptible to develop anxiety-like behaviors, probably through changes in the glutamatergic and GABAergic neurotransmission within the ventromedial and dorsomedial hypothalamic nucleus [39
], which demonstrated that a high-fat/high-sugar diet induces alterations in the function of the prefrontal cortex through changes in the expression of GABAergic parvalbumin-expressing inhibitory interneurons, which was associated with increased anxiety-like behaviors.
Maternal malnutrition has also been shown to play an important role in programming behavioral disorders, such as anxiety, associated with alterations in the neurodevelopment of the brain areas involved in behavior. However, most studies are focused on the hypothalamic–pituitary–adrenal (HPA) axis [1
]. The prefrontal cortex (PFC) provides executive control by coordinating cognitive, emotional and behavioral responses to threatening stimuli, however, the precise role of signaling within PFC, with respect to the stress of a maternal hypercaloric diet, has been minimally investigated.
Our results showed an overall activation of PFC glutamate signaling in the PP male group, with an increase in the mRNA levels of the glutamate synthesis enzyme Gls
, the glutamate transporter Slc1a1
, the ionotropic receptors NMDA type Grin1
, the ionotropic receptors AMPA type Gria1
, and the metabotropic receptors Grm3
. Several studies relate behavioral disorders in offspring, such as schizophrenia and anxiety-like behaviors, to alterations in GABAergic signaling (in involved areas such as HPA axis, hippocampus and cortex) induced by such perinatal insults as lipopolysaccharide (LPS), drugs and malnutrition [1
]. Contrarily, our data show no effect of perinatal and/or postnatal free-choice P diet on GABAergic signaling in PFC of adult offspring. Furthermore, our results showed a negative correlation between anxiety-related behavioral tests and the main altered genes of the glutamatergic system. Thus, an increase in the expression of the glutamatergic system is associated with less time spent in the central area of the open field test, and less time in, and number of entries into, the open arms of the elevated plus maze test; that is, greater anxiety.
So, results from the present study suggest that the impact of the maternal and/or postnatal P diet on anxiety levels in adult offspring relate to alterations in PFC glutamate levels, or its modulatory systems, including the endocannabinoid system (ECS), as we will discuss below.
The ECS plays an important role in various physiological functions, including neuroprotection, synaptic plasticity and energy homeostasis. ECS is a neuromodulator of the major excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmitter systems in the brain. In addition, the ECS plays a crucial role during critical periods of brain development, and its disruption by early stressful events, including metabolic disruptions, can lead to significant neuropsychiatric symptoms. [17
]. Our results are in agreement with previous studies that demonstrate alterations in cannabinoid signaling in the brain of adult offspring induced by maternal malnutrition [8
]. We found a decrease in the mRNA expression of the cannabinoid receptor Cnr1
in the PFC of adult animals born from mothers fed with a free-choice palatable diet, and who continued eating the P diet after weaning. Furthermore, the protein levels of CB1 are also affected by the maternal diet, with an overall decrease in its expression in males compared to the control group. The palatable diet consumed from the perinatal period to adulthood (PP groups) also induces a decrease in gene levels of Nape-pld
, the main synthesis enzyme of anandamide (AEA). The decrease of both Cnr1
in the PP groups could indicate a decrease in AEA, that would explain the increase in anxiety in these animals, since the anxiolytic role of this endocannabinoid has been demonstrated, and the pharmacological augmentation of central endogenous cannabinoid (eCB) signaling being a promising treatment for anxiety disorders [44
Our results also discard a potential diet-associated neuroinflammation factor in these observed long-term alterations, despite previous reports suggesting that a hypercaloric diet might result in neuroinflammation, thus contributing to behavioral disorders [40
]. Our results do not support this hypothesis, since we did not find alterations in the mRNA expression of several genes that would indicate the activation of inflammatory pathways, such as the toll-like receptor related to pathogen recognition and the activation of innate immunity Tlr4
, the cyclooxygenase key in prostaglandin biosynthesis Ptgs2
, the microglia/macrophage-specific calcium-binding protein Iba1
, the major intermediate filament proteins of mature astrocytes Gfap
, and the cannabinoid receptor Cnr2
, induced by the perinatal and/or postnatal P diet in the adult offspring PFC.
The perinatal and/or postnatal hypercaloric diet can exert a direct effect on cannabinoid signaling, decreasing cannabinoid tone, interrupting the negative feedback that this system exerts on glutamatergic neurotransmission, which is thus increased. There could also be a direct effect of diet on glutamatergic signaling, by increasing it. Both effects could explain the increase in the anxious behavior of PP animals. Although we have not addressed the impact of specific nutrients as potential sources for the effects observed, we must consider that the fatty acid composition of the diet might dysregulate the amount of endocannabinoid precursors [46
], and that maternal hypoproteic diets can also disrupt brain glutamatergic systems [47
In the present study we have found some inconsistencies between the genetic and protein results. We consider these differences to be a limitation of the study, due to the low number of samples analyzed by western blot. So, future extensive protein studies will be necessary to elucidate this controversy.