The main finding of the present study is that newborn rats exposed to a moderate caloric restriction during the entire pregnancy displayed alterations in endocannabinoids and/or endocannabinoid-related lipids, in brain structures involved in the regulation of metabolism and emotional and cognitive responses. Specifically, male and female offspring from diet-restricted dams exhibited decreased levels of the main endocannabinoids, their precursor and/or NAEs in the hypothalamus and, conversely, they showed enhanced content of these lipids in the hippocampus. This similar profile between males and females from calorie-restricted dams was not evident in the olfactory bulb, where the calorie-restricted female offspring presented decreased levels of AEA, their precursor (AA) and PEA. Moreover, these modifications were accompanied of underweight at birth, a common result when diet restriction is applied for the entire pregnancy or in the last phases of pregnancy [12, 13]. Interestingly, this finding has been widely associated to metabolic and behavioral abnormalities later in life [12–14].

The majority of animal research conducted to evaluate the effects of undernutrition in critical developmental windows has focused on investigating the deleterious effects of a severe gestational diet restriction in the offspring [12, 13]. In our study, we have adopted a moderate maternal calorie-restriction, that may have simulated better the reduction in food intake documented in some human studies, commonly associated to decreased weight gain during pregnancy [5, 6, 52], and could have prevented unnecessary effects in the animals. In rodents, this type of maternal restriction has been demonstrated previously to be enough to induce long-lasting alterations in offspring [11, 14, 53, 54]. Moreover, we have recently showed that the preconceptional and gestational exposure to a moderate calorie-restricted diet increases the risk of developing features of metabolic syndrome as well as behavioral abnormalities in the offspring [47, 55]. Importantly, in the present study, we have demonstrated for the first time that a moderate maternal caloric restriction applied during the entire pregnancy alters brain endocannabinoid and/or endocannabinoid-related lipid levels at birth in male and female offspring and reduces the weight at birth.

The modifications in the supply of nutrients to the fetus may have altered the intrauterine growth leading to inadequate size at birth in our study. Regarding the most important nutrients during intrauterine life, apart from glucose and aminoacids, the fatty acids and, particularly, the long-chain (LC) PUFAs, such as the arachidonic acid (AA) and docosahexaenoic acid (DHA) has been revealed as critical elements for a correct growth and neurodevelopment [37, 56]. The concentration of LC-PUFAs and their precursor depends on diet, fatty acid storage in the adipose tissue and endogenous synthesis, which requires adequate functionality of the enzymes involved in desaturation and elongation of essential fatty acids [57]. Therefore, to ensure an adequate fatty acid supply, the fetuses and new born animals depend on the mother nutritional status and the placenta functionality to obtain both essential fatty acids (EFAs) and long-chain fatty acids (LC-PUFAs). This is due to the limited capacity of the fetus to transform EFAs and the importance of depositing the PUFAs in key tissues, including the fetal brain, during intrauterine growth [57, 58]. Regarding the endocannabinoids, little is known about the fetal-maternal relationship in endocannabinoid content. It has been proposed that, although n-6 PUFA derivatives may be synthetized by the fetus in the tissues, the placenta may also transfer maternal endocannabinoids to the fetus by contributing to maintenance of the endocannabinoid basal tone [27]. Consequently, a maternal caloric restriction in our study may have had an impact on the levels of PUFAs and their derivatives, including the endocannabinoids, in the fetus, and leading to alterations in endocannabinoid and related lipid levels in different brain structures.

Indeed, we found decreased levels of endocannabinoids and/or endocannabinoid-related lipids in the hypothalamus of male and female offspring. Specifically, we found decreased AA levels in the hypothalamus of male offspring, without any change in the concentrations of AA-derived endocannabinoids (i.e., AEA and 2-AG). In contrast, the female offspring exhibited decreased levels of 2-AG in this brain region despite the unchanged concentrations of its precursor (AA). Apart from the reduced concentration of the LC-PUFAs after a caloric restriction, including the reduction of the AA, precursor of endocannabinoids, an alternative explanation to these findings may involve a sex-specific alteration in the activity and/or levels of the endocannabinoid metabolic enzymes. Therefore, in an attempt to maintain endocannabinoid and/or AA homeostasis, an increased synthesis of 2-AG and AEA could have lately determined the reduction of AA in male offspring. Conversely, an increased degradation of 2-AG may have determined its reduction and maintained the AA content in females unchanged. Further analysis of the expression and activity of enzymes responsible for biosynthesis and degradation of endocannabinoids has to be addressed to confirm this hypothesis. Another possibility is that an increased endocannabinoid transfer from mother, through placenta, might have also contributed to maintain an endocannabinoid tone in the hypothalamus of male offspring [27], despite the decreased AA availability and the depletion in maternal tissues due to undernutrition. This idea is supported by previous studies showing differences in male and females placentas after inadequate maternal diets [59, 60].

Our finding in male hypothalamus contrasts with a previous report [45], describing decreased hypothalamic endocannabinoid levels in male offspring coming from dams exposed to a 50% calorie-restricted diet during late pregnancy. However, in this interesting study and differently to ours, the endocannabinoid measurement was performed at weaning, and not at birth. Considering that the pups are independent from the maternal fatty acid stores at this developmental stage, the levels of endocannabinoids here may easily have reflected the metabolic status of the pups and, thus, their own fatty acid stores. Specifically, in this previous study the pups at weaning exhibited decreased body weight that was accompanied by a reduction of hypothalamic AEA. Although the body weight at birth was not reported in this study, similar types of restriction have been associated to decreased body weight at birth, which suggests that these animals did not exhibited a rapid catch-up growth. This phenomenon has been described after caloric restriction in pregnancy and/or small fetuses associated to hyperphagia [12, 13] and is known to induce a severely dysfunctional metabolic phenotype in the offspring later life [13, 61]. Therefore, the decreased hypothalamic anandamide described in the study of Matias et al. (2003) might have affected the appetite [62, 63] in these animals, leading to a lean phenotype at weaning and favoring complete recovery at adulthood. Although we did not evaluate the long-term effects on the offspring, the opposite might have occurred in our study. Particularly, we found decreased levels of OEA in the hypothalamus of male offspring from calorie-restricted mothers. Considering that this NAE is involved in the modulation of satiety [64], our data suggest a possible increased appetite in these animals, similar to previously reported in offspring exposed to undernutrition in pregnancy and undergoing to rapid catch-up growth during lactation and infant period and impaired metabolic phenotype at adulthood [13, 61].

Moreover, in female hypothalamus we found decreased levels of PEA, a NAE with anti-inflammatory and antiobesity properties [65, 66], suggesting the possibility of inflammatory status in female offspring, which has been associated to the development of metabolic and behavioral abnormalities [67–69].

The reduction of the levels of OEA and PEA in male and female offspring may be a consequence of a limited amount of the precursors required for their synthesis, particularly the oleic acid and/or palmitic acid, as previously proposed in adult animals [70, 71] and recently demonstrated in humans [72]. Additionally, the impairment of the activity and/or levels of the enzymes implicated in the synthesis and degradation of NAEs might also explain these results. Further research is needed to address these possibilities.

Concerning the possible interpretation to our findings in hypothalamus, it is important to note that previous studies have showed that the reduction in both AA and endocannabinoid levels in this brain structure at birth is associated to the development of metabolic disturbances at adulthood [47, 51]. Although alterations in endocannabinoids were not observed in male offspring in this brain region, the decreased levels of AA and their association with decreased birth at weight found in the present study might have promoted adverse consequences in the development of hypothalamus. This supposition takes into account that AA is a LC-PUFA involved in growth and brain development and is the precursor of the eicosanoids, which contribute to regulate cell proliferation, growth, immunity and inflammation [37].

We also evaluated the endocannabinoid levels in the hippocampus, a brain structure involved in modulating emotional responses and where the endocannabinoid system plays an important role in memory formation and neurogenesis associated to metabolism-dependent mechanisms [29, 73]. Intriguingly, we found increased hippocampal levels of AA and 2-AG at birth in male offspring from calorie-restricted dams, which is opposite to the findings in the hypothalamus. In the female hippocampus we also found increased concentration of AEA, AA and PEA levels. The increased endocannabinoid and AA levels found in our animals might reflect a fetal adaptation against the reduced availability of nutrients to preserve hippocampal development. This idea is supported by previous works showing fluctuating BDNF levels, a protein related to endocannabinoids [26], in different stages of the brain development of offspring from severely calorie-restricted dams [23]. Interestingly, a peak in 2-AG levels at PN1 in the whole brain has been described together with increased expression of the cannabinoid receptor type 1 (CB₁), suggesting the importance of endocannabinoids (i.e., 2-AG) and endocannabinoid synthesis precursors (i.e., AA) for an adequate brain development [46]. Considering this evidence, the increased 2-AG and AA levels at PN0 in male offspring may reflect a premature peak to prevent the deleterious effects on hippocampus development. In female offspring we found increased AEA and PEA levels. It is interesting to note that the enzyme fatty acid amide hydrolase (FAAH) degrades both AEA and PEA [74], which suggests that an eventual alteration in this metabolic enzyme may be implicated. Moreover, the increased levels of AA, 2-AG and AEA in male and female offspring suggest that implementing a moderate caloric restriction in rat previously well-nourished might have a modest impact on the fatty acid storage at the beginning of pregnancy, favoring the deposition of AA in hippocampus. In particular, it is interesting to note that these findings contrasts with the results recently described by our group in offspring from mothers exposed to the same caloric restriction during the preconceptional and gestational period, and with presumably decreased maternal stores. In this previous study, we observed decreased levels of AEA in hippocampus at birth in association to increased anxiety-related responses in adolescence [47], suggesting that the potential compensatory effect of increased endocannabinoids in the hippocampus is inverted in worse nutritional conditions. Further research is needed to confirm these possibilities.

Although the role of the endocannabinoid system in behavioral programming has not been well established yet, alterations in hippocampal endocannabinoid content are known to promote impaired emotional and cognitive responses. For instance, a decrease in the hippocampal 2-AG level has been correlated to anxiety-related responses [51, 75, 76], and the blockade of anandamide reuptake specifically in the hippocampus produces anxiolytic effects [77]. Furthermore, increased 2-AG in hippocampus was associated to mitigation of the cognitive alterations in severely undernourished mice supplemented with a diet rich in fish oil, an important source of n-3 PUFAs [78], although the opposite has been described in an animal model of schizophrenia [79]. In the context of nutritional programming, emotional responses and cognitive performance have been found to be affected after exposure to undernutrition during critical windows of development and/or in new born small for gestational age [14, 20, 80] and in a sex specific-manner [81, 82]. Despite this evidence, the increased levels of 2-AG, AEA and AA we found in the hippocampus are difficult to interpret, considering that CB₁ receptor activation by endocannabinoids may mediate bimodal opposite responses depending on the differential distribution of CB₁ in distinct neuronal populations [29].

Additionally, we measured endocannabinoid and NAE levels in the olfactory bulb of male offspring. The contribution of the endocannabinoid system has been revealed recently in this brain structure, where CB₁ receptor stimulation increases odor perception and food intake in fasted animals [48]. In male offspring, we did not detect any modification in the endocannabinoid levels in the olfactory bulb from calorie-restricted dams, even though these animals displayed higher levels of OEA in the hypothalamus, which was probably associated to disrupted hunger and/or feeding behavior in these animals. However and interestingly, the female offspring displayed decreased levels of AEA, AA and PEA in the olfactory bulb, suggesting alterations in feeding behavior and an inflammatory status. The findings in female offspring are in agreement to a previous report showing that prenatal adverse conditions (such as prenatal stress) can affect odor preference in a sex specific-manner, leading to alterations in odor preference in female offspring [83] and suggesting that the females might have increased vulnerability in this brain structure after exposure to adverse perinatal conditions.

The changes in endocannabinoid and/or endocannabinoid-related lipid levels found in the hypothalamus and hippocampus of male and female offspring from calorie-restricted dams raise several questions. On the one hand, as modifications in these lipid regulators were found in developing brain structures in association with decreased weight at birth, we cannot discard the possibility that these alterations might have long-lasting consequences in the offspring, as it has been previously reported [12–14]. Indeed, alterations in endocannabinoid signaling in brain structures involved in the modulation of metabolism and emotional responses may lead to inadequate neuronal wiring or subtle alterations in neuronal connectivity and favor vulnerability to diseases later in life [27]. Moreover, previous studies have shown that alterations in endocannabinoid signaling during early development after treatment with specific agonists/antagonists of the CB₁ cannabinoid receptors are associated to long-lasting neurochemical, endocrine and behavioral effects [39–43]. In support of this notion, we have recently reported changes in brain endocannabinoids and endocannabinoid-related lipids at birth after inadequate maternal diets in hypothalamus and hippocampus in association with metabolic and behavioral alterations [47, 51]. On the other hand, it is possible that some brain structures were protected from the effects of a moderate caloric restriction implemented only during pregnancy by the preferential uptake of the fetal tissues of specific LC-PUFAs, such as AA or n-3 PUFAs. It is interesting to note here that the n-3 PUFAs can affect the levels of endocannabinoids by decreasing their levels by competing for the metabolic enzymes [70, 84], or by increasing their levels depending on different circumstances [78, 85]. Additionally, the presence of sexual dimorphism mainly associated to the alterations found in olfactory bulb and the PEA levels in all the brain regions of females, suggest that the maternal calorie-restriction might have affected the male and female offspring through different mechanisms. Further investigations are needed to explore these possibilities.