- Top of page
- Materials and methods
Mild reduction in the protein content of the mother's diet from 25 to 8% casein, calorically compensated by carbohydrates, does not alter body and brain weights of rat pups at birth, but leads to significant enhancements in the concentration and release of cortical noradrenaline during early postnatal life. Since central noradrenaline and some of its receptors are critically involved in long-term potentiation (LTP) and memory formation, this study evaluated the effect of mild prenatal protein malnutrition on the α2C-adrenoceptor density in the frontal and occipital cortices, induction of LTP in the same cortical regions and the visuo-spatial memory. Pups born from rats fed a 25% casein diet throughout pregnancy served as controls. At day 8 of postnatal age, prenatally malnourished rats showed a threefold increase in neocortical α2C-adrenoceptor density. At 60 days-of-age, α2C-adrenoceptor density was still elevated in the neocortex, and the animals were unable to maintain neocortical LTP and presented lower visuo-spatial memory performance. Results suggest that overexpression of neocortical α2C-adrenoceptors during postnatal life, subsequent to mild prenatal protein malnutrition, could functionally affect the synaptic networks subserving neocortical LTP and visuo-spatial memory formation.
It has been reported that perinatal malnutrition and severe forms of prenatal malnutrition in the rat, in addition to decrease body and brain weights of pups results in functional changes of central noradrenergic systems, including increased activity of brain tyrosine hydroxylase (Shoemaker and Wurtman 1971; Miller et al. 1978; Marichich et al. 1979), increased concentration of noradrenaline in the whole brain and neocortex (Stern et al. 1975; Morgane et al. 1978; Soto-Moyano et al. 1995), increased release of noradrenaline in the neocortex (Soto-Moyano et al. 1994, 1995, 1998a,b, 1999), and decreased number of α and β adrenoceptors in total brain (Keller et al. 1982) and neocortex (Seidler et al. 1990). Together with these alterations in central noradrenergic profiles, on reaching adulthood, prenatally-malnourished rats on a 6% prenatal/25% postnatal casein diet exhibit learning disturbances, such as deficits in acquisition of alternation tasks (Tonkiss et al. 1990) and impaired visual discrimination learning (Tonkiss et al. 1991). Whether altered central noradrenergic function may be partly responsible for the deficits in cognitive processes showing previously malnourished adult animals has remained an elusive question. For example, together with changes in noradrenergic function, perinatally-malnourished animals also exhibit significant elevations of brain serotonin and 5-hydroxyindoleacetic acid, the main metabolite of serotonin, which may be directly correlated with corresponding increases in brain tryptophan (Morgane et al. 1978). In addition, 5-HT1A receptors are decreased in the hippocampus of malnourished rats during adulthood (Blatt et al. 1994), and dopamine levels have been shown to be diminished in the hippocampus of severely prenatal protein-malnourished rats (Kehoe et al. 2001). It is therefore likely that more than one neurotransmitter system could be involved in the learning disturbances appearing in severely malnourished animals.
In contrast to severe forms of maternal malnutrition, mild reduction of the protein content of the mother's diet, calorically compensated for by carbohydrates, results in apparently normal development in utero of fetuses, as assessed by normal maternal weight gain during pregnancy and normal body and brain weights of pups at birth (Resnick et al. 1982). However, this insidious form of protein maternal malnutrition, so-called hidden prenatal malnutrition (Resnick et al. 1982), results in altered noradrenergic function in the neocortex of the offspring, as revealed by increased concentrations and release of cortical noradrenaline during early postnatal life, followed by decreased cortical release of the neurotransmitter during adulthood (Soto-Moyano et al. 1998b). Together with decreased cortical noradrenaline release, the visual cortex of adult prenatally-malnourished animals shows altered electrophysiological indices, including decreased ability of callosal-cortical synapses to perform temporal summation (Soto-Moyano et al. 1998b). Changes in central noradrenaline activity after hidden prenatal malnutrition are presumably accompanied by changes in the number of brain adrenoceptors that are relevant for memory processing. However, this question has not yet been addressed in detail. The results presented here provide evidence that hidden prenatal malnutrition in rats results, during adulthood, in increased α2C adrenoceptor (α2C-AR) density in both the frontal and occipital cortices, as well as in impaired neocortical LTP and lower visuo-spatial memory performance.
- Top of page
- Materials and methods
A mild reduction in the protein content of the maternal diet did not significantly alter body and brain weights of pups at birth, indicating that the protein diet resulted in apparently normal fetal development as assessed by birth body and brain weights, similar to reports elsewhere (Morgane et al. 1978; Resnick et al. 1982). According to Resnick et al. (1982) and Morgane et al. (1993), fetal growth retardation and reductions in brain weight after prenatal malnutrition occur only after protein restriction, with maternal diets providing less than 6% casein.
[3H]-Rauwolscine binding revealed that α2C-ARs were already present in the frontal and occipital cortices of 8-day-old normal rats; afterwards, the number of binding sites increased slightly, as revealed by [3H]-rauwolscine binding at postnatal day 60. These results are in agreement with studies showing moderate increases in both α2C-AR mRNA expression and [3H]-rauwolscine binding during postnatal development of the rat cerebral cortex, the mRNA for the α2C-AR being already present at birth and [3H]-rauwolscine binding appearing only during the second postnatal week (Winzer-Serhan et al. 1997). In contrast, prenatally-malnourished pups at postnatal day 8 exhibited a threefold increase of α2C-AR density in both the frontal and occipital cortices, as revealed by [3H]-rauwolscine binding. This increase in α2C-AR density is similar to the overexpression of the α2C-AR subtype obtained by mutation of the α2C-gene in mice (Holmberg et al. 2003). To our knowledge, this is the first demonstration in rat pups of the enhancing effect of maternal protein malnutrition on the number of α2C-ARs in the cerebral cortex, which are known to develop postnatally in this animal species (Nicholas et al. 1993; Winzer-Serhan et al. 1997). As mentioned previously, it seems likely that [3H]-rauwolscine binding actually reflects α2C-AR density, since rauwolscine has a 20- to 30-fold higher affinity for the α2C-AR compared with the α2A adrenoceptor in rats and mice (Harrison et al. 1991; Link et al. 1992). Further support for this concept is provided by studies showing that deletion of the gene encoding the α2C-AR results in substantial decreases in [3H]-rauwolscine binding sites in the brain (Link et al. 1995). It should be noted, however, that the α2C-AR population could be slightly overestimated when assessed through [3H]-rauwolscine binding as the ligand may also detect some α2A-ARs in brain regions with high expression of this receptor subtype, such as the cerebral cortex and hippocampus, where 90% of α2-ARs belong to the α2A subtype and only 10% are of the α2C subtype, at least in adult mice (Bücheler et al. 2002). Changes in adrenoceptor density induced by other forms of malnutrition have already been reported. In fact, rats submitted to a low protein diet between days 14 of fetal life and 50 of postnatal age showed decreased whole brain α and β adrenoceptor binding at adulthood (Keller et al. 1982). Nevertheless, it is not possible to compare these data with those of our study, due to the different periods of development during which the nutritional injury occurred (partial prenatal plus postnatal malnutrition versus purely prenatal malnutrition) as well as to the different type of receptors evaluated (the whole population of α receptors versus the α2C-AR subtype). Other studies have shown that moderate prenatal malnutrition increases hippocampal kainate receptor density in adult rats (Fiacco et al. 2003) and striatal NMDA receptor binding in adult female rats (Palmer et al. 2004). Furthermore, it has been reported that severe protein restriction during gestation in rats increases the expression of microtubule-associated protein type 1 (MAP 1), which remains elevated until adulthood (Gressens et al. 1997). As has been pointed out in the literature, MAP 1B is abundant in the newborn rat brain (Schoenfeld et al. 1989) and is associated with neurite outgrowth (Dehmelt and Halpain 2004). Moreover, MAP 1B has been suggested to be important for synapse formation in the rat cerebral cortex (Kawakami et al. 2003). In contrast, MAP 1A is very low or absent in developing axonal fibers but increases during development and maturation of dendritic processes (Schoenfeld et al. 1989). These results, together with the present observations, support the idea that nutritional insults during fetal life may induce long-term postnatal changes in neurotransmitter receptor expression, and in other proteins that are involved in neuronal growth and development. At 60 days of age, α2C-AR density in the two cortical regions studied had decreased in prenatally-malnourished rats compared with 8-day-old prenatally-malnourished pups, although a 50% increase in [3H]-rauwolscine binding above the control values could still be detected. This suggests that changes induced by prenatal malnutrition in α2C-AR expression persist in the rat long after postnatal nutritional recovery, which could result in altered noradrenergic function in adult age.
Prenatal malnutrition in rats resulted in impaired neocortical LTP and decreased visuo-spatial memory performance in adult animals. The foregoing data suggest that prenatally-malnourished young adult rats were unable to maintain normal LTP in the frontal and occipital cortices in vivo, although alternative hypotheses involving increased threshold of neural elements excited in the cortex could also be possible. Also, the progressive decrease in the number of errors and time spent during task execution by malnourished animals indicated that they were able to learn the general strategies of the task. However, their scores were always higher than those of normal animals, indicating that protein malnutrition during fetal life adversely affected their ability to perform in the Olton maze in adulthood. Deficits induced by prenatal malnutrition in task acquisition in adult rats have already been reported but in those studies, moderate protein restriction to pregnant mothers (6% protein diet) had been used as the paradigm (Tonkiss et al. 1990). While it is tempting to speculate that the deficits in behavioral performance of rats in the Olton maze reported here are due to visuo-spatial memory deficits, a number of alternative interpretations are possible (for reviews see Cain and Saucier 1996; Cain 1998). For example, it is possible that prenatal protein malnutrition could generate some visual impairment that interfered with performance in the visuo-spatial memory test. In this regard, it has been reported that visual impairments can interfere with the apparent learning of mazes in rats (O'Steen et al. 1995; Spencer et al. 1995). However, there are no studies showing that prenatal malnutrition may result in some type of visual impairment at postnatal age. Another possibility is that malnutrition could affect visuo-spatial learning by altering anxiety levels or stress responses to the test situation. In this respect, it has been shown that prenatal malnutrition can affect the exploratory behavior and avoidance of adult rats in the elevated T-maze test, probably through reduction of anxiety (Almeida et al. 1996a,b). As another alternative, it is possible that prenatal malnutrition interferes with motor performance at adulthood, rather than producing a true learning impairment. In this regard, it has been shown that motor performance on a revolving drum of adult rats undernourished during gestation plus lactation did not differ from that of well nourished control rats (Tonkiss and Smart 1983). However, other studies suggest that mature rats born from 6 and 8% casein diet-restricted mothers, who continue the protein malnutrition paradigm during the lactation period, were hyperactive in the open field but tended to perform at control levels on the learning measurements in eight-arm radial maze testing (Wolf et al. 1986). It is therefore apparent that the question of whether prenatal protein malnutrition altered the performance in the Olton maze at adulthood by affecting visual-spatial memory and/or by generating sensorimotor disturbances remains unresolved, and further research would be necessary to elucidate this aspect.
Whether the effects of prenatal malnutrition on LTP and visuo-spatial memory are related to the marked postnatal increase in α2C-AR density observed in the neocortex of malnourished rats is unknown at present, although reported data have shown a relationship between overexpression of α2C-ARs and poorer navigation capacity in the water maze (Björklund et al. 2000). The functional consequences of increased α2C-AR expression in the neocortex of developing rats could be analyzed, keeping in mind the possible role of these receptors in the brain. In this regard, it has been shown that α2C-ARs are involved in many physiological processes, such as body temperature regulation, sensorimotor integration and cognitive functions, including modulation of the acoustic startle reflex and its pre-pulse inhibition, isolation-induced aggression, development of behavioral despair and spatial working memory, as well as modulation of dopamine and serotonin release (Sallinen et al. 1997, 1998a,b, 1999; Björklund et al. 1998, 1999, 2000; Tanila et al. 1999). In addition, α2C-ARs are involved in the presynaptic control of noradrenaline release from peripheral neurons (Hein et al. 1999). It has also been reported that this receptor subtype could play a minor role in the release of noradrenaline in the central nervous system (Bücheler et al. 2002). α2C-ARs seem to play a negative role in long-term memory formation, since α2C-overexpressing mice performed less well in the water maze (Björklund et al. 1998, 1999, 2000). Interestingly, α2C-overexpressing animals also performed less well in a visible platform water maze, suggesting that in addition to memory deficits, other sensorimotor disturbances could be present in these animals. The fact that overexpression of α2C-ARs leads to deficits in water-maze performance suggests that the remarkable increase in this receptor subtype in the neocortex of malnourished rats early in life could be involved in the decreased neocortical LTP and in the lower visuo-spatial memory shown in these animals when they reach adulthood. In this respect, it is worth pointing out that noradrenaline is crucially involved in the generation of brain regressive events during development (Wendlant et al. 1977; Blue and Parnavelas 1982; Caviness 1989), and that its concentration and release are significantly increased in the brain of neonates born from dams receiving a 7.2% protein diet (Stern et al. 1975; Morgane et al. 1978; Soto-Moyano et al. 1998b, 1999). Therefore, it can be argued that hyperactive central noradrenergic mechanisms induced by prenatal protein malnutrition, operating upon an unusually high number of neocortical α2C-ARs, could disrupt the developmental programming of several processes, including receptor expression of some neurotransmitter systems, axonal growth and synaptic network formation, thereby contributing to impair plasticity in the neocortex and/or to depress memory formation at later stages of development. The possibility that disturbances in neocortical LTP and visuo-spatial memory are mainly mediated by the increased α2C-AR population persisting until adulthood cannot be discarded, since Björklund et al. (1999) showed that the α2 adrenoceptor antagonist, atipamezole, fully reversed the deficit in platform finding and search strategy in genetically α2C-overexpressing mice submitted to a water maze navigation paradigm. Further studies investigating the effect of specific α2C-AR blocking agents and α2C-AR antisense targetting on neocortical LTP and memory performance in adult prenatally-malnourished adult rats would be helpful in elucidating these aspects.
Abnormalities in hippocampal function of malnourished animals as a factor contributing to the deficits in visuo-spatial learning seem to be less probable, on the basis that early functional alterations induced by prenatal malnutrition on electrophysiological properties of hippocampal neurons seem to disappear on reaching the adult age (Rushmore et al. 1998). However, caution must be taken regarding this question, since it is thought that nutritional rehabilitation could contribute to ameliorating long-term alterations in hippocampal plasticity and related learning and memory deficits, though by no means fully reversing them (Morgane et al. 2002). In contrast, a bulk of structural changes and functional disturbances are still present in the neocortex long after postnatal nutritional recovery (Morgane et al. 1993; Levitsky and Strupp 1995). These are probably related to the limited structural/functional recuperative capacity of the neocortex due, amongst other factors, to the absence of postnatal neurogenesis. Interestingly, it has been reported that lesions in several neocortical areas, including the prefrontal and occipital cortices, impair the performance in the water maze (Kolb et al. 1994; Compton et al. 1997; Espinoza et al. 1999; Hoh et al. 2003). This evidence is consistent with the long held view that the neocortex is an important site for neuronal changes that underlie learning and, in addition, may mechanistically link chemical/functional neocortical disorders induced by prenatal malnutrition in the rat neocortex, such as altered α2C-AR expression and synaptic potentiation, to the impaired visuo-spatial performance observed in these animals.
In summary, the present data show that mild prenatal protein malnutrition results in increased α2C-AR density in the rat cerebral cortex during postnatal life, together with decreased LTP and visuo-spatial performance at adulthood.