The neural basis of attentional alterations in prenatally protein malnourished rats.

Protein malnutrition during gestation alters brain development and produces specific behavioral and cognitive changes that persist into adulthood and increase the risks of neuropsychiatric disorders. Given evidence for the role of the prefrontal cortex in such diseases, it is significant that studies in humans and animal models have shown that prenatal protein malnutrition specifically affects functions associated with prefrontal cortex. However, the neural basis underlying these changes is unclear. In the current study, prenatally malnourished and control rats performed a sustained attention task with an unpredictable distractor, a task that depends on intact prefrontal cortical function. Radiolabeled 2-deoxyglucose was used to measure neural and brain network activity during the task. Results confirmed that adult prenatally malnourished rats were more distractible than controls and exhibited lower functional activity in prefrontal cortices. Thus, prefrontal activity was a predictor of task performance in controls but not prenatally malnourished animals. Instead, prenatally malnourished animals relied on different brain networks involving limbic structures such as the hippocampus. These results provide evidence that protein reduction during brain development has more wide-reaching effects on brain networks than previously appreciated, resulting in the formation of brain networks that may reflect compensatory responses in prenatally malnourished brains.

Prenatal protein level impacts homing behavior in Long-Evans rat pups.

OBJECTIVE: This study assessed the effect of varying prenatal protein levels on the development of homing behavior in rat pups. METHODS: Long-Evans rats were fed one of the four isocaloric diets containing 6% (n = 7 litters), 12% (n = 9), 18% (n = 9), or 25% (n = 10) casein prior to mating and throughout pregnancy. At birth, litters were fostered to well-nourished control mothers fed a 25% casein diet during pregnancy, and an adequate protein diet (25% casein) was provided to weaning. On postnatal days 5, 7, 9, 11, and 13, homing behaviors, including activity levels, rate of successful returns to the nest quadrant and latencies to reach the nest over a 3-minute test period were recorded from two starting positions in the home cage. Adult body and brain weights were obtained at sacrifice (postnatal day 130 or 200). RESULTS: Growth was impaired in pups whose mothers were fed a 6% or, to a lesser extent, a 12% casein diet relative to pups whose mothers were fed the 18 and 25% casein diets. The 6 and 12% prenatal protein levels resulted in lower activity levels, with the greatest reduction on postnatal day 13. However, only the 6% pups had reduced success and higher latencies in reaching the nest quadrant when compared with pups from the three other nutrition groups. Latency in reaching the nest quadrant was significantly and negatively associated with adult brain weight. DISCUSSION: Home orientation is a sensitive measure of developmental deficits associated with variations in prenatal protein levels, including levels of protein deficiency that do not lead to overt growth failure.

Prenatal protein malnutrition decreases KCNJ3 and 2DG activity in rat prefrontal cortex.

Prenatal protein malnutrition (PPM) in rats causes enduring changes in brain and behavior including increased cognitive rigidity and decreased inhibitory control. A preliminary gene microarray screen of PPM rat prefrontal cortex (PFC) identified alterations in KCNJ3 (GIRK1/Kir3.1), a gene important for regulating neuronal excitability. Follow-up with polymerase chain reaction and Western blot showed decreased KCNJ3 expression in the PFC, but not hippocampus or brainstem. To verify localization of the effect to the PFC, baseline regional brain activity was assessed with (14)C-2-deoxyglucose. Results showed decreased activation in the PFC but not hippocampus. Together these findings point to the unique vulnerability of the PFC to the nutritional insult during early brain development, with enduring effects in adulthood on KCNJ3 expression and baseline metabolic activity.

Early childhood malnutrition predicts depressive symptoms at ages 11-17.

BACKGROUND: We examined the prevalence of depressive symptoms in Barbadian youth with histories of infantile malnutrition and in a healthy comparison group and the extent to which the effect of malnutrition was mediated/moderated by maternal depression. METHODS: Depressive symptoms were assessed using a 20-item scale administered to youths (11-17 years of age) who had experienced an episode of protein-energy malnutrition (marasmus or kwashiorkor) during the first year of life and in a comparison group of healthy youths without a history of malnutrition. Their mothers completed the same questionnaire on the same test on three occasions when their children were 5-17 years of age at 2-5-year intervals. RESULTS: The prevalence of depressive symptoms was elevated among previously malnourished youth relative to healthy comparison children (p < .001). When youth depression scores were subjected to a longitudinal multiple regression analysis, adjusting for the effect of maternal depressive symptoms, significant effects due to the history of early childhood malnutrition remained and were not discernibly attenuated from an unadjusted analysis. We also found significant independent effects of maternal depressive symptoms on youth depressive symptoms. CONCLUSION: Early childhood malnutrition contributed independently to depressive symptoms in youths who experienced a significant episode of malnutrition in the first year of life. This relationship was not mediated or moderated by the effects of maternal depression. Whether the later vulnerability to depression is a direct effect of the episode of malnutrition and related conditions early in life or whether it is mediated by the more proximal neurobehavioral effects of the malnutrition remains to be determined.

Prenatal protein malnutrition induces a phase shift advance of the spontaneous locomotor rhythm and alters the rest/activity ratio in adult rats.

Evidence is accumulating for significant structural and functional changes within the central nervous system (CNS) following prenatal protein malnutrition. Included among the structures that are likely to be affected are the suprachiasmatic nuclei (SCN) involved in the regulation of locomotor activity, sleep-wake cycle, and drinking behavior. To determine the effects of prenatal protein malnutrition on the spontaneous activity rhythm, 24 h radiotelemetric measurements were recorded over an 8-day period. Male offspring of rats provided with protein-deficient (6% casein) or adequate (25% casein) diets for 5 weeks prior to mating and throughout pregnancy were studied. Well nourished rats displayed a rise in activity level during the first hour of the 12h light phase, whereas prenatally malnourished rats displayed this increase during the 12h dark phase, approximately 50 min before lights on, reflecting a significant phase advance in this group. In addition, cosinor analysis revealed that the alpha/rho relationship was affected in the previously malnourished group, the activity phase being shorter than in the well-nourished animals. These findings suggest changes in the regulatory systems controlling the locomotor activity rhythm as a consequence of prenatal protein malnutrition. Alterations in entrainment to the light-dark cycle, and/or in the coupling force of the circadian oscillators are all candidate mechanisms.

Birthdates and number of neurons in the serotonergic raphe nuclei in the rat with prenatal protein malnutrition.

The effect of prenatal protein deprivation on timing of neurogenesis and on number of neurons generated in the serotonergic dorsal (DR) and median raphe (MR) nuclei of the rat was studied. These neurons are of interest because their neurogenesis occurs during the period of malnutrition and their axonal projections participate in the earliest stages of brain development. In this study, dams were maintained on a 25% casein diet or a 6% casein diet 5 weeks prior to mating and throughout pregnancy. At birth, all pups were cross-fostered to dams on a 25% casein diet. Bromodeoxyuridine, a thymidine analog that is incorporated into nuclear deoxyribonucleic acid during the cell cycle synthetic phase, was used as a marker of neurogenesis. Bromodeoxyuridine was administered on either embryonic day 11, 12, 13 or 14. On postnatal day 30, serial sections of raphe nuclei were processed with bromodeoxyuridine immunocytochemistry to determine the number of raphe cells generated on each day and with Nissl stain to determine the total number of cells generated. There were no significant differences between the two diet groups in timing of generation or in total number of cells generated, indicating that neurogenesis of these early generated neurons appears unaffected by concomitant protein deprivation.

Development and modulation of GABA(A) receptor-mediated neurotransmission in the CA1 region of prenatally protein malnourished rats.

Prenatal protein malnutrition has been demonstrated to result in alterations in the serotonergic and GABAergic neurotransmitter systems in the rat hippocampus. In the present study, whole-cell patch clamp recordings of CA1 pyramidal cells were employed in an effort to gain insight into the specific cellular locus and functional consequences of the previously reported changes. Hippocampal slices were prepared from Sprague-Dawley rats whose dams were fed either a normal (25% casein) or low (6% casein) protein diet during pregnancy. The development of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) and their modulation by the benzodiazipine agonist zolpidem were compared in cells from the two nutritional groups at postnatal days 7, 14, 21 and >90. The modulation of mIPSCs by serotonin was also examined in cells from 21 day old rats. No significant differences were observed in the characteristics of mIPSCs in cells from control vs. prenatally protein malnourished rats at any of the ages studied, although there was a trend for a higher frequency of mIPSCs in adult (>p90) prenatally protein malnourished rats. At all ages, zolpidem produced a significant increase in the mean decay time of mIPSCs that was not significantly different in cells from the two nutritional groups. Serotonin application resulted in a significant increase in the frequency of mIPSCs in CA1 pyramidal cells but there was no significant difference between cells from the two nutritional groups in the characteristics of this effect. These data demonstrate that the previously observed alterations in the serotonergic and GABAergic systems that result from prenatal protein malnutrition do not have significant functional consequences at a single cell level in the CA1 region of the rat hippocampus as measured in vitro.

Prenatally protein-malnourished rats are less sensitive to the amnestic effects of medial septal infusions of chlordiazepoxide.

Evidence is mounting that prenatal protein malnutrition affects the physiological properties of the GABAergic neurotransmitter system in rats. To investigate the functional behavioral consequences of these changes, chlordiazepoxide (CDP, a positive modulator of the GABA(A) receptor) was applied directly to the medial septum and the amnestic response appraised. In adulthood, male offspring of rats provided with a protein-deficient diet (6% casein) for 5 weeks prior to mating and throughout pregnancy underwent stereotaxic surgery to implant steel cannulae aimed at the medial septum. After recovery, spatial learning performance in the submerged platform version of the Morris water maze task was assessed immediately following a 1 microl infusion of either artificial cerebrospinal fluid (aCSF), or one of three doses of CDP (15, 30 and 60 nmol). Well-nourished control rats demonstrated a robust amnestic response to intraseptal CDP. During task acquisition, well-nourished rats administered each of the doses exhibited significantly longer escape latencies than those given aCSF. On the probe trial (platform removed) a lower proportion of time was spent in the target quadrant (all three doses) at a greater average distance from the former platform location (30 and 60 nmol doses). In contrast, prenatally malnourished rats exhibited a muted sensitivity to CDP, most notable at the 30 nmol dose. These findings provide further support for functional changes within the GABAergic system consequent to malnutrition.

Maternal depressive symptoms affect infant cognitive development in Barbados.

This longitudinal study is part of a series examining the relationships between maternal mood, feeding practices, and infant growth and development during the first 6 months of life in 226 well-nourished mother-infant dyads in Barbados. In this report, we assessed maternal moods (General Adjustment and Morale Scale and Zung Depression and Anxiety Scales), feeding practices (scales describing breast-feeding and other practices associated with infant feeding in this setting), and infant cognitive development (Griffiths Mental Development Scales). Multivariate analyses, with and without controlling for background variables, established significant relationships between maternal moods and infant cognitive development. Infants of mothers with mild moderate depression had lower Griffiths scores than infants of mothers without depression. Maternal depressive symptoms and lack of trust at 7 weeks predicted lower infant social and performance scores at 3 months. Maternal moods at 6 months were associated with lower scores in motor development at the same age. Although no independent relationships emerged between feeding practices and infant cognitive development, the combination of diminished infant feeding intensity and maternal depression predicted delays in infant social development. These findings demonstrate the need to carefully monitor maternal moods during the postpartum period, in order to maximize the benefits of breast-feeding and related health programs to infant cognitive development.

Prenatal protein malnutrition results in increased frequency of miniature inhibitory synaptic currents in rat CA1 pyramidal cells.

There is growing evidence for an effect of prenatal protein malnutrition on the GABAergic neurotransmitter system in the rat hippocampus and associated structures. In the present study, we examined the functional electrophysiological consequences of observed alterations in GABA(A) and benzodiazepine receptor systems. Whole-cell patch clamp recordings of spontaneous and of miniature inhibitory postsynaptic currents (mIPSCs) generated by CA1 pyramidal cells were performed in in vitro hippocampal slices prepared from control and prenatally protein malnourished adult male rats. The characteristics of spontaneous synaptic currents were unaltered by the prenatal insult, as were the amplitudes and kinetics of GABA(A) receptor-mediated mIPSCs. The frequency of mIPSCs, however, was significantly increased in CA1 pyramidal cells in slices prepared from prenatally malnourished vs. control rats. The effect of the benzodiazepine receptor agonist chlordiazepoxide on the characteristics of mIPSCs was also examined and found to be the same in cells from both nutritional groups. The increased frequency of mIPSCs together with the lack of a change in amplitude, kinetics, or modulation by benzodiazepines of mIPSCs in response to prenatal protein malnutrition indicate a presynaptic locus of effect of this insult.

Prenatal protein malnourished rats show changes in sleep/wake behavior as adults.

Prenatal protein malnutrition significantly elevates brain levels of serotonin in rats, and these levels remain elevated throughout their lives. This biogenic amine is involved in the regulation of many physiological functions, including the normal sleep/wake cycle. The present study examined the effects of prenatal protein malnutrition on the sleep/wake cycle of freely moving adult rats. Six prenatally protein malnourished (6% casein) and 10 well-nourished (25% casein) male rats (90-120-day-old) were chronically implanted with a standard set of electrodes (to record cortical electroencephalogram, neck muscle electromyogram, electrooculogram, and hippocampal theta wave) to objectively measure states of sleep and wakefulness. Six-hour polygraphic recordings were made between 10.00 and 16.00 h; a time when the rats normally sleep. Prenatally malnourished rats spent 20% more time in slow wave sleep (SWS) compared to the well-nourished rats. The total percentage of time spent in rapid eye movement (REM) sleep was 61% less in prenatally malnourished rats compared to well-nourished control rats. These findings demonstrate the adverse consequences of prenatal protein malnutrition on the quality and quantity of adult sleep in rats. These sleep changes are potentially detrimental to normal social behavior and cognitive functions. Prenatally malnourished rats are an excellent animal model to study the role of endogenous serotonin in the regulation of the normal sleep/wake cycle.

Chlordiazepoxide-induced spatial learning deficits: dose-dependent differences following prenatal malnutrition.

The sensitivity of prenatally protein-malnourished rats to the amnestic properties of the benzodiazepine (BZ) receptor agonist, chlordiazepoxide (CDP), was studied in the male offspring of rats provided with a protein-deficient diet (6% casein) for 5 weeks prior to mating and throughout pregnancy. Rats were tested during acquisition of the submerged platform version of the Morris water maze task using three systemic doses of CDP (3.2, 5.6, and 7.5 mg/kg i.p.) at two ages (day 30 and day 90). At 30 days, prenatally malnourished rats showed less sensitivity to the amnestic effect of the 5.6-mg/kg dose when compared with well-nourished controls by displaying shorter swim paths during acquisition and a more selective search of the target quadrant upon removal of the platform (probe trial). At 90 days, prenatally malnourished rats again showed less sensitivity to CDP at a dose of 5.6 mg/kg, but more sensitivity to the 3.2-mg/kg dose (indicated on the probe trial). No obvious relationship was identified between the nutritional group differences in behavioral sensitivity to CDP at 90 days and their BZ receptor density in the hippocampus or medial septum. It can be concluded that prenatal malnutrition alters the amnestic response to CDP in a dose-dependent and developmentally specific manner, thus providing further support for functional changes within the GABAergic system subsequent to malnutrition.

Modulation of paired-pulse responses in the dentate gyrus: effects of prenatal protein malnutrition.

Since our major hypothesis is that prenatal protein malnutrition significantly affects hippocampal neuroplasticity, this study examined the effects of prenatal protein malnutrition on the modulation of dentate granule cell excitability in freely moving rats at 15, 30 and 90 days of age across the vigilance states of quiet waking (QW), slow-wave sleep (SWS) and rapid eye movement (REM) sleep. Using paired-pulse stimulation, the paired-pulse index (PPI), a measure of the type and degree of modulation of dentate granule cell excitability elicited by stimulation of the medial perforant path, was obtained for each vigilance state at each stage of development. Four specific measures of granule cell excitability were computed, namely, PPI using both population spike amplitude (PSA) and EPSP slope measures, absolute values of PSA(1) and EPSP(1) slope. PPI values obtained at 15, 30 and 90 days of age, however, were altered during normal ontogenetic development, but not by vigilance state. At 15 days of age, the malnourished group exhibits greater early inhibition of the PPI using the PSA measure at IPIs between 20 and 30 ms regardless of vigilance state, while at 30 days of age, the malnourished group exhibits greater facilitation at IPIs between 50 and 70 ms during QW and SWS, but not during REM sleep. In the control adult (PND90) and juvenile (PND30) animal, PSA(1) values are significantly higher during SWS than in QW or REM sleep. However, for the younger malnourished animals (PND15 and PND30), PSA(1) values were found to be significantly greater during REM sleep rather than SWS. Therefore, as the animal matures, there appears to be a shift in vigilance state dependent synaptic transmission through the hippocampal trisynaptic circuit from REM sleep to SWS in both control and malnourished animals, with the change occurring later in malnourished animals when compared to control ones. Furthermore, our findings suggests that prenatal protein malnutrition significantly alters modulation of dentate granule cell excitability (i.e., PPI values using the PSA measure) during the earlier stages of development but not in adulthood.

The effects of median raphé electrical stimulation on serotonin release in the dorsal hippocampal formation of prenatally protein malnourished rats.

Our previous work had shown an enhanced inhibition in the hippocampal formation of prenatally protein malnourished rats. We have also found a diminishment in 5-hydroxytryptamine (5-HT) fibers in the hippocampal formation of malnourished rats as well as increased levels of 5-HT in the brain. The purpose of the present study was to determine 5-HT release in the dorsal hippocampal formation following electrical stimulation of the median raphé nucleus (MRN) in unanesthetized prenatally malnourished rats. Stimulation of this nucleus at 20 Hz in malnourished rats resulted in a significantly diminished release of 5-HT compared to well-nourished rats. The latter group showed a lesser, though still significant, decrease in 5-HT release following raphé stimulation. Basal release of 5-HT prior to stimulation was significantly higher in malnourished rats as compared to well-nourished controls. This may be the result of a decreased density of 5-HT neurons leading to a diminished control of release. Stimulation of the MRN in behaving malnourished animals may markedly affect the recurrent negative feedback collaterals onto somatodendritic 5-HT(1A) and 5-HT(1D) autoreceptors thus enhancing the inhibitory effects of stimulation of the median raphé on 5-HT release. Studies are underway to examine the sensitivity of both the somatodendritic and terminal 5-HT autoreceptors in malnourished animals, in order to understand possible mechanisms for our findings.

Maternal moods predict breastfeeding in Barbados.

This study was designed to identify psychosocial variables affecting early infant feeding practices in Barbados. The sample included 93 healthy women and infants born at the Queen Elizabeth Hospital who were extensively evaluated 7 weeks, 3 months, and 6 months after birth. Maternal moods were assessed with the Zung Depression and Anxiety Scales and the General Adjustment and Morale Scale. Feeding practices were evaluated using a questionnaire developed for this population. The prevalence of mild depression in this population was 16% at 7 weeks and increased to 19% at 6 months, whereas there were very few cases of moderate-to-severe depression. Disadvantaged environmental conditions, including less information-seeking by the mother, lower family income, and poor maternal health, were closely associated with increased symptoms of depression and anxiety in all women. However, significant predictive relationships between mood and feeding practices remained even when the effects of the home environment were controlled. Specifically, depressive symptoms at 7 weeks postpartum predicted a reduced preference for breastfeeding at current and later infant ages. Conversely, feeding practices did not predict maternal moods at later ages. These findings have important implications for public policy dealing with programs promoting breastfeeding. Early interventions designed to treat mild postnatal depression should be instituted early in the postpartum period to improve the chances for successful breastfeeding.

Prenatal protein restriction increases sensitization to cocaine-induced stereotypy.

The present study characterized the total amount of stereotyped behavior following acute and repeated administration of cocaine in male and female prenatally protein malnourished rats. Adult offspring of female Sprague-Dawley rats fed either a low (6% casein) or adequate (25% casein) protein diet 5 weeks prior to mating and throughout their pregnancy were studied. Once every 3 days (for a total of six injections), half the rats from each nutritional treatment group (repeated exposure) were injected with cocaine (30 mg/kg, i.p.) and their total amount of stereotypy (rearing, forepaw treading, compulsive sniffing and head bobbing) monitored. The remaining rats received five saline injections followed by a cocaine injection on the last injection day (acute exposure group) and their behavioral response was also measured. Despite being slightly less sensitive to cocaine following their first injection, by the sixth injection, prenatally protein malnourished animals in the repeated-exposure group exhibited significantly greater sensitization to the psychomotor stimulant effects of cocaine than well-nourished controls. In the acute exposure groups, however, prenatally malnourished males, but not females, exhibited significantly more stereotypy than well-nourished subjects following a single cocaine injection. These findings have implications for characterizing addiction potential in the previously malnourished rats, as well as providing additional information regarding factors which can influence sensitization.

Influence of prenatal protein malnutrition on behavioral reactivity to stress in adult rats.

In Experiment 1, adult prenatally protein malnourished and well-nourished male and female rats were tested in an open field after having been subjected to a 15-day regimen of varied uncontrollable and inescapable mild stress (experimental group). Their responses were compared with rats that had not been subjected to the stress regimen (control group). In the control group, females with a history of prenatal malnutrition made significantly fewer entries into the center of the arena than did well-nourished females, suggesting that baseline differences in anxiety exist between the two nutritional groups of females. In addition to open field, die experimental group of animals was also tested in a forced swim test conducted at the beginning (Day 5) and at the end of the stress regimen (Day 15). Significant differences were observed between nutritional groups on Day 15 only: prenatally malnourished males exhibited a lower latency to immobility than well-nourished males, whereas the opposite effect was found in malnourished females. In Experiment 2, separate groups of males were exposed to forced swim on two different occasions without the stress regimen between exposures. A somewhat different pattern of findings was generated. There was no significant difference in the latency to immobility between malnourished and well-nourished rats on the second forced swim. However, malnourished animals showed greater total immobility than the well-nourished controls in the second exposure to forced swim, providing further support for the interpretation that the malnourished males were less affected than well-nourished ones, or adapted more readily to the stress regimen in Experiment 1. Overall these results suggest that the relationship between prenatal malnutrition and stress depends on the level of stress (acute vs. chronic), the type of behavioral measure used to assess its effects, as well as gender.

Impaired Response of Serum IGF-I Levels to Puberty in Previously Malnourished Adolescents.

Serum levels of IGF-I were measured in Barbadian children, aged 9-15 years, half of whom had experienced protein-energy malnutrition limited to the first year of life. Despite current nutritional adequacy, menarche was delayed more than one year in the girls with a history of early malnutrition and their IGF-I levels failed to show the 60% postmenarchic increase seen in the controls. In addition, the IGF-I levels of boys and girls with prior malnutrition in infancy were not significantly correlated with current anthropometric measurements, whereas IGF-I values of control boys and girls were significantly correlated for almost every growth parameter. Infantile malnutrition may cause an alteration in hypothalamic function resulting both in delayed onset of hypothalamic pituitary functions needed for puberty, and in an impaired growth hormone-IGF-I response.

Differential Effects of Prenatal Protein Malnutrition and Prenatal Cocaine on Radial Arm Maze Performance in Adult Male Rats.

The effects of prenatal cocaine and protein malnutrition were examined on acquisition of the radial arm maze in adult male Sprague-Dawley rats whose mothers were provided with a 6% casein, a 25% casein or a standard chow diet and cocaine (30mg/kg) or saline injections beginning 5 weeks prior to mating and continuing to parturition. Rats were tested using an 8-arm radial maze with 4 baited arms and were required to collect all 4 food pellets within 5 min to complete a trial. Subjects were tested for 1 trial/day until they met criterion for successful acquisition of the task. Criterion was attained when the rat collected 3 out of the 4 food pellets within their first 4 arm entries within a trial (while still completing the trial) with this level of performance being maintained for 3 consecutive trials. The results showed dissociation between the effects of prenatal protein malnutrition and prenatal cocaine. Prenatally protein malnourished adult male rats required a greater number of trials to criterion, made more reference memory (but not working memory) errors, and required a longer time to complete each trial when compared with control males. However, rats with prenatal cocaine exposure showed no significant impairments in the radial arm maze. These results contrast with our previous findings using the Morris maze task in which adult male subjects exhibited impaired acquisition following prenatal cocaine while there were no effects following prenatal protein malnutrition. Thus, the radial arm maze and water maze procedures appear to engage different processes that are differentially sensitive to the prenatal insults.

Effect of Prenatal Protein Malnutrition on Birthdates and Number of Neurons in the Rat Locus Coeruleus.

The effect of prenatal protein deprivation on the timing of neurogenesis and on the number of neurons generated in the locus coeruleus of the rat was studied. These neurons are of interest as their axon projections are involved in the earliest stages of cerebral cortical development. Dams were maintained on a 25% casein diet or a 6% casein diet five weeks prior to mating and the diets continued throughout the pregnancy. At birth, all pups were cross-fostered to dams on a 25% casein diet. BrDU, a thymidine analog that is incorporated into the nuclear DNA during the synthetic phase of the cell cycle, was used as a marker of the generation period. It was administered intraperitoneally (25 mg/kg body weight) on embryonic day 10, 11, 12, 13, or 14. On postnatal day 30, the brain stems were processed with BrDU immunocytochemistry to determine the relative number of neurons generated on each day, and with Nissl stain to determine the total number of neurons generated in the two groups. There were no significant differences between the two diet groups in the timing of their generation or in the total number of neurons generated, indicating a preservation of neurogenesis of these early generated neurons in these malnourished rats.