The role of milk fat globule membranes in behavior and cognitive function using a suckling rat pup supplementation model

Milk fat globule membranes (MFGM) surround droplets delivering lipids to the breast-fed infant and are enriched with glycoproteins upon exocytosis out of the mammary cell. MFGM is typically removed during processing of cow milk for the manufacture of infant formula. Recent clinical trials have found that formula supplemented with bovine MFGM improved cognition. Here, we aimed to explore the mechanisms behind this improved developmental outcome. Sprague–Dawley rats were bred, and their litters were manipulated to either 10 pups to represent normal growth (N) or 16 pups to represent restricted growth (R) per dam. From postnatal day (PD) 2, pups were supplemented daily by oral gavage with MFGM or nonfat milk (NFM, control) at 100 mg/kg body weight. Pups were supplemented until PD13 and killed or supplemented until PD21 and then later exposed to cognitive testing (T-maze and passive avoidance). R NFM-supplemented female rats had lower T-maze scores than the N NFM females. The R MFGM-supplemented animals, however, did not show lower cognitive scores. Restricted growth and treatment affected the passive avoidance test scores. At PD14, MFGM was shown to increase mRNA expression of genes involved in brain function in both N and R animals, including brain-derived neurotrophic factor [1.51-fold change (fc) N, 1.36 fc R] and St8 alpha-N-acetyl-neuraminide alpha-2,8-sialytransferase 4 (1.62 fc N) (P<.01). Our findings suggest that MFGM plays a role in later cognitive development by early up-regulation of genes involved in brain function.     

Endogenous clock gene expression in the suprachiasmatic nuclei of previsual newborn rabbits is entrained by nursing

The rabbit is particularly suitable for investigating the development of mammalian circadian function. Blind at birth, the pups are only visited by the mother to be nursed once every 24 h for about 3 min and so can be studied largely without maternal interference. They anticipate the mother's visit with increased behavioral arousal and with a rise in body temperature, both of which represent endogenous circadian rhythms. We now report that in newborn pups the suprachiasmatic nuclei of the hypothalamus (SCN; the main circadian pacemaker in mammals) show endogenous 24‐h rhythmicity in the expression of the clock genes Per1, Per2, and Bmal1. Pups nursed from postnatal days 1 to 7 and fasted to day 9 showed the same rhythms of clock gene expression as normally nursed controls. We also report that these rhythms are entrained by nursing. Pups killed on postnatal days 3–4 showed the same rhythms in gene expression as pups in the previous experiment, whereas littermates subsequently nursed from postnatal days 4 to 7 with nursing delayed 6 h showed a corresponding shift in the diurnal pattern of clock gene expression. Consistent with this, two groups of pups implanted with telemetric thermal sensors and nursed 6 h apart had daily patterns in body temperature synchronized with the two different nursing times. We conclude that the expression of clock genes associated with the newborn rabbit's circadian system is entrained by nonphotic cues accompanying nursing, the exact nature of which now needs to be clarified. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Maternal dietary tryptophan deficiency alters cardiorespiratory control in rat pups

Malnutrition during pregnancy adversely affects postnatal forebrain development; its effect upon brain stem development is less certain. To evaluate the role of tryptophan [critical for serotonin (5-HT) synthesis] on brain stem 5-HT and the development of cardiorespiratory function, we fed dams a diet ～45% deficient in tryptophan during gestation and early postnatal life and studied cardiorespiratory variables in the developing pups. Deficient pups were of normal weight at postnatal day (P)5 but weighed less than control pups at P15 and P25 (P < 0.001) and had lower body temperatures at P15 (P < 0.001) and P25 (P < 0.05; females only). Oxygen consumption (Vo(2)) was unaffected. At P15, deficient pups had an altered breathing pattern and slower heart rates. At P25, they had significantly lower ventilation (Ve) and Ve-to-Vo(2) ratios in both air and 7% CO(2). The ventilatory response to CO(2) (% increase in Ve/Vo(2)) was significantly increased at P5 (males) and reduced at P15 and P25 (males and females). Deficient pups had 41-56% less medullary 5-HT (P < 0.01) compared with control pups, without a difference in 5-HT neuronal number. These data indicate important interactions between nutrition, brain stem physiology, and age that are potentially relevant to understanding 5-HT deficiency in the sudden infant death syndrome.     

Littermate Influence on Infant Growth in Mice: Comparison of SJL/J and ICR as Cotransferred Carrier Embryos

In mice, a minimum number of healthy embryos is required to trigger and maintain pregnancy. Therefore, when recovering mouse embryos from a limited litter, one useful technique is to transfer carrier ICR embryos along with the embryos of interest, a technique referred to as cotransfer. In this study, we examined suitable mouse strains for cotransfer with C57BL/6J (B6) embryos in regards to the maintenance of pregnancy, number of pups born, intrauterine growth, and postnatal growth. Because the coat color of B6 is black, we compared two white coat-colored strains, SJL/J and ICR. Cotransfer of SJL/J and ICR embryos had similar effects on maintenance of pregnancy, number of pups born, and intrauterine growth. However, the postnatal growth of B6 mouse pups cotransferred and grown with SJL/J pups was better than for B6 mouse pups cotransferred and grown with ICR pups, suggesting competition among littermates. These results demonstrate that cotransfer of SJL/J embryos will be useful not only as carrier embryos with B6-background embryos but also as a model system to examine littermate competition.     

The effect of late prenatal and/or early postnatal zinc deficiency on the development and some biochemical aspects of the cerebellum and hippocampus in rats

In rats, late prenatal and/or early postnatal zinc deficiency results in behavioural anomalies in adult animals, but not in overt dysmorphogenesis of the central nervous system. Cerebellar and hippocampal development occurs mainly in the first three weeks postnatally and zinc accumulates specifically in the mossy fibres of the hippocampus during this period.In the present investigation, rat pups were suckled by dams fed a zinc-deficient (<0.5 mg/kg) diet either from day 19 of pregnancy or from parturition. Control animals were restricted-fed the same diet supplemented with 100 mg zinc/kg. Studies were performed on pups either on day 18 postpartum in the case of animals fed the experimental diets from parturition, or on day 20 for pups which received treatment from day 19 of gestation.Cerebellar and hippocampal weights were lower in pups suckling from zinc-deficient dams but zinc levels were not affected in either organ, although histological evidence suggested less zinc in the hippocampal mossy fibres. Incorporation of H-thymidine into cerebellar and hippocampal DNA was not affected by maternal zinc status, nor was the activity of the zinc metalloenzyme alkaline phosphatase.The activity of the myelin-marker enzyme 2′, 3′-cyclic nucleotide 3′-phosphohydrolase was substantially lower in both regions of the brain in zinc deprived pups, especially in the hippocampus. Activity of the zinc metalloenzyme L-glutamic acid dehydrogenase was also diminished in both tissues from 20-day-old pups and in the hippocampi of 18-day-old animals.The data suggest that cerebellar and hippocampal DNA synthesis is not seriously affected by late prenatal and/or early postnatal zinc depletion, but that the activities of two enzymes associated with neural function are. The possibility is raised that these defects may be associated with the behavioural changes observed in rats subjected to zinc impoverishment during the period of maximal cerebellar and the hippocampal development.     

Comparative Effects of Ethanol and Malnutrition on the Development of Catecholamine Neurons: Changes in Neurotransmitter Levels

Abstract: The comparative effects of exposure to ethanol and malnutrition on the concentrations of tyrosine and catecholamines in whole brain and selected regions of brain have been studied in the developing rat. These animals were the offspring of optimally nourished rats (control pups), of rats fed a diet with 35% of the calories supplied by ethanol (ETOH pups), or of animals fed a diet calorically equivalent to the latter but lacking ethanol (iso-caloric, 1C pups). These diets were administered to dams either during the last week of gestation (prenatal) or during lactation (postnatal). Tyrosine levels were elevated prior to birth in the prenatal ETOH or IC pups or at 1 and 2 weeks of age in postnatal ETOH or 1C pups as compared with values found in the control offspring. Dopamine concentration in whole brain was significantly lower in prenatal ETOH pups than in prenatal IC pups at 3 weeks of age. Levels in the brains of postnatal ETOH pups were lower than control values, but not relative to animals exposed to 1C diet. Investigation of corpus striatum showed a significant decrease in dopamine concentration compared with control or IC pup values as a result of postnatal exposure to ethanol. Norepinephrine levels in the whole brain of prenatal ETOH pups were consistently 30–40% lower than either control or matched 1C pups during development. At 3 weeks of age, the norepinephrine levels in the hypothalamus of animals exposed to ethanol pre or postnatally were 30–60% lower than values in the corresponding region in either control or 1C pups. In the rat model described, ethanol caused a decrease in catecholamine levels, perhaps solely by affecting the norepinephrine neurons.     

Postnatal development of the denervated lung in normoxia, hypoxia, or hyperoxia.

We asked whether lung innervation was essential for the normal postnatal development of the lung in conditions of normoxia, hypoxia, or hyperoxia. Litters of newborn rats were assigned to a normoxic [inspired oxygen partial pressure (PIO2) = 150 Torr, eight litters], hypoxic (PIO2 = 100 Torr, nine litters), or hyperoxic (PIO2 = 360 Torr, nine litters) group. Each litter consisted of 12 pups. Two days after birth, one-third of the litter had the vagus and sympathetic trunk cut in the neck on the left side [left denervated (L)], one-third was denervated on the right side (R), and one-third was sham-operated (S). From day 3, all pups were exposed to the designed PIO2, until day 8 or days 21-22. Almost all rats, whether S, R, or L, survived in normoxia and hyperoxia, whereas in hypoxia survival at day 22 of R and L was approximately 60-65%. Body growth was the same in S, R, and L and less in hypoxia than in normoxia or hyperoxia. At days 8 and 22, hematocrit and hemoglobin concentration, heart and lung dry and wet weights, and lung DNA content did not differ among S, R, and L, whether the pups were raised in normoxia, hypoxia, or hyperoxia. At days 21-22, aerobic metabolism and breathing pattern, both measured during air breathing, as well as compliance of isolated lungs, were also similar among S, R, and L for each of the conditions in which the pups were raised.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential metabolism of brown adipose tissue in newborn rabbits in relation to position in the litter huddle

Competition for resources can contribute importantly to the early development of individual differences in behavioral and physiological phenotypes. In newborn rabbits, littermates compete for thermally favorable positions within the litter huddle. As brown adipose tissue (BAT) is the principal site of thermogenesis in such altricial young, we investigated differences in rabbit pups’ growth and morphological differences in BAT associated with position within the huddle. We formed three treatment groups (7 litters/group): GI—birth (pups killed at birth); GII—chronic thermal challenge (pups killed after exposure to a moderately cold environmental during postnatal days 1–3); GIII—acute thermal challenge (as for GII but pups killed after an additional 30 min exposure to a very cold environment on postnatal day 3). Interscapular BAT was removed at death for histological analysis, and triglyceride concentrations measured in serum. Pups occupying central positions in the huddle had higher skin temperatures, obtained more milk, and were more efficient at converting this into body mass, than pups occupying peripheral positions. There was no significant difference in BAT morphology or triglyceride concentrations between pups at birth, nor between central and peripheral pups chronically exposed to moderate cold until postnatal day 3. However, during acute cold exposure at this age, peripheral pups were less able to maintain their body temperature, they depleted BAT fat reserves almost completely, and they had lower serum concentrations of triglycerides than central pups. These findings confirm the contribution of early sibling relations to individual differences in growth and metabolic processes associated with thermoregulation in newborn rabbits.     

Characterizing the functional significance of the neonatal rat vibrissae prior to the onset of whisking

The present series of experiments assessed how information from the whiskers controls and modulates infant rat behavior during early learning and attachment. Passive vibrissal stimulation can elicit behavioral activity in pups throughout the first two postnatal weeks, although orienting to the source of stimulation is evident only after ontogenetic emergence of whisking. In addition, while pups were capable of demonstrating learning in a classical conditioning paradigm pairing vibrissa stimulation with electric shock, no corresponding changes were detected in the anatomy of the barrel cortex as determined by cytochrome oxidase (CO) staining. Finally, the role of whiskers in a more naturalistic setting was determined in postnatal day (PN)3-5 and PN11-12 pups. Our results showed that both nipple attachment and huddling were disrupted in whisker-clipped PN3-5 pups but only marginally altered in PN11-12 pups. Together, these results suggest that the neonatal whisker system is behaviorally functional and relevant for normal mother-infant interactions, though it lacks the sophistication of a mature whisker system that evokes very specific and directed responses.     

Characterizing the functional significance of the neonatal rat vibrissae prior to the onset of whisking

The present series of experiments assessed how information from the whiskers controls and modulates infant rat behavior during early learning and attachment. Passive vibrissal stimulation can elicit behavioral activity in pups throughout the first two postnatal weeks, although orienting to the source of stimulation is evident only after ontogenetic emergence of whisking. In addition, while pups were capable of demonstrating learning in a classical conditioning paradigm pairing vibrissa stimulation with electric shock, no corresponding changes were detected in the anatomy of the barrel cortex as determined by cytochrome oxidase (CO) staining. Finally, the role of whiskers in a more naturalistic setting was determined in postnatal day (PN)3-5 and PN11-12 pups. Our results showed that both nipple attachment and huddling were disrupted in whisker-clipped PN3-5 pups but only marginally altered in PN1I 1-12 pups. Together, these results suggest that the neonatal whisker system is behaviorally functional and relevant for normal mother-infant interactions, though it lacks the sophistication of a mature whisker system that evokes very specific and directed responses.     

Demonstration of a quiescent period for feeding controls in the developing rat

Feeding behaviour of rats during development was assessed by weighing pups before and after a 4 h feeding session. During the first postnatal week, fasted pups gained significantly more weight than fed pups. This difference disappeared during the second week, but reappeared during the third week and persisted through the fourth week. In another series, pups were weighed at 2 and 4 h after beginning feeding. This showed that fasted pups aged 6 days compensate by suckling longer than fed pups. At 10 and 14 days of age there were no differences between fed and fasted pups at either 2 or 4 h, but from 16 days onward, fasted pups had eaten significantly more than fed pups at both times. A control experiment showed that the lack of compensation by fasted pups aged 10-14 days did not reflect lack of availability of milk. Video-analysis of suckling behaviour at ages 6, 10 and 15 days provided further evidence for lack of feeding controls during the second postnatal week: at 6 and 15 days fasted pups spent more time actively sucking than did fed pups. Whereas at 10 days, there were no differences between fed and fasted pups. It is concluded that feeding controls are present during the first postnatal week, become quiescent during the second week and reappear during the third week.     

Thyrotropin releasing hormone in the pancreas of newborn rats from streptozotocin-treated mothers

The effect of maternal diabetes (induced by i.p. injections of 40-50 mg/kg BW Streptozotocin on the day of mating) on TRH in the pancreas of newborn rats was studied. Determination of peptide alpha amidation activity and TRH precursor level on the day of birth revealed decreased biosynthesis of TRH resulting in profoundly (10 times) lower pancreatic TRH and TRH-OH concentrations in pups of diabetic rats. Pancreatic His-Pro-diketopiperazine (His-Pro-DKP) remained unaffected by maternal diabetes. The depression of pancreatic TRH was less profound 24 h later, and even elevated TRH was measured in the pancreas of pups of diabetic mothers on postnatal day 5. Short term postnatal starvation or nursing of intact pups by the diabetic foster mother did not affect pancreatic TRH. It could be postulated that postnatal TRH development in the rat pancreas is retarded by maternal diabetes, while His-Pro-DKP remains unaltered.     

Oxygen consumption and survival prediction in neonatal rats exposed to prenatal hypervitaminosis A

Fetal exposure to excess vitamin A results in a highly variable degree of lung pathology and high neonatal mortality in the Long-Evans rat. The present study evaluated O2 consumption in newborn of vitamin A-treated, vehicle-treated, and untreated pregnancies on five consecutive postnatal days beginning with the day of delivery (D0). Pregnant female rats were treated by gavage with 160,000 USP units of retinyl acetate dissolved in 0.5 ml corn oil on days 15 through 19 of gestation. Vehicle and undisturbed controls were run concurrently. All animals delivered spontaneously, and the pups were tattooed and individually tested in a closed system consisting of three chambers submerged within a thermostatically controlled water bath at 33 degrees C. Vitamin A-exposed pups, as a group, have significantly lower QO2 (ml O2 consumed/min/kg body weight) values than controls through postnatal day 2 (p less than 0.05). By days 3 and 4 of age, the mean QO2 values of surviving vitamin A-treated pups were similar to those of controls. A QO2 of 30 or greater on day 0 appears to be critical for early neonatal survival of vitamin A-exposed pups, as 87% of the pups with initial QO2 less than 30 died prior to day 4. Oxygen consumption rates in teratogen-exposed pups exhibiting low QO2 on day 0 rarely reached normal levels. In contrast, the occasional control pup with such low initial levels were well within normal limits (means +/- 1 SD) by the following day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of prenatal spaceflight on vestibular responses in neonatal rats.

Ten pregnant Norway rats (Rattus norvegicus) were flown for 11 days on board the NASA space shuttle from gestational day 9 (launch) until gestational day 20 (landing) of the rats' 22-day pregnancy. After the birth of the pups, vestibular responses were analyzed from postnatal day (P) 0 until P5. In the first test, P0 neonates were supported on a platform in a side-lying position. Skyward head movements (i.e., movements performed against the gravity vector) were more frequent than head movements toward Earth in both flight and control neonates. In the second test, the contact-righting reflex, composed of stereotyped movements that rotate the body from supine to prone on a solid surface, was analyzed in P0 neonates. The frequency and latency of contact-righting responses did not differ in flight and control neonates. In the third test, vestibular head righting, with tactile and proprioceptive cues removed, was tested in neonates on P1, P3, and P5 by using a water-immersion test. Righting responses were observed less frequently in P1 and P3 flight neonates compared with controls. However, this deficit was transient, as evidenced by complete response recovery on P5. Collectively, these findings provide evidence for a selective disruption of vestibular-mediated responses after prenatal exposure to spaceflight.     

Attenuation of NF-κβ mediated apoptotic signaling by tocotrienol ameliorates cognitive deficits in rats postnatally exposed to ethanol

Ethanol-induced damage in the developing brain may result in cognitive impairment including deficits on neuropsychological tests of learning, memory and executive function, yet the underlying mechanisms remain elusive. In the present study we investigated the protective effect of tocotrienol against cognitive deficit, neuroinflammation and neuronal apoptosis in rat pups postnatally exposed to ethanol. Pups were administered ethanol (5g/kg, 12% v/v) by intragastric intubation on postnatal days 7, 8 and 9. Ethanol-exposed pups showed significant memory impairment in Morris water maze task as evident from increase in escape latency and total distance travelled to reach the hidden platform. Time spent in target quadrant, % total distance traversed in target quadrant and frequency of appearance in target quadrant was also significantly decreased in ethanol exposed pups in probe trial. Poor memory retention was exhibited by ethanol-exposed pups in elevated plus maze test also. Impaired cognition was associated with significantly enhanced acetylcholinesterase activity, increased neuroinflammation (oxidative-nitrosative stress, TNF-α, IL-1β and TGF-β1) and neuronal apoptosis (NF-κβ and Caspase-3) in different brain regions of ethanol-exposed pups. Co-administration with tocotrienol significantly ameliorated all the behavioral, biochemical and molecular alterations in the different brain regions of ethanol exposed pups. The current study thus demonstrates the possible involvement of NF-κβ mediated apoptotic signaling in cognitive deficits associated with postnatal ethanol exposure in rats and points to the potential of tocotrienol in the prevention of cognitive deficits in children with fetal alcohol spectrum disorders (FASDs).     

Activation of dopamine D1 receptors blocks phencyclidine-induced neurotoxicity by enhancing N-methyl-D-aspartate receptor-mediated synaptic strength

Early postnatal blockade of NMDA receptors by phencyclidine (PCP) causes cortical apoptosis in animals. This is associated with the development of schizophrenia-like behaviors in rats later in life. Recent studies show that the mechanism involves a loss of neurotrophic support from the phosphoinositol-3 kinase/Akt pathway, which is normally maintained by synaptic NMDA receptor activation. Here we report that activation of dopamine D1 receptors (D1R) with dihydrexidine (DHX) prevents PCP-induced neurotoxicity in cortical neurons by enhancing the efficacy of NMDAergic synapses. DHX increases serine phosphorylation of the NR1 subunit through protein kinase A activation and tyrosine phosphorylation of the NR2B subunit via Src kinase. DHX enhances recruitment of NR1 and NR2B, but not NR2A, into synapses. DHX also facilitated the synaptic response in cortical slices and this was blocked by an NR2B antagonist. DHX pre-treatment of rat pups prior to PCP on postnatal days 7, 9 and 11 inhibited PCP-induced caspase-3 activation on PN11 and deficits in pre-pulse inhibition of acoustic startle measured on PN 26-28. In summary, these data demonstrate that PCP-induced deficits in NMDA receptor function, neurotoxicity and subsequent behavioral deficits may be prevented by D1R activation in the cortex and further, it is suggested that D1R activation may be beneficial in treating schizophrenia.     

Gestational zinc deficiency amplifies the regulation of metallothionein induction in adult mice

To determine if prenatal zinc deficiency has a persistent effect on metallothionein (MT) regulation, Swiss-Webster mice were mated and fed a diet containing either control (100 micrograms Zn/g) or low levels of zinc (5 micrograms Zn/g) from Day 7 of gestation to parturition. After birth all mice were given the control diet. Liver zinc and MT levels were 50% lower in newborn pups from dams fed the low zinc diets than in control pups. In control pups, liver zinc and MT concentrations were relatively stable during the first week of postnatal life. In contrast, in pups prenatally deprived of zinc, liver levels of zinc and MT increased such that by Day 3 of postnatal life, the levels were not significantly different from controls. At Day 56, serum IgM concentrations were significantly lower in the low zinc offspring. Liver zinc concentrations in the two groups of mice were similar at Day 70 postnatal, and in both groups liver MT levels were below detection limits. However, when Day 70 mice were given zinc injections to stimulate MT synthesis, the prenatally zinc deprived offspring showed markedly higher liver MT levels than did control mice given similar injections, despite similar liver zinc concentrations in the two groups. These results show that prenatal zinc deficiency has pronounced effects on postnatal MT metabolism which can persist into adulthood.     

Postnatal recruitment of brown adipose tissue is induced by the cold stress experienced by the pups. An analysis of mRNA levels for thermogenin and lipoprotein lipase.

In order to investigate the postnatal recruitment process, gene expression in the brown adipose tissue of rat pups was followed during the first 20 h of life. In normal pups, the level of mRNA coding for the uncoupling protein thermogenin increased markedly but gradually within the first 24 h. Lipoprotein lipase and actin mRNA levels were relatively low and remained constant. In pups exposed to thermoneutral temperature (35 degrees C) for the first 12 h after birth, no increase in thermogenin mRNA or lipoprotein lipase mRNA was observed, whereas in pups exposed to 28 degrees C a clear increase in both thermogenin and lipoprotein lipase mRNA levels was found. Actin mRNA levels were not affected by the environmental temperature under these circumstances. It was concluded that the postnatal recruitment in brown adipose tissue is a consequence of the cold stress experienced by the newborn pups. Thus, postnatal recruitment is not ontogenically predetermined.     

Maternal entrainment of tau mutant hamsters.

Maternal entrainment of the circadian wheel-running activity rhythm was examined in Syrian hamsters heterozygous for a single gene mutation (tau) that affects the free-running period of circadian rhythms. Heterozygous tau pups were born to and raised by wild-type mothers under constant dim light. The pups' wheel-running activity was recorded after weaning on postnatal day 18 or 24. Pups weaned on day 18 had an average free-running period of 21.70 hr, demonstrating that the tau phenotype was fully expressed at this age. Using the activity onset of the postnatal free-running rhythms as a phase reference, we estimated the phase relationships between the pups and their mothers on days 18 and 24. In contrast to results with wild-type pups, the activity rhythms of tau pups were not in phase with the rhythms of their wild-type mothers; that is, activity onsets of mothers and pups did not coincide. The pups did, however, show synchrony among themselves, indicating that they had been exposed to a synchronizing signal sometime during development. It is likely that this synchronizing signal was provided by the mothers, since pups from different litters showed phase relationships similar to those of their mothers. Thus the mothers provided a signal that was sufficient to cause entrainment, despite the 2-hr difference in free-running period between the mothers and pups. Although the pups' activity rhythms appeared to have been entrained by the mothers, they were clearly free-running by postnatal day 18. The mechanism for entrainment is lost during the course of development, despite continued interaction between the mothers and pups.