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.

Regulation of heat shock gene transcription in neuronal cells.

The heat shock protein (HSP) molecular chaperones are the primary cellular defense against damage to the proteome, initiating refolding of denatured proteins and regulating degradation after severe protein damage. Many neurodegenerative disorders involve aberrant protein folding and protein damage, which accumulates in an age-dependent manner. Ageing is associated with the decrease in activity of the heat shock transcription factors (HSF) that regulate HSP gene transcription. Neuronal cells seem particularly vulnerable in this sense as HSF activity and HSP expression are relatively weak in such cells and motor neurons appear to require input of HSP secreted from adjacent glial cells to maintain adequate molecular chaperone levels. It may be significant that motor neurons have been shown to be the sensitive cells in the ageing of Drosophila and C. elegans and that these organisms may acquire extended lifespans with over-expression of small heat shock proteins and HSF1. HSF1 transcriptional activity has been discussed in neuronal cells, concentrating on the regulation and activity of HSF1 and HSF2 and their role in HSP expression, during neurodegenerative diseases and as mediators of cell survival.

Spatial learning deficits induced by muscimol and CL218,872: lack of effect of prenatal malnutrition.

The sensitivity of prenatal protein malnourished rats to the amnestic properties of the direct GABAA receptor agonist muscimol and the selective benzodiazepine (BZ) receptor agonist, CL218,872, 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. At postnatal day 90, rats were tested during acquisition of the submerged platform version of the Morris water maze task using four systemic doses of muscimol (0.1, 0.3, 1.0 and 1.8 mg/kg i.p.) or three systemic doses of CL218,872 (1.0, 3.2, and 5.6 mg/kg i.p.). In a dose dependent manner both drugs impaired acquisition of the task and impaired accuracy of the search pattern on the probe trial (platform removed). However, neither drug dissociated the performance of the two nutritional groups. These data are important in light of previous findings of differential behavioral effects of the non-specific BZ agonist, chlordiazepoxide (CDP), on spatial learning and on drug discrimination in prenatally malnourished rats and in the context of previous findings of reduced sensitivity to the anxiolytic effects of non-specific BZ receptor agonists across a wide variety of models of malnutrition. The present findings also support the concept that prenatal malnutrition does not affect the global functioning of the GABAA receptor, but fundamentally alters the way in which a subset of GABAA receptors (i.e. those containing the alpha2, alpha3 and/or the alpha5 but not the alpha1 subunit) is modulated by BZs.

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.

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.

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.

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.

Prenatally malnourished female but not male rats show increased sensitivity to MK-801 in a differential reinforcement of low rates task.

A reduced behavioral sensitivity to drugs acting on central GABAergic, serotonergic, opioid and cholinergic systems has previously been identified in predominantly male malnourished animals. The present study sought to compare the effects of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 on responding maintained by a differential reinforcement of low rates (DRL-18s) operant schedule in two groups of rats with different prenatal nutritional histories (well-nourished and protein restricted). The schedule required that the rats space their responses at least 18 s apart in order to obtain food reinforcement (timing behavior). After training to a stable high proficiency, MK-801 was administered to female rats (Experiment 1) at doses of 0 (saline), 0.004, 0.008, 0.016, 0.024 or 0.032 mg/kg (doses expressed as the free-base). MK-801 produced dose-dependent decreases in the percentage efficiency of responding and the number of rewarded responses, with dose-dependent increases in the number of responses emitted. Prenatal malnutrition significantly shifted the inter-response time (IRT) curve to the left, relative to that of the well-nourished controls, leading to a significantly lower efficiency and a lower number of reinforcers, at an MK-801 dose of 0.016 mg/kg. In Experiment 2, the effect of MK-801 on DRL performance was compared between male and female rats after prenatal malnutrition. In general, females proved more sensitive to MK-801 than males. Consistent with Experiment 1, a significantly greater drug impairment was observed in prenatally malnourished females compared with well-nourished females, albeit at a slightly higher dose (0.032 mg/kg). Prenatal malnutrition did not alter the drug response in male rats. These findings suggest that the behavioral response to NMDA blockade is augmented in adult female, but not male, rats after prenatal malnutrition.

Prenatal malnutrition-induced changes in blood pressure: dissociation of stress and nonstress responses using radiotelemetry.

A link between prenatal malnutrition and hypertension in human populations has recently been proposed. Rat models of prenatal malnutrition have provided major support for this theory on the basis of tail-cuff measurements. However, this technique requires restraint and elevated temperature, both potential sources of stress. To determine the effect of prenatal protein malnutrition on blood pressure under nonstress conditions, 24-hour radiotelemetric measurements were taken in the home cage. Male rats born to dams fed a 6% casein diet for 5 weeks before mating and throughout pregnancy were studied in early adulthood (from 96 days of age). During the waking phase of their cycle but not the sleep phase, prenatal malnutrition gave rise to small but significant elevations of diastolic blood pressure and heart rate compared with well-nourished controls. Direct effects of stress on blood pressure responses were determined in a second experiment using an olfactory stressor. Prenatally malnourished rats showed a greater increase in both systolic and diastolic pressures compared with well-nourished controls during the first exposure to ammonia. A different pattern of change of cardiovascular responses was also observed during subsequent presentations of the stressor. These findings of a small baseline increase in diastolic pressure consequent to prenatal malnutrition, but an augmented elevation of both systolic and diastolic pressures after first exposure to stress, suggest the need to reevaluate interpretation of the large elevations in blood pressure previously observed in malnourished animals using the stressful tail-cuff procedure.

Effects of diet on sensitization to cocaine-induced stereotypy in female rats.

The progressive increase in cocaine-induced stereotyped behavior that accompanies repeated cocaine injections (sensitization) was examined in rats consuming different diets. Adult female Sprague-Dawley rats were fed one of three diets: low protein (6% casein), adequate protein (25% casein), or a standard chow diet. Following 1 week of adaptation to the diets, the rats were injected every 3-4 days with either cocaine (30 mg/kg, IP) or saline, and the total amount of stereotypy was measured over a 90-min interval following each of four injections. Cocaine-induced stereotypy peaked at 40-50 min following each injection, after which it declined for all diet groups. With repeated injections, the total amount of stereotypy increased in all diet groups. By the fourth injection, the low protein diet group (6% casein) exhibited a slower onset and a possibly prolonged duration of cocaine-induced stereotypy when compared with the two adequate protein diet groups (25% casein and chow). Interestingly, the rats in the two purified diet groups (6% casein and 25% casein) exhibited significantly more stereotypy across injections than those in the chow diet group. Weight differences did not explain the differences in stereotypy present among the diet groups. This study concludes that diet significantly alters the pattern of cocaine-induced stereotypy in female rats, especially after repeated exposure.

Development of spatial navigation following prenatal cocaine and malnutrition in rats: lack of additive effects.

The effects of prenatal cocaine exposure and protein malnutrition on the development of spatial navigation were assessed in rats. Sprague-Dawley dams were fed a low-protein (6% casein), adequate protein (25% casein), or a laboratory chow diet prior to mating and throughout pregnancy. Within each diet group, rats received either cocaine injections (30 mg/kg i.p. two times per week prior to mating and then 30 mg/kg s.c. daily from day 3 to 18 of pregnancy) or saline injections. All litters were fostered on the day of birth to saline-injected mothers fed either the 25% casein diet or the chow diet. Gestation length was decreased by prenatal cocaine exposure whereas litter size was reduced by prenatal malnutrition. On postnatal days 21, 25, 30, or 70, rats were tested for their ability to locate a submerged platform in a Morris water maze. In well-nourished rats, prenatal cocaine increased the mean distance swum during acquisition over days 21-30, a difference that was abolished in rats with prenatal malnutrition. In the absence of drug exposure (saline groups), prenatal malnutrition was itself associated with longer swim paths. Neither prenatal insult affected the accuracy of the spatial navigation at these ages, as determined by their search pattern when the platform was removed. On postnatal day 25, rats raised on the chow diet exhibited superior performance to that of rats raised on the 25% casein diet, but by day 30 these two well-nourished groups were comparable. At day 70, prenatal cocaine impaired spatial performance on the first session, in well-nourished rats only. Thus, these results provide no support for the hypothesis that prenatal cocaine and protein malnutrition combine to produce a greater effect on behavioral development than either insult alone.

Differential effects of prenatal protein malnutrition and prenatal cocaine on a test of homing behavior in rat pups.

The effects of prenatal cocaine exposure and protein malnutrition on orientation to home nest material was assessed in rat pups. Sprague-Dawley dams were fed a diet of low protein content (6% casein), and isocaloric diet of adequate protein content (25% casein, control), or a laboratory chow diet prior to mating and throughout pregnancy. Within each diet group, rats received either cocaine injections (30 mg/kg IP 2 times per week prior to mating and the 30 mg/kg SC daily from day 3 to 18 of pregnancy) or saline injections. All litters were fostered on the day of birth to control mothers fed an adequate diet. On postnatal days 7, 9, and 11, a single pup from each litter (n = 11-15 per treatment) was tested repeatedly in a clean test cage for the rapidity of approach, and level of attraction to their own home (nest) bedding compared with fresh bedding. Prenatal malnutrition and prenatal cocaine exposure each gave rise to independent effects on performance, based upon factor analysis. Prenatal malnutrition, but not prenatal cocaine increased the time taken for rat pups to approach their nest bedding, reduced the time spent on this bedding, decreased the number of entries into the sector containing the home bedding and reduced pup weight. Prenatal cocaine, but not prenatal malnutrition, produced a reduction in activity, but had no effect on pup weight. The lower activity level was most pronounced on postnatal day 7. Surprisingly, interactive effects of prenatal cocaine and prenatal malnutrition were not observed on any behavior examined. Nevertheless, the co-existence of drug addiction and malnutrition in human populations raises the possibility that some of the effects generally attributed to drug exposure may, in fact, be due to malnutrition.

Prenatal protein malnutrition affects exploratory behavior of female rats in the elevated plus-maze test.

To study the effects of prenatal protein deficiency in the exploration of the elevated plus-maze, an ethological procedure was used. Female rats were provided with 25% (control) or with 6% (low-protein) casein diets before and during pregnancy. After birth eight pups in each litter (six males and two females) were fostered to a control mother. After weaning (21 days of age) all animals received a lab chow diet until behavioral testing began at 70 days of age. Individual prenatally malnourished (n = 12) and well-nourished (n = 12) females were placed at the center of the elevated plus-maze and allowed to explore for a 5-min session. One session was given per day for 6 consecutive days. The following variables were recorded: percentage of open arm entries; percentage of time spent in open arms; total arm entries; time in the center platform; latency to first open arm entry; number of attempts to enter an open arm; number of rearings; number of head-dips. The results showed a significant effect of malnutrition on six behaviors (percent open arm entries, percent time spent in open arms, attempts to enter open arms, rearings, head-dips, and latency to first open arm entry) and a significant diet by session interaction on two behaviors (attempts to enter open arms and head-dips). These results indicate increased exploration of the open arms in prenatally malnourished as compared with well-nourished control rats, suggestive of lower anxiety and/or a higher impulsiveness in these animals.

Prenatal protein malnutrition affects the social interactions of juvenile rats.

The effects of prenatal protein malnutrition on juvenile social behavior was investigated in male and female rats. Animals were provided with 25% (control) or 6% (low protein) casein diets before and during pregnancy. After birth eight pups in each litter (six males and two females) were fostered to lactating control mothers. After weaning (21 days of age) all animals received a lab chow diet until behavioral testing began at 45 days of age. To assess social interaction, pairs of rats of the same gender, consisting of one malnourished and one control rat, were placed in a familiar rectangular arena on 3 consecutive days. Playful social behavior (pin), nonplayful social behaviors (anogenital sniff, walk-over, side-mount, and allogroom), and nonsocial behavior (rear) were recorded in 10-min sessions. Prenatal malnutrition significantly decreased both playful and nonplayful social behaviors, and increased nonsocial rearing. No significant gender differences were observed. The finding that early social behavior is altered by prenatal malnutrition opens the possibility that such changes may play an important role in determining some of the later behavioral differences described in the adult animal.

Prenatal protein malnutrition affects avoidance but not escape behavior in the elevated T-maze test.

An elevated T-maze was used to study the effects of prenatal protein deficiency on inhibitory avoidance and escape behaviors. Female rats were provided with a 25% (control) or a 6% (low protein) casein diets before and during pregnancy. After birth, eight pups in each litter (six males and two females) were fostered to a lactating well-nourished mother. After weaning (21 days of age) all animals received a lab chow diet. Behavioral testing of these offspring began at 70 days of age. To assess inhibitory avoidance, prenatally malnourished and control rats were placed individually at the end of an enclosed arm in an elevated T-maze (one enclosed and two open arms) and the time taken to emerge from this arm was recorded. The same procedure was repeated in 2 subsequent trials given at 30-s intervals. Thirty seconds after the last of these trials, the rat was placed at the end of one open arm and the time taken to withdraw from this arm was measured, thus estimating escape latency. To assess retention, inhibitory avoidance and escape were measured again 72 h later. Prenatally malnourished males and females did not significantly increase avoidance latency from trials 1-3, in contrast to male and female controls. Only control female rats significantly reduced their avoidance latency on the retention test. No significant differences in escape latency were found between diet groups. These results suggest that prenatal malnutrition results in a reduction of anxiety, and that there are gender-specific responses to this test.

Effect of prenatal protein deprivation on postnatal granule cell generation in the hippocampal dentate gyrus.

The effect of prenatal malnutrition, produced by protein deprivation, on postnatal neurogenesis of granule cells in the fascia dentata of the rat hippocampal formation was examined by injecting tritiated thymidine on P8 and P15 and sacrificing the pups on P30, or by injecting on P30 and sacrificing on P90. The number of labeled granule cells was significantly decreased in prenatally malnourished rats injected on P8, and unaffected in those injected on P15. In contrast, the number of labeled granule cells in prenatally malnourished rats was significantly increased in animals injected in P30. The study shows that prenatal malnutrition significantly alters the postnatal pattern of granule cell neurogenesis in rat hippocampal formation and that the effect persists despite nutritional rehabilitation at birth.