Impaired social cognition caused by perinatal protein malnutrition evokes neurodevelopmental disorder symptoms and is intergenerationally transmitted.

Nutritional inadequacy before birth and during postnatal life can seriously interfere with brain development and lead to persistent deficits in learning and behavior. In this work, we asked if protein malnutrition affects domains of social cognition and if these phenotypes can be transmitted to the next generation. Female mice were fed with a normal or hypoproteic diet during pregnancy and lactation. After weaning, offspring were fed with a standard chow. Social interaction, social recognition memory, and dominance were evaluated in both sexes of F1 offspring and in the subsequent F2 generation. Glucose metabolism in the whole brain was analyzed through preclinical positron emission tomography. Genome-wide transcriptional analysis was performed in the medial prefrontal cortex followed by gene-ontology enrichment analysis. Compared with control animals, malnourished mice exhibited a deficit in social motivation and recognition memory and displayed a dominant phenotype. These altered behaviors, except for dominance, were transmitted to the next generation. Positron emission tomography analysis revealed lower glucose metabolism in the medial prefrontal cortex of F1 malnourished offspring. This brain region showed genome-wide transcriptional dysregulation, including 21 transcripts that overlapped with autism-associated genes. Our study cannot exclude that the lower maternal care provided by mothers exposed to a low-protein diet caused an additional impact on social cognition. Our results showed that maternal protein malnutrition dysregulates gene expression in the medial prefrontal cortex, promoting altered offspring behavior that was intergenerationally transmitted. These results support the hypothesis that early nutritional deficiency represents a risk factor for the emergence of symptoms associated with neurodevelopmental disorders.

Maternal protein malnutrition induces autism-like symptoms in rat offspring.

Objective: We tested the correlation between maternal protein malnutrition and autistic-like symptoms using behavioral tests in rodents that measure main behavioral characteristics observed in humans with autism spectrum disorder (ASD). Methods: Pregnant female rats were fed a normal diet or a hypoproteic diet during gestation and lactation periods. The litters were weighed every 3 days during lactation, and the offspring were tested in behavioral tasks during infancy (postnatal day (PND) 5: quantification of ultrasonic vocalizations; PND 13: homing behavior test) and adolescence (PND 30-32: open field, hole-board, play social behavior, and object recognition tests) in order to capture the prevalence of some of the core and associated symptoms of ASD. Results: Litters of the hypoproteic diet group had a lesser weight gain during lactation. In addition, pups of dams fed with a hypoproteic diet vocalized less compared to those fed with a normal diet, and they showed impaired social discrimination abilities in the homing behavior test. In adolescence, both male and female offspring of the hypoproteic diet group showed no impairment in locomotor activity; however, they exhibited stereotypic behavior in the hole-board test and a decrease in social play behaviors. Male offspring showed increased interest in exploring a familiar object rather than a novel object. Conclusion: Our results show that maternal protein malnutrition in rats causes offspring behaviors that resemble core and associated ASD symptoms.

Developmental Changes in Oligodendrocyte Genesis, Myelination, and Associated Behavioral Dysfunction in a Rat Model of Intra-generational Protein Malnutrition.

Impairments in oligodendrocyte development and resultant myelination deficits appear as a common denominator to all neurological diseases. An optimal in utero environment is obligatory for normal fetal brain development and later life brain functioning. Late embryonic and early postnatal brains from F1 rat born to protein malnourished mothers were studied through a combination of immunocytochemical and quantitative PCR assay for analyzing the relative expression of platelet-derived growth factor receptor-α (PDGFRα), myelin-associated glycoprotein (MAG), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) to determine oligodendrocyte genesis, differentiation, maturation, and myelination. Myelin integrity and corpus callosum caliber was assessed by Luxol fast blue (LFB) staining, whereas grip strength test and open field activity monitoring for behavioral evaluation in F1 rats. We demonstrate that intra-generational protein deprivation results in drastically low PDGFRα+ oligodendrocyte precursor (OPC) population and significantly reduced expression of myelin protein genes resulting in poor pre-myelinating and mature myelinating oligodendrocyte number, hypo-myelination, and misaligned myelinated fibers. LFB staining and MOG immunolabeling precisely revealed long-term changes in corpus callosum (CC) caliber and demyelination lesions in LP brain supporting the behavioral and cognitive changes at early adolescence and adulthood following maternal protein malnutrition (PMN). Thus, intra-generational PMN negatively affects the oligodendrocyte development and maturation resulting in myelination impairments and associated with behavioral deficits typically mimicking clinical hallmarks of neuropsychiatric disorders. Our results further strengthen and augment the hypothesis "Impaired gliogenesis is a big hit for neuropsychiatric phenotype."

Protein malnutrition during pregnancy alters maternal behavior and anxiety-like behavior in offspring.

OBJECTIVE: The aim of this study was to evaluate the effects of protein malnutrition during pregnancy on maternal behavior, on the early behavior in pups by ultrasonic vocalizations (USVs) emission, and on the behavior of offspring in adulthood in an elevated T-maze. METHODS: Pregnant female rats were fed a normal protein-powdered diet (22% casein; control) or a low-protein (hypoproteic) diet (6% casein; protein restriction) during the first 2 weeks of pregnancy. On the fifth postpartum day (PND5), the number of USV was rated. On PND7, maternal behavior was assessed. Male offspring in adulthood were evaluated for behavioral performance in an elevated T-maze. RESULTS: Our results demonstrated that a hypoproteic diet during early pregnancy increased the maternal behavior, increased the number of USV by pups, and reduced the inhibitory avoidance responses in an elevated T-maze during adulthood. In addition, there was a reduction in weight gain of rats during pregnancy and of offspring during lactation. CONCLUSION: In conclusion, the data found in our study suggest that the increase in USV emitted by pups due to hypoproteic diet during pregnancy accentuated maternal behavior. In addition, an increase in maternal care promoted the reduction in anxiety-like behavior in adult male offspring.

Stress-induced changes in extracellular dopamine and serotonin in the medial prefrontal cortex and dorsal hippocampus of prenatally malnourished rats.

Prenatal protein malnutrition continues to be a significant problem in the world today. Exposure to prenatal protein malnutrition increases the risk of a number of neuropsychiatric disorders in adulthood including depression, schizophrenia and attentional deficit disorder. In the present experiment, we have examined the effects of stress on extracellular serotonin (5-HT) and dopamine in the medial prefrontal cortex and dorsal hippocampus of rats exposed in utero to protein malnutrition. The medial prefrontal cortex and dorsal hippocampus were chosen as two limbic forebrain regions involved in learning and memory, attention and the stress response. Extracellular 5-HT and dopamine were determined in the medial prefrontal cortex and dorsal hippocampus of adult male Sprague-Dawley rats using dual probe in vivo microdialysis. Basal extracellular 5-HT did not differ between malnourished and well-nourished controls in either the medial prefrontal cortex or the dorsal hippocampus. Basal extracellular dopamine was significantly decreased in the medial prefrontal cortex of malnourished animals. Restraint stress (20 m) produced a significant rise in extracellular dopamine in the medial prefrontal cortex of well-nourished rats but did not alter release in malnourished rats. In malnourished rats, stress produced an increase in 5-HT in the hippocampus, whereas stress produced a decrease in 5-HT in the hippocampus of well-nourished rats. These data demonstrate that prenatal protein malnutrition alters dopaminergic neurotransmission in the medial prefrontal cortex as well as alters the dopaminergic and serotonergic response to stress. These changes may provide part of the bases for alterations in malnourished animals' response to stress.

A panic experimental model: validation of a complex operant behavioral method in undernourished rats, with desipramine to provide a template effect profile.

INTRODUCTION: Clinical studies have shown that some antidepressants may be more efficient than benzodiazepines to alleviate anxiety associated with panic disorders; however, operant conflict procedures in rats developed so far seem not particularly able to model human anxiety sensitive to antidepressant treatments. Previous panic models with learned responses did not statistically subtract the effect of confounding factors from the variable of interest. METHODS: Undernourished rats were selected due to their behavioral and neurobiological resemblance to human patients suffering from panic disorder. The Geller-Seifter paradigm represented the stressful environmental condition in adult life. Desipramine (10 mg/kg/day) or saline were administered IP during 7 days under a cross over design (N=10). Five daily 15 min-operant sessions were carried out on each experiment. Unpunished, unrewarded and punished operant behavioral periods were identical both in their duration and in their reward system (the FR1 schedule) in order to measure response suppression, which has not been considered in previous studies with the Geller-Seifter paradigm. The dependent variable was the difference between comparable unpunished and punished periods. RESULTS: A significant Diet x Drug interaction was observed in the dependent variable, which represented the level of "suppression/suppression release" induced by treatments. DISCUSSION: Compared to control rats, deprived rats showed a significant and selective anticonflict effect of desipramine on the stressful and complex operant performance. The animal model of perinatally protein-deprived rats along with the Geller-Seifter's operant behavioral paradigm may represent a more sensitive approach to model human anxiety sensitive to antidepressant treatments by considering the combined impact of both early biological trauma and adult learned experiences under the same design.

Postnatal protein malnutrition affects inhibitory avoidance and risk assessment behaviors in two models of anxiety in rats.

Protein malnutrition induces structural, neurochemical and functional alterations in the central nervous system, leading to alterations in behavioral function. In order to study the effects of early protein malnutrition on inhibitory avoidance and escape behaviors we used the elevated T-maze (ETM), while the risk assessment behaviors were evaluated by the canopy stretched attend posture (SAP) test. Rat pups were fed by lactating females receiving 16% (control) or 6% (malnourished) protein diets during the lactation period. After weaning the animals received the same diets until 49 days of age, when all animals started receiving a lab chow diet. Behavioral tests were started at 70 days of age. ETM results showed lower inhibitory avoidance in malnourished animals, without differences in escape behavior. SAP test results showed higher exploration and lower risk assessment behaviors in malnourished animals compared to control. These results suggest that malnourished animals are less anxious and/or more impulsive as measured by these two animal models and that malnutrition seems to affect differently behavioral strategies underlying fear and anxiety responses.

Parabrachial lesions disrupt responses of rats to amino acid devoid diets, to protein-free diets, but not to high-protein diets.

Normal rats "reduce" intake of diets that lack an essential amino acid (THR-DEV), are protein free (PO%), or contain a high proportion of protein (P75%). We tested the importance of the parabrachial nuclei (PBN) in signaling such adjustments of food intake by placing electrophysiologically guided lesions in these nuclei at points that responded to gustatory stimuli. When fed the THR-DEV diet, rats with PBN lesions (PBNx) decreased their food intake significantly less than the controls (78.5 vs. 44.4%). When put on a P0% diet, PBNx animals decreased their intake only 8% compared with 23% for our CONT group. When put on a P75% diet, however, both groups decreased their intake in an equivalent amount. These experiments show that the PBN is involved in the learned aversion to an amino acid devoid diet.

Release of hypothalamic norepinephrine during MSG intake in rats fed normal and nonprotein diet.

Effects of monosodium L-glutamate (MSG) solution (0.06 M) on interstitial levels of norepinephrine (NE) were measured in the lateral hypothalamus (LH). Wistar male rats, housed in standard operant boxes, were fed either normal or nonprotein diet for 3 days. Beside a daily bar-mediated drinking session (75 min), animals were without access to fluids. Microdialysates, collected from the LH during the 75 min of drinking, were analyzed using high-pressure liquid chromatography. No significant responses of the LH NE to the drinking of distilled water, MSG, NaCl (0.06 M), and glucose solution (0.6 M) were found in normally fed rats. However, a specific decline in LH NE release was detected during MSG solution-drinking in rats fed nonprotein diet. As MSG preference indicates protein intake, it is possible that LH NE is, at least partially, one of the brain signals that relate MSG preference to dietary protein intake.

Behavioral effects of protein deprivation and rehabilitation in adult rats: relevance to morphological alterations in the hippocampal formation.

In the present study we have analyzed the behavioral and neuroanatomical effects of protein deprivation in adult rats. Starting at 2 months of age, animals were maintained on 8%-casein diet either for 8 months (malnourished group), or for 6 months followed by a 2-month period of nutritional rehabilitation (17%-protein diet, rehabilitated group). Malnourished rats exhibited reduced emotional reactivity and impaired habituation in the open field. In a water maze, these animals did not differ from controls during training, but showed retention deficits on the probe trial. However, working memory, sensorimotor abilities and passive avoidance behavior were not significantly impaired in malnourished rats. The performance of rehabilitated group was similar to that of the control group throughout behavioral testing. Postmortem morphological analysis revealed that the total number of neurons in the granular layer of the dentate gyrus, and in CA3 and CA1 hippocampal fields was reduced in protein-deprived and rehabilitated rats relative to controls. In addition, it was found that protein deprivation caused a 30% loss of synapses established between mossy fibers and dendrites of CA3 pyramidal cells, whereas nutritional rehabilitation resulted in a reversal of this effect. These results show that prolonged malnutrition in adult rats produces marked loss of hippocampal neurons and synapses accompanied by substantial impairments of hippocampal-dependent behaviors. The fact that nutritional rehabilitation results in restoration of the total number of hippocampal synapses and parallel amelioration of the behavioral impairments suggests that the mature CNS possesses a remarkable potential for structural and functional recovery from the damage induced by this type of dietary 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.

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.

Perinatally protein-deprived rats and reactivity to anxiolytic drugs in the plus-maze test: an animal model for screening antipanic agents?

Adult rats submitted to a protein deprivation schedule at perinatal age (from 14th day of fetal life until 50 days of age) and then recovered on balanced chow (D rats) were assayed in the elevated plus-maze test for anticonflict effects of diazepam and drugs with therapeutic efficacy in panic disorders as compared with controls (C rats). Diazepam and alprazolam showed a similar anticonflict effect in D rats than in C rats. In contrast, buspirone, which was ineffective in C rats at a wide dosage range, showed a significant anticonflict effect on D rats at 0.3 mg/kg. Neither propranolol, desipramine, nor phenelzine treatment (10 mg/kg/day during 3-7 days) induced anticonflict effect in C rats. Conversely, these treatments fostered a significant and selective anxiolytic effect on D rats. Such results underscore long-lasting alterations caused by early undernutrition, namely, changes in reactivity to the drugs assayed. In addition, perinatally deprived rats may represent a useful animal model for studying potential antipanic agents.

Corticotropin-releasing factor modulates dietary preference in nutritionally and physically stressed rats.

In order to evaluate the action of central nervous system Corticotropin-Releasing Factor (CRF) in the control of feeding behavior the present studies employed a dietary self-selection task sensitive both to overall appetite as well as preferential intake of familiar versus unfamiliar foods. Prior to the diet selection test, one group of nutritionally stressed animals was fed a protein deficient diet in order to increase the preference for unfamiliar foods relative to nutritionally replete subjects. Both CRF (0.05 and 0.5 micrograms ICV) and physical restraint (30 min) attenuated selectively the consumption of a novel food choice by deficient animals without affecting concurrent intake of familiar food. Further, CRF administration did not alter water intake or consumption of either diet by the replete control group suggesting that the peptide produced a stress dependent, enhanced response to novelty without a general effect on appetite. The CRF antagonist, alpha-helical CRF9-41 (1, 5 and 25 micrograms ICV), increased familiar diet consumption in nutritionally deficient subjects without affecting the self-selection pattern or replete controls. Chlordiazepoxide (5 mg/kg) also increased selectively the intake of familiar food suggesting that this action is the anxiolytic complement of the effect of stress in this paradigm. The CRF antagonist (5 and 25 micrograms) reversed the anorexia produced by CRF (0.5 micrograms) as well as that induced by restraint stress. These results favor a direct role for endogenous CRF systems in coordinating the behavioral responses to dietary stress.

Prenatal protein malnutrition and maternal behavior in Sprague-Dawley rats.

Female Sprague-Dawley rats were provided with an adequate (25% casein) or low (6% casein) protein diet, fortified with methionine, starting 5 wk prior to gestation and continuing until the birth of their pups. At birth, the offspring were fostered to well-nourished lactating dams as follows: in Experiment 1, litters contained eight prenatally malnourished pups; in Experiment 2, litters consisted of four pups with prenatal malnutrition and four well-nourished pups; control litters for both experiments contained eight well-nourished pups. In Experiment 1, the time spent in passive nursing and pup grooming was significantly higher for dams rearing previously malnourished pups than for those rearing control litters. In Experiment 2, mixed litters were groomed less frequently than control litters, but there was no significant difference between groups for any other measure. In both experiments, the prenatally malnourished pups showed similar body weight deficits relative to well-nourished pups. These findings underscore the usefulness of mixed litters in studying the later effects of prenatal malnutrition because later deficits in brain function are less likely to be attributable to changes in the early rearing environment.

Protein deficiency magnifies social influence on the food choices of Norway rats (Rattus norvegicus).

The food choices of protein-deprived juvenile rats were more profoundly affected by interaction with conspecifics than were the food choices of protein-replete juvenile rats. When choosing among four different-flavored, protein-deficient diets, protein-deprived rats ate significantly more of the diet eaten by a conspecific demonstrator than did protein-replete rats. These data suggest that the food choices of the relatively less successful members of a population are most affected by social interaction. Consequently, the mean effect of social interaction on diet selection in a population of Norway rats is likely to be positive.

Olfactory self-selection of protein-containing foods.

When one unflavored, nonprotein diet was available in two differently scented bins, rats fed a protein-free diet over four days ate more from the bin smelling of gluten, ovalbumin, yeast or fibrin, but not soy, casein or lactalbumin, than from the bin smelling of butter. Rats fed a protein-containing diet over the same four-day period had no such preference. This result demonstrates that protein-deprived rats can use odor cues in making their selection of certain proteins. Since the direction, speed, and size of preference for these protein odors, excepting soy, are remarkably similar to those previously observed when rats actually consumed the proteins, olfactory stimuli appear to elicit appropriate protein selection responses independently of other protein quality variables such as taste, texture or nutrient composition.

Behavioral effects of severe and moderate early malnutrition.

Rats with a history of prenatal and early postnatal undernutrition (6 or 8% casein diets) were "nutritionally rehabilitated" at weaning, and were compared to well-fed animals (25% casein) at maturity. The severely-malnourished (6%) animals were hyperactive in the open field and when tested in a stabilimeter. They also appeared to be highly active during the early trials in 8-arm radial maze testing where they made more arm entries and re-entry errors than the well-fed rats. In terms of trials to criterion, however, their scores on the radial maze and on a spatial alternation task fell within normal limits. The moderately-malnourished (8%) rats tended to perform at control levels on the learning measures, but these rats were not as active as the 6% rats on the measures of activity. Brain size and weight differences among the three groups of rats also are presented and discussed.

Severe early malnutrition and DRL performance in the rat.

Rats with a history of severe early malnutrition (6% casein) were compared to well-fed control animals on an ascending series of DRL values ranging from 5 to 60 seconds. The 6% rats who were dietarily-rehabilitated at weaning did not differ from control animals in efficiency, responses per reinforcement or response rate. In contrast, rats chronically exposed to 6% diets performed so poorly during training with continuous reinforcement that they did not advance to even the first DRL (5-sec) condition. These findings show that severely-undernourished rats can perform within normal limits on even high DRL values, provided they are well trained and that they have adequate nutritional rehabilitation.