Prenatal protein malnutrition alters the proportion but not numbers of parvalbumin-immunoreactive interneurons in the hippocampus of the adult Sprague-Dawley rat.

Prenatal protein malnutrition alters the structure and function of the adult rat hippocampal formation. The current study examines the effect of prenatal protein malnutrition on numbers of parvalbumin-immunoreactive (PV-IR) GABAergic interneurons, which are important for perisomatic inhibition of hippocampal pyramidal neurons. Brain sections from prenatally protein malnourished and normally nourished rats were stained for parvalbumin and PV-IR neurons were quantified using stereology in the dentate gyrus, CA3/2 and CA1 subfields, and the subiculum for both cerebral hemispheres. Results demonstrated that prenatal malnutrition did not affect the number of PV-IR interneurons in the hippocampus. Since prenatal protein malnutrition reduces total neuron numbers in the CA1 subfield (1), this results in an altered ratio of PV-IR interneurons to total neuronal numbers (from 1:22.9 in controls to 1:20.5 in malnourished rats). Additionally, there was no hemispheric asymmetry of either PV-IR neuron numbers or ratio of PV-IR:total neuron numbers.

Aging increases retinal vascular lesions characteristic of early diabetic retinopathy.

The goal of this study was to determine whether aging induces retinal vascular lesions that are similar to those seen in diabetic retinopathy. Female rats were randomly divided into four groups; each group represented a time point and consisted of four non-diabetic rats and four diabetic rats. At time points of 3, 12, 18, or 22 months of age, retinas were isolated and subjected to retinal trypsin digestion (RTD) for isolation of retinal capillary networks. Blood glucose, body weight, and hemoglobin A1c (HbA1c) levels were monitored throughout the study. One RTD from each animal was stained with Hematoxylin and Periodic Acid Schiff's (PAS) reagent to analyze acellular capillaries and pericyte loss, while the contralateral RTD from each animal was subjected to TUNEL assay to assess apoptosis in the retinal vascular cells. The numbers of acellular capillaries and pericyte loss were significantly increased between the 12 vs. 18 month groups, and the 18 vs. 22 month groups. Similarly, acellular capillaries and pericyte loss increased with aging in the diabetic rat retinas; however, the appearance of acellular capillaries and pericyte loss occurred at 3 months of diabetes. TUNEL assay showed increased apoptosis associated with acellular capillaries and pericyte loss in both normal, aged rats and diabetic rats. In conclusion, retinal vascular lesions that develop in aged rat retinas have striking similarities to those of diabetic rat retinas. The breakdown of normal vascular architecture with aging appears to have resemblance with the anatomical and histological lesions associated with diabetic retinopathy.

Prenatal malnutrition alters diazepam-mediated suppression of ultrasonic vocalizations in an age dependent manner.

The sensitivity of prenatally malnourished rats to the ultrasonic vocalization (USV) suppressant effect of diazepam (a non-specific benzodiazepine (BZ) receptor agonist) was investigated. Male offspring of dams provided with a protein deficient diet (6% casein) for 5 weeks prior to mating and throughout pregnancy were compared to the offspring of mothers provided with a diet of adequate protein content (25% casein). At postnatal day 7 or 11, pups were injected with vehicle or one of five doses of DZ (0.03, 0.1, 0.3, 1 or 3mg/kg) 30 min after removal from their dam. Thirty minutes later they were subjected to 2 min of cooling on a 20 degrees C surface and their USVs were quantified. DZ dose-dependently suppressed USV at both ages. At P7, the USV suppressant effect of DZ was the same for both groups. However, by P11 the prenatally malnourished rats showed significantly greater suppression of USV by 0.03 and 0.1mg/kg DZ than well-nourished controls. These differences were not related to degree of temperature loss or body weight. Thus, differential sensitivity to BZ receptor agonists develops in the second postnatal week in prenatally malnourished rats. This reflects either an altered program of development of the GABAergic system, or adaptive, compensatory changes in the GABAergic system in response to more extensive functional disturbances in the developing brain.

Genome-wide scan for quantitative trait loci influencing spatial navigation and social recognition memory in Dahl rats.

The genetic determinants of learning and memory have been difficult to unravel because of the complex inheritance of these forms of cognitive behavior encompassing multiple genetic and environmental factors. Indeed, genes that can account for strain and individual variations in learning and memory are largely unknown. Here we report a genome-wide scan for quantitative trait loci (QTLs) affecting spatial learning and memory and social recognition memory in an F2 population derived from Dahl rats. We detected five QTLs on chromosomes 1, 8, 11, 17, and 20 affecting spatial acquisition performance and five QTLs on chromosomes 2, 3, 9, and 20 influencing spatial accuracy (once information about the target location had been acquired). None of these QTLs overlap, indicating the existence of independent genetic determinants for these two distinct behavioral components of spatial navigation. Moreover, five QTLs affecting social recognition memory were detected, two on chromosome 9 and three on chromosome X. The chromosomal regions linked to social recognition memory performance in the rat are syntenic to regions that have been linked to autism in humans. Thus our results could have paradigmatic value in guiding the experimental investigation of similar pathways in genetic susceptibility to this disorder, which results in profound impairments in social behavior.

Prenatal malnutrition and sleep states in adult rats: effects of restraint stress.

Independently, prenatal malnutrition and psychological/physical stress have been shown to affect sleep architecture in adult rats. As malnutrition and stress commonly co-exist in malnourished human populations, the objective of the present study was to ascertain the combined effects of these two insults by examining sleep-wake parameters following a brief restraint stress in prenatally protein malnourished rats. The male offspring of rats provided with a protein deficient diet (6% casein) for 5 weeks prior to mating and throughout pregnancy were implanted with recording electrodes beginning at postnatal day 90. Polygraph recordings were obtained to quantify sleep states during the first 4 h of the dark phase of the cycle on 2 consecutive days. The first followed a 24-h habituation session to the recording chamber (baseline). The second occurred at the same time of day but followed 20 min of restraint stress in a Plexiglas tube. During baseline, prenatally malnourished rats spent more time in rapid eye movement sleep (REMS) in the first 2 h after "lights off" (block 1), and greater amounts of wakefulness (W) with a corresponding reduction in slow wave sleep (SWS) in the second two hours (block 2), as compared with controls. Following stress, the sleep architecture of both groups of rats remained unaltered in block 1 relative to their baseline day. In block 2, both groups exhibited significant reductions in SWS and REMS with significantly greater reductions being expressed in the prenatally malnourished group (most dramatically, REMS was completely eliminated). These findings suggest that sleep disturbances may be more severe in those malnourished human populations subjected to acutely stressful experiences.

Inactivation of RNA Viruses by Gamma Irradiation: A Study on Mitigating Factors.

Effective inactivation of biosafety level 4 (BSL-4) pathogens is vital in order to study these agents safely. Gamma irradiation is a commonly used method for the inactivation of BSL-4 viruses, which among other advantages, facilitates the study of inactivated yet morphologically intact virions. The reported values for susceptibility of viruses to inactivation by gamma irradiation are sometimes inconsistent, likely due to differences in experimental protocols. We analyzed the effects of common sample attributes on the inactivation of a recombinant vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein and green fluorescent protein. Using this surrogate virus, we found that sample volume and protein content of the sample modulated viral inactivation by gamma irradiation but that air volume within the sample container and the addition of external disinfectant surrounding the sample did not. These data identify several factors which alter viral susceptibility to inactivation and highlight the usefulness of lower biosafety level surrogate viruses for such studies. Our results underscore the need to validate inactivation protocols of BSL-4 pathogens using "worst-case scenario" procedures to ensure complete sample inactivation.

X-linked loci influence spatial navigation performance in Dahl rats.

Elucidation of natural genetic variations underlying strain or individual differences in cognitive function has remained elusive. Here we report the identification of two genetic loci that influence spatial navigation in Dahl rats. In the Morris water maze test, Dahl R rats exhibited efficient spatial navigation, whereas Dahl S rats displayed poor spatial navigation (accuracy). Analysis of F1 male progeny of reciprocal crosses between Dahl S and Dahl R strains implicated the X chromosome with the impairment in spatial navigation observed in Dahl S rats. Quantitative trait locus (QTL) analysis of an (RXS) F2 male population phenotyped for spatial navigation detected two QTLs on chromosome X influencing spatial navigation performance. One QTL (Nav-1, centered at DXRat21, significant for linkage) influenced acquisition performance without affecting spatial accuracy performance; and the second QTL (Nav-2, centered at DXRat25, significant for linkage) affected spatial accuracy performance with no detectable effect on acquisition performance. Our results demonstrate X linkage of spatial navigation performance in Dahl rats and provide evidence for the existence of independent genetic determinants for defined behavioral components of spatial navigation.

Effects of prenatal protein malnutrition and acute postnatal stress on granule cell genesis in the fascia dentata of neonatal and juvenile rats.

Although postnatal genesis of granule cells in the hippocampal fascia dentata is known to be influenced by prenatal protein deprivation or by stress, the combined effects of prenatal protein malnutrition and stress on these cells are unknown. This study was designed to examine this combined effect. Well-nourished and prenatally malnourished pups on postnatal day 7 (P7) were stressed by maternal separation and reduction of body temperature and on postnatal day 30 (P30) by immobilization with restraint. Bromodeoxyuridine (BrDU) was injected at the time of stress, and 2 h later, the numbers of immunolabeled cells were quantified by standard stereological techniques. In comparison to controls, prenatally malnourished rats showed a significantly lower number of cells tagged in the fascia dentata on P7 (p < or =0.05), and a significantly higher number of cells (p < or =0.05) on P30. In both age groups, control rats exposed to acute stress showed a significantly decreased number of cells tagged in the fascia dentata (p < or =0.05). In contrast, neurogenesis in malnourished rats was not significantly affected by acute stress at either age. Thus, the pattern of neurogenesis in the fascia dentata and its response to stress has been fundamentally altered by prenatal protein deprivation.

Prenatal protein malnutrition enhances stimulus control by CDP, but not a CDP/THIP combination in rats.

In the present study, the effects of prenatal protein malnutrition on stimulus control exerted by the benzodiazepine (BZ), chlordiazepoxide (CDP) and the GABA-A receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) were characterized. The adult, male offspring of female Sprague-Dawley rats fed either low (6% casein) or adequate (25% casein) protein diets 5 weeks prior to mating and throughout pregnancy served as subjects. Subjects were first trained to discriminate CDP (8.0 mg/kg ip) from saline using drug discrimination procedures. Once a criterion level of performance was achieved, generalization tests were performed to lower doses of CDP (4.0, 2.0, 1.0, 0.5 and 0.25 mg/kg) and then to several doses of THIP (10.0, 7.5, 5.6 and 3.2 mg/kg). Lastly, the ability of a single dose of THIP (3.0 mg/kg) to enhance discriminative control by several low doses of CDP (4.0, 2.0, 1.0 and 0.5 mg/kg) was assessed. Although both diet groups acquired the original CDP/saline discrimination at the same rate, malnourished rats exhibited significantly more generalization to low doses of CDP than their well-nourished counterparts. Neither diet group exhibited significant generalization to THIP nor a difference in THIP's ability to enhance the CDP cue. These results suggest that a subject's sensitivity to the stimulus properties of drugs can be selectively modified by prenatal malnutrition.

MDMA pretreatment leads to mild chronic unpredictable stress-induced impairments in spatial learning.

3,4-Methylenedioxymethamphetamine (MDMA) is a drug of abuse worldwide and a selective serotonin (5-HT) neurotoxin. An important factor in the risk of drug abuse and relapse is stress. Although multiple parallels exist between MDMA abuse and stress, including effects on 5-HTergic neurotransmission, few studies have investigated the consequences of combined exposure to MDMA and chronic stress. Therefore, rats were pretreated with MDMA and exposed 7 days later to 10 days of mild chronic unpredictable stress (CUS). MDMA pretreatment was hypothesized to enhance the effects of CUS leading to enhanced 5-HT transporter (SERT) depletion in the hippocampus and increased anxiety and cognitive impairment. Whereas MDMA alone increased anxiety-like behavior on the elevated plus maze, CUS alone or in combination with MDMA pretreatment did not increase anxiety-like behavior. In contrast, MDMA pretreatment led to CUS-induced learning impairment in the Morris water maze but not an enhanced depletion of hippocampal SERT protein. These results show that prior exposure to MDMA leads to stress-induced impairments in learning behavior that is not otherwise observed with stress alone and appear unrelated to an enhanced depletion of SERT.

Asymmetry of neuron numbers in the hippocampal formation of prenatally malnourished and normally nourished rats: a stereological investigation.

There is considerable evidence for lateralization of hippocampal function and hemispheric asymmetry in humans. In the rat, studies have reported asymmetries in the thicknesses of layers, the volumes of hippocampal subfields, and the density of cells at specific points along the septotemporal axis. To determine if there is an asymmetry of neuron numbers and whether prenatal malnutrition affects any asymmetries, 90-day old male Sprague-Dawley rats that were either normally nourished or malnourished prenatally were perfused with 4% paraformaldehyde and the brains cut into 30-micro m sections. One interrupted series of sections through the entire hippocampus was analyzed stereologically to estimate the total number of neurons in the hilus of the dentate gyrus, the CA3/CA2 stratum pyramidale (SP), the CA1 SP, and the SP of the prosubiculum/subiculum of both hemispheres. Significant asymmetries (P < 0.05) were found in the CA1 and CA3/CA2 subfields, with the right hemisphere containing 21 and 6% fewer neurons, respectively. Malnutrition reduced neuron numbers in the CA1 subfield by 12%, but did not alter the hemispheric asymmetry. Our findings agree with previous reports of left dominant asymmetries in the rat brain and suggest that this may result from differences in total numbers of neurons.

Effect of prenatal protein malnutrition on numbers of neurons in the principal cell layers of the adult rat hippocampal formation.

Malnutrition has been associated with a variety of functional and anatomical impairments of the hippocampal formation. One of the more striking of these is widespread loss of hippocampal neurons in postnatally malnourished rats. In the present study we have investigated the effect of prenatal malnutrition on these same neuronal populations, neurons that are all generated during the period of the dietary restriction. In prenatally protein deprived rats, using design-based stereology, we have measured the regional volume and number of neurons in the hilus of the dentate gyrus and the pyramidal cell layers of CA3, CA2, CA1, and the subiculum of 90-day-old animals. These results demonstrated a statistically significant reduction of 20% in neuron numbers in the CA1 subfield, while numbers in the other subfields were unchanged. There was a corresponding significant reduction of 22% in the volume of the CA1 subfield and a significant 14% decrease in the volume of the pyramidal layer of the subiculum. The change in volume of the pyramidal layer of the subiculum without neuron loss may reflect loss of CA1 afferent input to the pyramidal layer. Although the effect of nutritional deprivation on the neuronal population appears to be different in pre- and postnatal malnutrition, both dietary paradigms highlight the vulnerability of key components of the hippocampal trisynaptic circuit (consisting of the dentate granule cell mossy fibers projection to CA3 pyramids and the CA3 projection to the CA1 pyramids), which is an essential circuit for memory and learning.

Modulation of learning and memory in Dahl rats by dietary salt restriction.

The Dahl rat represents a robust animal model of salt-sensitive hypertension, with Dahl S rats being salt sensitive and Dahl R rats (the Dahl S counterparts) being salt resistant for the development of hypertension. Here we evaluate the effect of reduced dietary salt intake on learning and memory in the Dahl rat model. Salt restriction produced a significant impairment in social transmission of food preference and social recognition memory in Dahl S rats without affecting spatial learning. In contrast, social transmission of food preference and social recognition memory remained unaffected in Dahl R rats, whereas navigation performance was significantly improved. This effect on learning and memory was not generalized because sodium restriction did not influence object recognition memory in either Dahl S or Dahl R rats. The significant decrement in select cognitive functions in Dahl S rats produced by salt restriction are in sharp contrast to the well known positive effect of dietary salt restriction in alleviating high blood pressure and associated target organ complications, suggesting that caution must be exercised when weighing the benefits of salt restriction in improving cardiovascular health in salt-sensitive hypertension against the potential undesirable effects of reduced cognitive function.

Prenatal protein malnutrition in rats alters the c-Fos response of neurons in the anterior cingulate and medial prefrontal region to behavioral stress.

Prenatal protein malnutrition affects brain development and behavior despite dietary rehabilitation from birth. Behavioral alterations include abnormal responses to stressors. To explore what brain regions mediate this altered response, we used immunocytochemistry to c-Fos protein, a transcription factor marking neuronal activation. Controls (25% casein diet) and prenatally malnourished (6% casein) adult rats were subjected to 20min of restraint stress or were unstressed. Plasma corticosterone levels were monitored before and after stress. Paired comparisons of corticosterone levels confirmed that both groups showed a significant post-stress increase. Three hours after onset of stress, rats were perfused with paraformaldehyde. Brain sections were immuno-stained together for c-Fos. Since anterior cingulate and medial prefrontal cortex modulate stress responses, labeled neurons in this region were quantified using unbiased stereology. A 2-way ANOVA of neuron numbers demonstrated a strong effect of stress and a stress by nutrition interaction. Post-hoc comparisons showed that stress significantly increased the number of c-Fos labeled neurons in both nutrition groups. Within the stress condition, prenatally malnourished rats showed a significantly greater number of c-Fos positive neurons than well-nourished rats. These results suggest that neurons in anterior cingulate and medial prefrontal regions respond excessively to restraint stress in prenatally malnourished rats.

Ultrasonic call characteristics of rat pups are altered following prenatal malnutrition.

The male offspring of rats provided with a protein deficient diet (6% casein) for 5 weeks prior to mating and throughout pregnancy were subjected to a brief period of isolation and cooling at postnatal Days (P)7, 9, and 11, and their ultrasonic vocalizations were compared with those of well-nourished pups. Calls were categorized into 12 different types based upon their sonographic patterns. Although call rates were equal, the call characteristics of the prenatally malnourished pups differed significantly from those of well-nourished controls. At P7, their mean peak sound frequency (irrespective of call type) was significantly higher, and constant frequency calls were of both higher frequency and longer duration. Over the age range studied, prenatally malnourished pups emitted a smaller variety of calls, with significantly fewer ascending frequency vocalizations while producing either significantly fewer (P9) or greater (P11) descending frequency calls. Altered crying patterns have been related to brain damage in human babies, with more abnormal cries being associated with more severe neurological impairment. Therefore, the present results most likely reflect altered central nervous system development and function. Ultrasonic vocalization characteristics in rat pups may provide a useful early marker of the severity of disturbance to the development of the central nervous system following an insult, and offer the potential for predicting the degree of functional and behavioral deficits later in life.