Effects Of Perinatal Food Restriction On Neurobehavior Development As Well As Learning And Memory In Rats

ObjectiveFood restriction can induce the beneficial effects. It extends life span, reduces and retards the incidence of several diseases, increases learning and memory ability and attenuates the toxicological effects of drugs. Howerver, perinatal food restriction induces intrauterine growth restriction (IUGR), with neonatal babies at decreased body weight, childhood stunting and poor school achievement Perinatal food restriction or malnutrition can also induce neurodevelopmental delay, abnormal social behaviors and impaired spatial learning and memory ability besides decreased body weight and childhood stunting. Because perinatal malnutrition exerts harmful effects on human life and health, we studied the effect of perinatal food restriction on neurobehavioral development (neurodevelopmental milestones) and congnition ability. Our studies help further to recognize human fetal IUGR and provide beneficial information on developing neuroprotective therapies of human fetal IUGR. To address this question, a model of perinatal 50% food restriction (FR50) was induced in Wistar rats. The effect of perinatal FR50 on neurobehavioral development was observed, including physical growth and neurological reflexes. Since impaired or delayed neurobehavioral development is usually related to later recognition behaviors in young and adults, we studied spatial learning and memory abilities of adult offspring rats. In addition, in order to explore the possible mechanism of impaired spatial learning and memory abilities, we also observed synaptic plasticity and the density of nitric oxide synthase neurons in the hippocampus.MethodsThe first part of the experiments:Establishment of animal model with perinatal FR50. To study the effect of perinatal malnutrition on neurobehavioral development and spatial learning and memory abilities, establishment of animal model with perinatal FR50 was necessary. Generally, there are two ways, one is to restrict protein content, the other is to restrict food content. Due to difficulties of rat chow preparation on protein restriction, we apply the way of restricting food content. According to the extent of food restriction, there are three ways:slight, mild and severe food restriction. In the present study, we used the mild food restriction. Two or three healthy female rats (body weight 250-280 g,14-week old) were paired with one male (2-3:1) until mating was confirmed by observation of a copulatory plug or the presence of sperm in a vaginal rinse. The day that mating was confirmed and recorded as embryonic day 0 (E0). Each dam was singly housed. The day of birth was identified as postnatal day 0 (PDO). Afterwards, The pregnant rats were assigned radomly into two groups:the control group (n=18) and the model group (n=16). In the control group, dams had free access to diet and water during gestation and lactation (average amount of food consumed by control was recorded). In the model group, dams received normal food intake from gestation day 0 to 6, and 50% of the daily food intake of control mothers from gestation day 7 until the postnatal day 21. Water were available ad libitum in both control and FR50 rats. The body weight of dams was measured on El, E7, E14 and E21. Dams delivered spontaneously and the day of delivery was designated as PDO. The body weight of offspring rats was measured on PD1, PD7. PD10, PD14 and PD21. The result showed that FR50 reduced the body weight of newborn pups and the body weight of FR50 dams during E14 and E21. The present model mimics perinatal malnutrition and models fetal IUGR. The present study helps to further explore neurobehavioral development and cognition ability.The second part of the experiments:Effects of perinatal food restriction on neurobehavioral development in newborn Wistar rats. Gender was distinquish between male and females according to the anogenital distance. Neurodevelopmental assessment typically includes analysis of physical growth and neurological reflexes. The parameters of physical growth include timing of pinna detachment (PD2-PD5), eye opening (PD14-PD17), teeth eruption (PD8-PD12) and hair growth (PD11-PD14). Neurological reflexes include righting reflexes (PD3-PD7), negative geotaxis (PD6-PD10) and cliff avoidance reflex (PD4-PD9). The changes of neurobehavior development were observed between control and FR50 offsprings.The third part of the experiments:Effects of perinatal food restriction on spatial learning and memory ability as well as long-term potentiation in hippocampal CA1 area in adult offspring rats. On PD70, spatial learning and memory ability of rats was tested in Morris water maze (MWM). MWM task consists of two phases, place navigation test and spatial probe performance. In place navigation test, the animals were subjected to two sessions (morning and afternoon) of four trials per day for 5 consecutive days. In each trial, rats were gently released into water, facing the wall of the tank, at one of the four starting points. The trial starting points were the middle of each quadrant edge. Rats were allowed to swim freely until they climbed on the platform. The swimming time to reach the platform (escape latency from start to goal) for each trial was recorded. If they failed to locate the platform within 120s, they were placed on it for 15 seconds. After the place navigation test, the platform was removed and the same rats were used in a spatial probe test to determine memory retention. Rats were released into water at the position opposite to the target quadrant and allowed to swim for 120s. The time percent in target quadrant and the number of platform location crossings were measured. Afterwards, the in vivo field excitatory postsynaptic potential (fEPSP) was observed in the hippocampal CA1 area. Adult wistar rats were implanted with a monopolar recording electrode in the stratum radiatum of the CA1 area (3.4 mm posterior to bregma and 2.5 mm lateral to the midline), and a bipolar stimulating electrode in the Schaffer collaterals of the dorsal hippocampus (4.2 mm posterior to bregma and 3.5 mm lateral to midline) via holes drilled through the skull. The optimal depth of the electrode in the stratum radiatum of the CA1 area of the dorsal hippocampus was determined by the maximal response to the Schaffer collateral pathway stimulus. The fEPSP slope was recorded at frequence of 0.033Hz (30 s interval) by delivering a single current pulse (0.2 ms in duration) to the Schaffer collateral pathway. The stimulus intensity was adjusted to give an fEPSP amplitude of 50% of maximum. Each experiment consisted of a baseline measurement taken for 30 min, followed by a further measurement of evoked responses (120 min) after high frequency stimulation (HFS) application. HFS consisted of ten trains of 20 stimuli at 200Hz with 2 s intertrain interval, with the same stimulation intensity (0.033Hz) used for the basline recordings. fEPSP slope was analysed by Clampfit 9.0 softwale.The forth part of the experiments:Perinatal food restriction impaired spatial learning and memory behaviour and decreased the density of nitric oxide synthase neurons in the hippocampus of adult male rat offspring. Excitory neurotransmitters, such as glutamic acid, are necessary to learning and memory as well as consolidation and maintenance of LTP. Nitric oxide (NO) facilitates LTP in postsynaptic neuronal cells by promoting release of glutamic acid as a retrograde messenger. Therefore, the density of nitric oxide synthase neurons in the hippocampus of adult male rat offspring was studied using immunohistochemistry method. The aim is to study the possible mechanisms of impairment of spatial learning and memory ability by detecting the change of NO.Results1) Perinatal food restriction delayed physical growth, such as pinna detachment, hair growth, eruption of incisor teeth and eye opening. 2) Perinatal food restriction delayed neurological reflexes in surface righting reflex and cliff avoidance reflex. In addition, it also exhibited a delay in achieving negative geotaxis response in male pups but not in female pups.3) Perinatal food restriction induced spatial learning and memory deficits in adult male rat offspring.4) Perinatal food restriction impaired synaptic plasticity in hippocampal CA1 area of adult male rat offspring.5) Perinatal food restriction decreased the density of nitric oxide synthase neurons in the hippocampus (CA1 area, CA3 area and dentate gyrus) of adult male rat offspring.Conclusion1) Perinatal food restriction delayed neurobehavior development in newborn rats.2) The effect of perinatal food restriction on the development of cerebellum is gender-dependent.3) Perinatal food restriction impaired synaptic plasticity in hippocampus by reducing the content of NO. Finally, it induced spatial learning and memory deficits.