Effects And Mechanisms Of Pre And Postnatal Stimulation On Fearful Behavior Of Rat Offspringe

BackgroundIt is well known that the relationship between mother and fetus is significant, and many factors during pregnancy can lead to physical malformations or behavioral dysfunctions. In the past few years, the effects of social-psychological factors during pregnancy have gradually aroused the attention of the public. In order to study the effect of prenatal stress on their offspring, many kinds of animal models have been made, and many studies have shown that prenatal stressed (PS) offspring exhibited more fearful behavior in behavioral tests. Furthermore, as showing behavioral, biochemical and anatomical similarities to anxious humans, the PS rat has become an animal model to study pathological anxiety. It is obviously that exploring the pathogenesis and prevention of abnormal fearful behavior of PS rat maybe contribute to the prevention and treatment of human anxiety disorders. Besides, the effects of environmental enrichment during pregnancy on fearful behavior of their offspring have been less documented and remain questionable.The amygdala is a key component of the neural circuitry of fear—both innate and learned in humans and in simpler vertebrate experimental animals. Cellular and molecular mechanisms of conditioned fear are focused on the interaction between positive glutamine pathway and inhibitory GABAergic pathway, involving in the electrophysiological changes, gene expression and synthesis of protein. Although GABA interneurons account for a small portion of neurons, they play a crucial role in inhibiting the excitatory neurons and regulating the complex interactions among principal cells, moreover, inhibitory postsynaptic potentials elicited from GABA interneurons has a robust effect of feed-forward and feedback.GRP and GRP receptor (GRPR) are distributed throughout the mammalian central nervous system. Gastrin-releasing peptide receptors (GRPR) have an important role in regulating amygdala-dependent, fear-related learning. GRPR-deficient mice showed decreased inhibition of principal neurons by interneurons in slices, enhanced long-term potential (LTP); these mice performed normally in hippocampus-dependent Morris maze, but showed greater and more persistent long-term fear memory in Pavlovian auditory cue fear conditioning, an amygdala-dependent task, and contextual fear conditioning which depend both on the amygdala and the hippocampus, which provide genetic evidence that GRP and its neural circuitry operate as a negative feedback regulating fear memory. However, both intrahippocampal infusion and microinjected into the basolateral amygdala of the bombesin/gastrin-releasing peptide antagonist RC-3095 impair formation of aversive memory, indicating that GRP system(s) can significantly enhance fear memory. In conclusion, the relationship between GRP, GRPR and conditioned fear memory remain questionable.Environmental enrichment (EE) is defined as a combination of "complex inanimate objects and social stimulation". The importance of the environment in brain regulation, behavior and physiology has long been recognized in the biological, social and medical sciences. Animals maintained under enriched conditions clearly have better memory, learning abilities and less anxiety-like behavior than those housed under standard conditions. However, the effects of environmental enrichment on the expression of GRPR in the brain have been less documented.Objective1. To investigate the effects of different stimulation during pregnancy on fearful behavior of offspring at 25, 45 and 60 days of age in the open field test and defensive withdrawal test.2. To investigate the relationship between fearful behavior and the number of GRPR positive cells, levels of GRPR mRNA and protein in the amygdala and hippocampus of prenatal stressed offspring.3. To investigate the effect of postnatal environmental enrichment on the number of GRPR positive cells, expression of GRPR mRNA and protein in the amygdala and hippocampus of PS offspring, and to explore the molecular mechanisms of environmental enrichment.Method1. On gestational days 11, primigravid female rats were randomly assigned to three groups: enriched group, stressed group and the control group, n=4 per group. All animals were individually housed. Prenatal treatments were performed daily on gestational days 11-21. The enriched dams were kept in specially designed cages (80 cm long×60 cm wide×40 cm high) that were equipped with a running wheel, a shelter, plastic color toys and small constructions such as chain and swing, tunnels. Throughout the enrichment periods, the shelter and running wheel were kept in the cage, while the toys and constructions were changed once a week. The stressed dams were put into a narrow animal holder and exposed to bright light for 45 min three times a day (starting from 1000, 1300 and 1600 h). The control dams were left undisturbed. At birth, the litters were randomly culled to 10 pups within 24 h post delivery. The offspring were left undisturbed together with their mother in standard cages. Male and female offspring were weaned at postnatal day 21(P21), and only male offspring (n=16 per group) were used in the present study. Open field test and the defensive withdrawal test were performed at P25, P45 and P60.2. From P21, 20 pups of control or stressed dams (CC group, SC group) were kept in standard Plexiglas cages (60 cm long×40cm wide×25cm high, n = 4 per cage) and left undisturbed. The same number pups from control or stressed dams (CE group, SE group) were kept in specially designed cages (n = 10 per cage). Throughout the enrichment periods (P21-P60), the shelter and running wheel were kept in the cage, while the toys and constructions were changed once a week. Also once a week, the feeding boxes and water bottles were moved to different cage points to encourage foraging and explorative behaviors. On P60, all pups were tested in the defensive withdrawal and then sacrificed. Six rats per group used in the study of GRPR staining and Nissl staining received an overdose of chloral hydrate (400mg/kg, i.p.) and perfused with different solution. The other part was sacrificed by decapitation and the cortical portion, amygdala and hippocampus were dissected on ice. These brains were used in the study of Western blot and quantitative real-time detection PCR.Results1. Effects of different stimulation during pregnancy on fearful behavior of their offspring at 25,45 and 60 days of age.Open-field testPS offspring at any age had greater central grille time, decreased total cross number and stand number than their corresponding controls (all p<0.05) , showing that prenatal stress inhibited exploratory behavior of their offspring in novel circumstance. On the contrary, the prenatal enriched offspring at 25 days of age had increased total cross number (p<0.05) and shorter central grille time (p<0.05) than their controls, however, the difference between enriched and control groups disappeared at postnatal day 45 and 60, indicating enriched environment during pregnancy can only enhanced exploratory behavior of their infancy offspring.Defensive-withdrawal testWhen compare to the controls, PS offspring had greater latencies at P25, greater latencies and decreased number of exits from the chamber at P45, and greater latencies, decreased number of exits from the chamber and increased time in the chamber with restraint at P60 (all p <0.05), which indicating that PS offspring had more fearful behavior than their corresponding controls, and the fearful behavior was enhanced with increasing of age. On the contrary, the prenatal enriched offspring at 25 days of age had shorter latencies and increased number of exits from the chamber than their controls(all p <0.05), but the difference between enriched and control groups disappeared at postnatal day 45 and 60, indicating enriched environment during pregnancy decreased fearfulness and enhanced exploratory behavior of their infancy offspring.2. Effects of enriched environment treatment on fearful behavior of PS offspringPS offspring showed less fearfulness after maintained under enriched conditions, which was measured as a shorter latency and increased number of exits from the chamber without restraint, and was less affected by restraint stress(all p <0.05), which indicate that enriched environment treatment can reverse the abnormal behavior of PS offspring.3. Effects of pre and postnatal circumstance on the number of GRPR -positive cells in cortical portion, amygdala and hippocampusWhen compared to the control pups (CC group and SC group) respectively, pups maintained under enriched environment (CE group and SE group) exhibited significantly increase in density of GRPR -positive cells in amygdala nuclei and hippocampus areas (all p <0.001), but there were no significant difference between CE and SE groups or SC and CC groups (all p>0.05). No differences were found in density of GRPR -positive cells in the cortex containing M1, M2, RSA and RsGb areas among all groups [F(3,143)=0.34, p>0.05].4. Effects of pre and postnatal circumstance on the expression of GRPR mRNA in cortical portion, amygdala and hippocampusGRPR gene expression in the amygdala and hippocampus of CE group were up-regulated 136.47±48.75 and 146.30±37.46 when compared to CC groups (all p <0.001), and GRPR gene expression in the amygdala and hippocampus increased 129.25±39.51 and 151.74±43.38 fold respectively in SE group comparing with that in SC groups (1.22±0.39, 0.98±0.21) (allp<0.001). However, there were no significant difference between CE group and SE group (all p>0.05) or SC group and CC group (all p>0.05), besides, no differences were found in GRPR gene expression in the cortex containing Ml, M2, RSA and RsGb areas among all groups [F(3,39)=1.42, p>0.05].5. Effects of pre and postnatal circumstance on GRPR protein in cortical portion, amygdala and hippocampusGRPR protein expression in the amygdala and hippocampus of CE groups were up-regulated 1.06±0.12 and 1.29±0.23 when compared to CC groups(0.29±0.04, 0.29±0.04) (all p <0.001), and GRPR protein expression in the amygdala and hippocampus of SE groups (0.97±0.12,1.39±0.29) were increased when compared with that of SC groups (0.28±0.03, 0.28±0.05) (all p<0.001). However, there were no significant difference between CE group and SE group (all p>0.05) or SC group and CC group (all p>0.05), besides, no differences were found in GRPR protein expression in the cortex containing M1, M2, RSA and RsGb areas among all groups [F(3,31)=0.30, p>0.05].Conclusions1. Our studies provided more evidence that prenatal stress (put into a narrow animal holder and exposed to bright light) enhance fearfulness of their offspring, which can be significantly increased following acute restraint, and with increasing age, the fearful behavior was enhanced. These findings in PS rats are similar to the data from humans showing that the onset of pathological anxiety often manifests at a young age, persists throughout adolescence and continues into adulthood, exhibiting increased fearfulness, manifesting as avoidance of anxiety provoking situations. So this animal model can be used as a model for the study of anxiety disorders.2. We found for the first time that environmental enrichment during pregnancy only can decrease fearfulness and enhance exploratory behavior of their infancy offspring. Further research will be needed to elucidate the mechanism.3. We found for the first time that prenatal stress has no effect on the number of GRPR -positive cells, the expression of GRPR gene and protein in the cortical, amygdala and hippocampus of their offspring, the mechanism need to be explored.4. We found for the first time that enriched environment treatment can reverse the abnormal behavior of PS offspring, and increase the expression of GRPR in the amygdala and hippocampus, indicating GRP and its neural circuitry operate as a negative feedback regulating fear memory.5. In conclusion, the present studies provided evidence that GRPR and its neural circuitry operate of GABA interneurons as a negative feedback regulating fear memory. Postnatal environmental enrichment can reverse the enhanced fearfulness in adult PS offspring, which might be in part mediated by modulation of GRPR in the amygdala and hippocampus. Therefore, enriched environment treatment may become a new method to cure anxiety disorders. Besides, our study also suggested congenital defect of GRP and/or GRPR may lead to the onset of anxiety disorders.