Iron Deficiency In Early Development Alters Behaviors And Dopamine D5 Receptor Expression In Rats

Iron deficiency (ID) is a common disorder in pediatric patients. Clinical studies demonstrate that iron deficiency is associated with apathy, irritability, anxiety, hypoactivity, and decreased cognition and attention. Iron deficiency in early development is associated with retardation in growth and cognitive development, and the effects on cognition may be irreversible.Iron plays an important role in the perinatal brain development. Brain iron deficiency in early development has multiple consequences in neurobiology including hypomyelination, impared dopaminergic function and delayed neuromaturatian. Iron deficiency in early life alters the course of behavioral and cognitive development in humans, leading to decreased physical activity and responsiveness to the environment. These behavioral changes are partly related to dopaminergic function in nigrostriatal and mesocorticolimbic pathways. Earlier studies show that iron deficiency decreased densities of D1 and D2 receptors in the caudate-putamen, as well as D2 receptor and dopamine transporter (DAT) densities in the nucleus accumbens. The changes in DA biology induced by perinatal iron deficiency are not completely normalized despite replenishment of iron status later.The effects of iron deficiency on behavior and dopamine receptor have been widely investigated in humans and in experimental animals.The dopamine receptor subtypes are divided into two major subclasses: the D1-like and D2-like receptors, The D1-like family (D1 and D5 receptors) has been involved in the regulation of motor activity at limbic system and is related to cognition. The D1 and D5 receptors share a 80% identity in their transmembrane domains. DA has -10 times higher affinity for the D5 receptor than the D1 receptor. D5 receptor can be found in rostral forebrain cortex, hippocampus, striatum and substantia nigra as well as thalamus. So far, it has not been possible to pharmacologically differentiate D1 and D5 receptors. There is still a question about distinct roles of D1 and D5 dopamine receptors in their function and clinical manifestation. This study was designed to investigate the effects of early Iron deficiency on dopamine D5 receptor expression, to determine whether Iron deficiency in early development produces behavioral changes not reversible with subsequent iron repletion, and to examine the relation between D5 receptor expression and behavioral changes.Materials and MethodMaterialsSprague-Dawley (SD) rats were obtained from the Center for Experimental Animal Center of Zhejiang University school of medicine. Iron sufficient diet (40mg iron/kg) and iron deficient diet (3-6mg iron/kg) were purchased from Harlan Teklad(USA), Madison, WI. The mouse anti-dopamine D5 receptor monoclonal antibody was purchased from CHEMICON International, Inc. USA.Method1, Establishment of Animal ModelsBefore mating, 16 SD female rats were treated with an iron sufficient diet for a week to regulate iron status. Then female rats were randomly assigned to two experimental diet groups. Iron deficient group (ID) (n=8) received an iron deficient diet (3-6mg iron/kg) and the control group (CN) (n=8) received an iron sufficient diet (40mg iron/kg). ID (n=70) and CN (n=85) pups were weaned at 21 days of age (P21) and fed the same diet as their dams. All the pups were treated with an iron sufficient diet at 25 days of age (P25). Blood samples of dams for hemoglobin and hematocrit analysis were obtained by thigh venous puncture.2, Behavioral AssessmentsThe level of development was assessed and recorded at days P6, P9, P12, P15, P18 and P21. The test for development assessment included forelimb hanging test, surface righting reflex and negative geotaxis reflex. Upper extremity sensorimotor function was assessed with vibrissae-evoked forelimb placing test, which is sensitive to nigrostriatal dopamine tract impairments, at P24 and P29. At P15, P25 and P35, pups received the open field test.3, Immunofluorescence assay (IFA)Whole brain of pups was removal and stored to take 8um frozen section for immunofluorescent staining at P15, P25 and P35. Dopamine D5 receptor expression levels in the two groups were evaluated under the fluorescene microscope.4, Statistical analysisAll values are expressed as mean ± SD. All the data were evaluated by theSPSS/PC+ software package. Significance of differences between groups were calculated using unpaired T-test. Difference between age in the same group was analyzed by one-way ANOVA test. P<0.05 was considered as statistically significant.Results1, Results of Animal Models(1) No differences was observed in the body weight of dams between the two groups (P>0.05). The ID dams showed a significant decrease in the hemoglobin and hematocrit levels at 20 days of gestation (G20) than GO; the level also decreased compared to that of the CN dams (P<0.001).(2) As the pups aged, the weights of ID pups were lower than that recorded for age-matched CN pups (P<0.05).2, Behavior(1) ID pups performed worse than age-matched CN pups in the development assessment tests, including forelimb hanging test, surface righting reflex and negative geotaxis reflex (P<0.05).(2) ID pups had reduced bilateral vibrissae-evoked forelimb placing after weaning as compared to age-matched CN pups. (P<0.05)o(3) ID pups at P15 and P25 showed significantly decreased levels of standing exploration and spontaneous groom in a new environment than age-matched controls (P<0.05). Total distance traveled by pups was also significantly lower in ID pups at P25 (P<0.001). At P35, total distance traveled by ID pups was still significantly lower than that by age-matched CN (P<0.05).3, Immunofluorescence assay(1) Dopamine D5 receptor expression in the basal ganglia was significantly lower in ID pups compared to that in age-matched CN pups at P15, P25 and P35.(2) D5 receptor expression for ID pups was significantly higher at P25 than P15 (P<0.001). ID pups at P35 showed an increase of D5 receptor expression compared to the pups at P25 (P<0.05).(3) D5 receptor expression for CN pups was significantly higher at P25 than P15 (P<0.05). CN pups at P35 showed no difference in D5 receptor expression compared to the pups at P25 (P>0.05).Conclusions1, The pups from perinatal iron-deficient dams showed a significantly retardation ingrowth and development.2, Iron deficiency in early development of the pups lead to more or less impairmentsincluding regulation of motor coordination, reflect development, sensorimotorfunction, emotion and cognition. And part of the changes in sensorimotor functionand cognition that vary with duration of perinatal iron deficiency are not completelynormalized despite replenishment of iron status.3, Dopamine D5 receptor expression follows a time-varying course of developmentin the rat brain.4, Iron deficiency in early development reduced the expression of dopamine D5receptor in rat brain. D5 receptor expression in basal ganglia peaked later with adelay the pups with iron deficiency in early development. The changes in D5receptor expression that vary with duration of early iron deficiency are notcompletely normalized despite replenishment of iron status.