Mu opioid receptor knockout mouse: Phenotypes with implications on restless legs syndrome.

Restless legs syndrome (RLS) is characterized by an irresistible need to move the legs while sitting or lying at night with insomnia as a frequent consequence. Human RLS has been associated with abnormalities in the endogenous opioid system, the dopaminergic system, the iron regulatory system, anemia, and inflammatory and auto-immune disorders. Our previous work indicates that mice lacking all three subtypes of opioid receptors have a phenotype similar to that of human RLS. To study the roles of each opioid receptor subtype in RLS, we first used mu opioid receptor knockout (MOR KO) mice based on our earlier studies using postmortem brain and cell culture. The KO mice showed decreased hemoglobin, hematocrit, and red blood cells (RBCs), with an appearance of microcytic RBCs indicating anemia. Together with decreased serum iron and transferrin, but increased ferritin levels, the anemia is similar to that seen with chronic inflammation in humans. A decreased serum iron level was also observed in the wildtype mice treated with an MOR antagonist. Iron was increased in the liver and spleen of the KO mice. Normal circadian variations in the dopaminergic and serotoninergic systems were absent in the KO mice. The KO mice showed hyperactivity and increased thermal sensitivity in wakefulness primarily during what would normally be the sleep phase similar to that seen in human RLS. Deficits in endogenous opioid system transmission could predispose to anemia of inflammation and loss of circadian variations in dopaminergic or serotonergic systems, thereby contributing to an RLS-like phenotype.

Motor restlessness, sleep disturbances, thermal sensory alterations and elevated serum iron levels in Btbd9 mutant mice.

Restless legs syndrome (RLS), also known as Willis-Ekbom disease, is a sensory-motor neurological disorder with a circadian component. RLS is characterized by uncomfortable sensations in the extremities, generally at night or during sleep, which often leads to an uncontrollable urge to move them for relief. Recently, genomic studies identified single-nucleotide polymorphisms in BTBD9, along with three other genes, as being associated with a higher risk of RLS. Little is known about the function of BTBD9 or its potential role in the pathophysiology of RLS. We therefore examined a line of Btbd9 mutant mice we recently generated for phenotypes similar to symptoms found in RLS patients. We observed that the Btbd9 mutant mice had motor restlessness, sensory alterations likely limited to the rest phase, and decreased sleep and increased wake times during the rest phase. Additionally, the Btbd9 mutant mice had altered serum iron levels and monoamine neurotransmitter systems. Furthermore, the sensory alterations in the Btbd9 mutant mice were relieved using ropinirole, a dopaminergic agonist widely used for RLS treatment. These results, taken together, suggest that the Btbd9 mutant mice model several characteristics similar to RLS and would therefore be the first genotypic mouse model of RLS. Furthermore, our data provide further evidence that BTBD9 is involved in RLS, and future studies of the Btbd9 mutant mice will help shine light on its role in the pathophysiology of RLS. Finally, our data argue for the utility of Btbd9 mutant mice to discover and screen novel therapeutics for RLS.

Iron supplementation dose for perinatal iron deficiency differentially alters the neurochemistry of the frontal cortex and hippocampus in adult rats.

BACKGROUND: Long-term prefrontal cortex (PFC)- and hippocampus-based cognitive deficits are the sequelae of perinatal iron deficiency, despite iron supplementation starting in the newborn period. Whether high-dose iron supplementation prevents these deficits is yet to be determined. METHODS: Perinatal iron deficiency was induced in rat pups using a low-iron (3 mg/kg diet) diet during gestation until postnatal day (P)8. Iron was supplemented using a standard (40 mg/kg diet) or a 10-fold higher (400 mg/kg diet) iron-containing diet until P21. PFC and hippocampal neurochemistry was determined using in vivo (1)H nuclear magnetic resonance (NMR) spectroscopy at 9.4 Tesla on P90. RESULTS: Both standard and 10-fold higher iron supplementation doses corrected anemia and brain iron deficiency by P21. The neurochemical profile of the PFC in both supplementation groups was comparable with the control group. In the hippocampus, standard-dose iron supplementation resulted in lower concentrations of N-acetylaspartate (NAA) and phosphoethanolamine (PE) and higher concentrations of N-acetylaspartylglutamate (NAAG) and glycerophosphocholine + phosphocholine (GPC + PC). High-dose iron supplementation resulted in lower PE and higher GPC + PC concentrations. CONCLUSION: The iron supplementation dose for perinatal iron deficiency differentially alters the neurochemical profile of the PFC and hippocampus in adults. The neurochemical changes suggest altered glutamatergic neurotransmission, hypomyelination, and abnormal phospholipid metabolism in the formerly iron-deficient (FID) hippocampus.

Systems genetic analysis of the effects of iron deficiency in mouse brain.

Iron regulation in the brain is both necessary and highly complex. Too little or too much iron can compromise neurological function, yet we still do not know all of the regulatory processes. In our research, we seek to identify genes and gene networks underlying individual differences in brain iron regulation. To this end, we fed mice from 20+ inbred strains a diet low in iron from weaning to 4 months of age. At sacrifice, we measured iron content in the ventral midbrain (VMB). The VMB contains the substantia nigra, a region particularly vulnerable to iron imbalance. The results showed high, inter-strain variability in dietary iron reduction, from almost no loss to more than 40 % vs. control. When we performed quantitative trait loci (QTL) analysis, we observed a significant area on chromosome 2. Within this QTL, we selected glial high-affinity glutamate transporter 1 (Glt1) as the leading candidate. Expression of this gene is both correlated with VMB iron and is also cis-modulated by local sequence variants that segregate in the BXD family. VMB expression differences of Glt1 in six strains covary with differential susceptibility to VMB iron loss.

Ceruloplasmin deficiency results in an anxiety phenotype involving deficits in hippocampal iron, serotonin, and BDNF.

Ceruloplasmin (Cp) is a ferroxidase involved in iron metabolism by converting Fe(2+) to Fe(3+), and by regulating cellular iron efflux. In the ceruloplasmin knockout (CpKO) mouse, the deregulation of iron metabolism results in moderate liver and spleen hemosiderosis, but the impact of Cp deficiency on brain neurochemistry and behavior in this animal model is unknown. We found that in contrast to peripheral tissues, iron levels in the hippocampus are significantly reduced in CpKO mice. Although it does not cause any discernable deficits in motor function or learning and memory, Cp deficiency results in heightened anxiety-like behavior in the open field and elevated plus maze tests. This anxiety phenotype is associated with elevated levels of plasma corticosterone. Previous studies provided evidence that anxiety disorders and long-standing stress are associated with reductions in levels of serotonin (5HT) and brain-derived neurotrophic factor (BDNF) in the hippocampus. We found that levels of 5HT and norepinephrine (NE), and the expression of BDNF and its receptor trkB, are significantly reduced in the hippocampus of CpKO mice. Thus, Cp deficiency causes an anxiety phenotype by a mechanism that involves decreased levels of iron, 5HT, NE, and BDNF in the hippocampus.

Systems genetic analysis of multivariate response to iron deficiency in mice.

The aim of this study was to identify genes that influence iron regulation under varying dietary iron availability. Male and female mice from 20+ BXD recombinant inbred strains were fed iron-poor or iron-adequate diets from weaning until 4 mo of age. At death, the spleen, liver, and blood were harvested for the measurement of hemoglobin, hematocrit, total iron binding capacity, transferrin saturation, and liver, spleen and plasma iron concentration. For each measure and diet, we found large, strain-related variability. A principal-components analysis (PCA) was performed on the strain means for the seven parameters under each dietary condition for each sex, followed by quantitative trait loci (QTL) analysis on the factors. Compared with the iron-adequate diet, iron deficiency altered the factor structure of the principal components. QTL analysis, combined with PosMed (a candidate gene searching system) published gene expression data and literature citations, identified seven candidate genes, Ptprd, Mdm1, Picalm, lip1, Tcerg1, Skp2, and Frzb based on PCA factor, diet, and sex. Expression of each of these is cis-regulated, significantly correlated with the corresponding PCA factor, and previously reported to regulate iron, directly or indirectly. We propose that polymorphisms in multiple genes underlie individual differences in iron regulation, especially in response to dietary iron challenge. This research shows that iron management is a highly complex trait, influenced by multiple genes. Systems genetics analysis of iron homeostasis holds promise for developing new methods for prevention and treatment of iron deficiency anemia and related diseases.

Behavior and monoamine deficits in prenatal and perinatal iron deficiency are not corrected by early postnatal moderate-iron or high-iron diets in rats.

Developmental iron deficiency anemia (IDA) causes brain and behavioral deficits in rodent models, which cannot be reversed when treated at periods equivalent to later infancy in humans. This study sought to determine whether earlier iron treatment can normalize deficits of IDA in rats and what iron dose is optimal. The offspring of dams with IDA during gestation were cross-fostered at postnatal d (P) 8 to dams receiving diets with 1 of 3 iron concentrations until weaning (P21): 0.003-0.01 g/kg [totally iron deficient (TID)]; 0.04 g/kg [formerly iron deficient (FID-40)]; or 0.4 g/kg (FID-400). Always iron-sufficient control dams (CN-40) received a 0.04-g/kg iron diet. At P21, TID pups received a 0.01 g iron/kg diet; all others received a 0.04 g iron/kg diet. Hematocrit and brain iron and monoamine concentrations were assessed at P21 and P100. Pup growth, development, activity, object recognition, hesitancy, and watermaze performance were evaluated. Regional brain iron was restored by iron treatment. Regional monoamine and metabolite concentrations were elevated in FID-40 rats and reduced in FID-400 and TID rats compared with CN-40 rats. FID-40 offspring had motor delays similar to TID during lactation and FID-400 rats had elevated thigmotaxis similar to TID rats at P25 and P100 in the spatial watermaze. In conclusion, iron treatment at P8 in rats did not normalize all monoamine or behavioral measures after early IDA. Moderate iron treatment improved adult behavior, but higher iron treatment caused brain and behavioral patterns similar to TID in the short and long term.

Iron chelation down-regulates dopamine transporter expression by decreasing mRNA stability and increasing endocytosis in N2a cells.

Cell surface expression of the dopamine transporter (DAT) is determined by the relative rates of its internalization and recycling. Changes in the cellular labile iron pool (LIP) affect many cellular mechanisms including those that regulate DAT trafficking. In this study, we analyzed DAT expression and posttranslational modifications in response to changes in cellular iron in transfected neuroblastoma cells (N2a). Iron chelation by desferrioxamine (DFO) altered DAT protein levels by decreasing the stability of DAT mRNA. Increased phosphorylation and ubiquitination of this transporter protein following DFO treatment were also observed. Cellular iron depletion elevated protein levels of the early endosomal marker Rab5. Moreover, confocal microscopy studies showed increased localization of DAT into the endosomal compartment in DFO-treated cells compared to control. Together, these findings suggest that cellular iron depletion regulates DAT expression through reducing mRNA stability as well as an increasing in endocytosis.

Interrelationships between tissue iron status and erythropoiesis during postweaning development following neonatal iron deficiency in rats.

Dietary iron is particularly critical during periods of rapid growth such as in neonatal development. Human and rodent studies have indicated that iron deficiency or excess during this critical stage of development can have significant long- and short-term consequences. Since the requirement for iron changes during development, the availability of adequate iron is critical for the differentiation and maturation of individual organs participating in iron homeostasis. We have examined in rats the effects of dietary iron supplement following neonatal iron deficiency on tissue iron status in relation to erythropoietic ability during 16 wk of postweaning development. This physiological model indicates that postweaning iron-adequate diet following neonatal iron deficiency adversely affects erythroid differentiation in the bone marrow and promotes splenic erythropoiesis leading to splenomegaly and erythrocytosis. This altered physiology of iron homeostasis during postweaning development is also reflected in the inability to maintain liver and spleen iron concentrations and the altered expression of iron regulatory proteins in the liver. These studies provide critical insights into the consequences of neonatal iron deficiency and the dietary iron-induced cellular signals affecting iron homeostasis during early development.

Genetic analysis of iron-deficiency effects on the mouse spleen.

Iron homeostasis is crucial to many biological functions in nearly all organisms, with roles ranging from oxygen transport to immune function. Disruption of iron homeostasis may result in iron overload or iron deficiency. Iron deficiency may have severe consequences, including anemia or changes in immune or neurotransmitter systems. Here we report on the variability of phenotypic iron tissue loss and splenomegaly and the associated quantitative trait loci (QTLs), polymorphic areas in the mouse genome that may contain one or more genes that play a role in spleen iron concentration or spleen weight under each dietary treatment. Mice from 26 BXD/Ty recombinant inbred strains, including the parent C57BL/6 and DBA/2 strains, were randomly assigned to adequate iron or iron-deficient diets at weaning. After 120 days, splenomegaly was measured by spleen weight, and spleen iron was assessed using a modified spectrophotometry technique. QTL analyses and gene expression comparisons were then conducted using the WebQTL GeneNetwork. We observed wide, genetic-based variability in splenomegaly and spleen iron loss in BXD/Ty recombinant inbred strains fed an iron-deficient diet. Moreover, we identified several suggestive QTLs. Matching our QTLs with gene expression data from the spleen revealed candidate genes. Our work shows that individual differences in splenomegaly response to iron deficiency are influenced at least partly by genetic constitution. We propose mechanistic hypotheses by which splenomegaly may result from iron deficiency.

A postweaning iron-adequate diet following neonatal iron deficiency affects iron homeostasis and growth in young rats.

Iron deficiency is among the most prevalent of nutrient-related diseases worldwide, but the long-term consequences of maternal and neonatal iron deficiency on offspring are not well characterized. We investigated the effects of a postweaning iron-adequate diet following neonatal iron deficiency on the expression of genes involved in iron acquisition and homeostasis. Pregnant rats were fed an iron-adequate diet (0.08 g iron/kg diet) until gestational d 15, at which time they were divided into 2 groups: 1) a control group fed an iron-adequate diet, and 2) an iron-deficient group fed an iron-deficient diet (0.005 g iron/kg diet) through postnatal d (P) 23 (weaning). After weaning, pups from both dietary treatment groups were fed an iron-adequate diet until adulthood (P75). Rat pups that were iron deficient during the neonatal period (IDIA) had reduced weight gain and hemoglobin concentrations and decreased levels of serum, liver, and spleen iron on P75 compared with rats that were iron sufficient throughout early life (IA). IDIA rats developed erythrocytosis during postweaning development. Further, hepatic expression of hepcidin in IDIA rats was 1.4-fold greater than in IA rats, which paralleled an upregulation of IL-1 expression in the serum. Our data suggest that an iron-adequate diet following neonatal iron deficiency induced an inflammatory milieu that affected iron homeostasis and early growth and development.

Iron-deficiency and dopaminergic treatment effects on RLS-Like behaviors of an animal model with the brain iron deficiency pattern of the restless legs syndrome.

BACKGROUND: Brain iron deficiency (BID), especially for the substantia nigra (SN), without peripheral iron deficiency (ID) has been well documented as a ubiquitous finding for restless legs syndrome (RLS) patients. This close association suggests the biology of RLS BID can produce RLS symptoms. Association, however, cannot establish such a direct relationship. Instead, the BID of RLS could be experimentally produced to determine if it then produces significant RLS-like biological or behavioral features. Forward genetics approach led to identification from the BXD strains the BXD40 females (BXD40f) as a putative animal model for the RLS BID. The BXD40f on an iron-sufficient diet have a lower iron in the VMB (containing the SN) during the active but not inactive period. This was not found for the other BXD strains evaluated. The BXD40f on an ID diet uniquely have even greater reduced VMB but not peripheral iron, matching the RLS BID pathophysiology. A prior report found that the BXD40f on an iron-sufficient diet had an RLS-like behavior of increased activity occurring only in the last part of the active period that was not present in the other strains without the low VMB iron. This increased activity matches the circadian pattern of symptoms in RLS patients with increased urge or drive to move in the last part of the day. This study asks first: if you decrease the VMB iron by an iron deficient diet do the RLS-like behaviors worsen; and second will the dopaminergic treatments effective for RLS also reduce the worsened RLSlike behaviors. METHODS: In sum, 13 BXD40f mice post weaning were randomly assigned for 100 days to either a iron-sufficient diet (n = 6) or an ID diet (N = 7). They were then evaluated for 24-h activity in their home cage using implanted G2 EMitter telemetry device. At 3 h before the end of the active period IP doses were given every other day of either: saline (vehicle only), 12.5 mg levodopa, 25 mg levodopa, 0.5 mg quinpirole, or 1 0.0 mg quinpirole. RESULTS: The ID compared to irons-sufficient diet produced earlier onset of the RLS-like behavior matching the earlier onset of symptoms with increasing severity of RLS. The dopaminergic treatments significantly reduced the RLS-like behavior. Added analyses of the RLS-like behaviors as decreased resting times showed similar results to activity increases. CONCLUSIONS: These data demonstrate both that The BXD40f provide a useful animal model of RLS and also strongly support the hypothesis that the biology of RLS BID can produce RLS symptoms.

Developing a behavioral model of Restless Legs Syndrome utilizing mice with natural variances in ventral midbrain iron.

BACKGROUND: The primary symptoms of Restless Legs Syndrome (RLS) are circadian-dependent, leading to increased activity or decreased rest, especially at night. The primary pathology in RLS is brain iron insufficiency despite normal systemic iron stores. Natural variances in brain and peripheral iron concentrations across recombinant inbred (RI) murine strains provide a biological model of RLS. The question is whether these RI mice strains show a behavioral analog to circadian-dependent clinical phenotype of RLS. METHODS: The home cage activity of eight female RI strains was measured over a 72-h period. The ratio of the average activity in the last 2 h of the active period relative to that in the total 12-h active period (late active period activity ratio, LAPAR) was the primary outcome variable. The relation of average LAPAR scores to measures of ventral midbrain (VMB) iron was evaluated across strains in this study. RESULTS: RI strain 40 (LAPAR = 1.28) and RI strain 21 (LAPAR = 1.02) were the only strains to show an increased activity in the last part of the active period. ANOVA showed the increased activity was significantly greater during the last 2 h compared to the preceding 10 h of the active phase only for the RI strain 40. Average LAPAR across the eight strains did not significantly correlate with the VMB iron content (r = -0.27, p < 0.10) but did correlate with changes in VMB iron with iron deficiency (r = 0.71, p < 0.05) and diurnal change in VMB iron (r = 0.65, p < 0.05). CONCLUSION: The female RI strain 40 mice exhibited a distinct end-of-active-period behavior analogous to circadian-dependent clinical phenotype of RLS.

Diurnal cycle influences peripheral and brain iron levels in mice.

Iron movement between organ pools involves a dynamic equilibrium of iron efflux and uptake, and homeostatic mechanisms are likely involved in providing iron to cells and organs when required. Daily iron levels in the plasma pool fluctuate with the diurnal cycle, but clear explanations regarding the objectives and regulation of the flux are lacking. The association between diurnal cycle and iron flux is relevant in the disease of restless legs syndrome (RLS), where individuals display diurnal deficits in motor control, have impaired brain iron metabolism, and perhaps altered iron uptake from the plasma pool. The goal of the present study was to examine diurnal variations in peripheral and regional brain iron to evaluate iron flux between organs in iron-sufficient and iron-deficient mice. In mice fed control diet, liver iron was elevated 30-40%, and plasma iron was reduced 20-30% in the active dark period compared with the inactive light phase. Dietary iron deficiency eliminated this variation in liver iron in male and female mice and in plasma iron in male mice. Reductions in ventral midbrain and nucleus accumbens iron and ferritin were apparent in iron-deficient mice during both diurnal phases, but only during the light phase was an approximately 25% reduction in whole brain iron observed, suggesting different brain iron requirements between phases. These data demonstrate that iron flux between organs is sensitive to diurnal regulatory biology. Importantly, variations in brain iron may have temporal implications regarding neural functioning and may contribute to the diurnal cycle-dependent symptoms of RLS.

Dopamine D2 receptor expression is altered by changes in cellular iron levels in PC12 cells and rat brain tissue.

Iron deficiency anemia in early life alters the development and functioning of the dopamine neurotransmitter system, but data regarding the specific effects of brain iron loss on dopamine D(2) receptor regulation are lacking. Cell culture and animal models were employed in this study to determine whether D(2) receptor expression is altered when cellular iron levels are depleted. Endogenous D(2) receptor-expressing PC12 cells exposed to increasing concentrations of the iron chelator desferrioxamine (25-100 micromol/L) exhibited dose-dependent decreases in total D(2) receptor protein concentrations (20-65%), but there were minimal effects on D(2) receptor mRNA levels. When iron-deficient cells were repleted with ferric ammonium citrate for 24 h, D(2) receptor protein densities were similar to control. Dietary iron deficiency for 6 wk in weanling rats also reduced regional iron concentrations by nearly 50% in the ventral midbrain and caudate but did not affect D(2) receptor mRNA levels in the ventral midbrain. Iron deficiency significantly reduced membrane D(2) receptor protein levels by >70% in caudate, whereas cytosolic concentrations showed only 25% losses. D(2) receptor protein densities and regional iron concentrations were restored within 2 wk of dietary iron repletion. These results support the concept that D(2) receptor gene expression is not significantly changed by iron deficiency, whereas dopamine receptor trafficking is affected and is likely related to known dopamine system alterations in iron deficiency.

Acoustic startle response is disrupted in iron-deficient rats.

Diurnal effects on motor control are evident in the human disease of Restless Leg Syndrome (RLS), which is purported to be linked to brain iron deficiency as well as alterations in dopaminergic systems. Thus, we explored the relationship between daily rhythms, the onset of motor dysregulation and brain iron deficiency in an animal model of iron deficiency. Male and female weanling Sprague-Dawley rats consuming control (CN) or iron-deficient (ID) diets were examined weekly for acoustic startle response (ASR) and prepulse inhibition (PPI) for a 5-week period. Iron deficiency reduced the magnitude, but not timing, of the ASR at specific time points. ASR was elevated 60% at the onset of the dark cycle relative to the median of the light cycle in male CN and ID rats. The respective elevation was 400% and 150% in female CN and ID rats during the first 2 weeks of testing. The diurnal cycle of ASR response was attenuated by 3 weeks of testing in both dietary treatment groups. PPI was not affected by iron deficiency, sex, diurnal cycle or the interaction between these factors. These results thus demonstrate that iron deficiency moderately alters ASR signaling although the inhibitory pathways of ASR do not appear to be affected.

Effects of Axial Vibration on Needle Insertion into the Tail Veins of Rats and Subsequent Serial Blood Corticosterone Levels.

Blood collection is commonplace in biomedical research. Obtaining sufficient sample while minimizing animal stress requires significant skill and practice. Repeated needle punctures can cause discomfort and lead to variable release of stress hormones, potentially confounding analysis. We designed a handheld device to reduce the force necessary for needle insertion by using low-frequency, axial (forward and backward) micromotions (that is, vibration) delivered to the needle during venipuncture. Tests with cadaver rat-tail segments (n = 18) confirmed that peak insertion forces were reduced by 73% on average with needle vibration. A serial blood-sampling study was then conducted by using Sprague-Dawley rats divided into 2 groups based on needle condition used to cause bleeds: vibration on (n = 10) and vibration off (n = 9). On 3 days (1 wk apart), 3 tail-vein blood collections were performed in each subject at 1-h intervals. To evaluate associated stress levels, plasma corticosterone concentration was quantified by radioimmunoassay and behavior (that is, movement and vocalization) was scored by blinded review of blood-sampling videos. After the initial trial, average corticosterone was lower (46% difference), the mean intrasubject variance trended lower (72%), and behavioral indications of stress were rated lower for the vibration-on group compared with the vibration-off group. Adding controlled vibrations to needles during insertion may decrease the stress associated with blood sampling from rats--an important methodologic advance for investigators studying and assessing stress processes and a refinement over current blood sampling techniques.

Preweaning iron deficiency increases non-contingent responding during cocaine self-administration in rats.

Iron deficiency (ID) is the most prevalent single-nutrient deficiency worldwide. There is evidence that ID early in development (preweaning in rat) causes irreversible neurologic, behavioral, and motor development deficits. Many of these effects have been attributed to damage to dopamine systems, including ID-induced changes in transporter and receptor numbers in the striatum and nucleus accumbens. These mesolimbic dopaminergic neurons are, in part, responsible for mediating reward and thus play a key role in addiction. However, there has been relatively little investigation into the behavioral effects of ID on drug addiction. In 2002, we found that rats made ID from weaning (postnatal day 21) and throughout the experiment acquired cocaine self-administration significantly more slowly than controls and failed to increase responding when the dose of the drug was decreased. In the present study, we assessed addiction for self-administered cocaine in rats with a history of preweaning ID only during postnatal days 4 through 21, and iron replete thereafter. The results showed that while ID did not affect the number of cocaine infusions or the overall addiction-like behavior score, ID rats scored higher on a measure of continued responding for drug than did iron replete controls. This increase in responding, however, was less goal-directed as ID rats also responded more quickly to the non-rewarded manipulandum than did control rats. Thus, while ID early in infancy did not significantly increase addiction-like behaviors for cocaine in this small study, the pattern of data suggests a possible underlying learning or performance impairment. Future studies will be needed to elucidate the exact neuro-behavioral deficits that lead to the increase in indiscriminate responding for drug in rats with a history of perinatal ID.

Low brain iron effects and reversibility on striatal dopamine dynamics.

Iron deficiency (ID) in rodents leads to decreased ventral midbrain (VMB) iron concentrations and to changes in the dopamine (DA) system that mimic many of the dopaminergic changes seen in RLS patient where low substantia nigra iron is a known pathology of the disease. The ID-rodent model, therefore, has been used to explore the effects that low VMB iron can have on striatal DA dynamics with the hopes of better understanding the nature of iron-dopamine interaction in Restless Legs Syndrome (RLS). Using a post-weaning, diet-induced, ID condition in rats, the No-Net-Flux microdialysis technique was used to examine the effect of ID on striatal DA dynamics and it reversibility with acute infusion of physiological concentrations of iron into the VMB. This study replicated prior findings by showing that the ID condition is associated with increased extracellular striatal DA, reduced striatal DA uptake, and blunted DA-2-receptor-agonist feedback enhancement of striatal DA uptake. Despite the increase in extracellular striatal DA, intracellular striatal DA, as determined in tissue homogenates, was decrease in the ID rat. The study's key finding was that an infusion of physiological concentrations of iron into the VMB reversed the ID-induced increase in extracellular striatal DA and the ID-induced decrease in intracellular striatal DA but had no effect on the ID-induced changes in DA uptake or on the blunted DA-uptake response to quinpirole. In summary, the ID-rodent model provides highly reproducible changes in striatal DA dynamics that remarkably parallel dopaminergic changes seen in RLS patients. Some but not all of these ID-induced changes in striatal DA dynamics were reversible with physiological increases in VMB iron. The small changes in VMB iron induced by iron infusion likely represent biologically relevant changes in the non-transferrin-bound labile iron pool and may mimic circadian-dependent changes that have been found in VBM extracellular iron.

Systems analysis of genetic variation in MPTP neurotoxicity in mice.

We analyzed genetic variation in severity of neuronal damage using the known dopaminergic neurotoxicant, MPTP, as a prototypical chemical denervation agent. Male mice from ten members of the BXD family of recombinant inbred strains received 12.5 mg/kg MPTP s.c. (vs. saline) and 48 h later brains were taken for multiple related biochemical analyses. Striatal dopamine (DA) and its metabolites, DOPAC and HVA, and serotonin and its metabolite, 5-HIAAA, were analyzed by HPLC. DA turnover was assessed using DOPAC/DA and HVA/DA ratios. Striatal tyrosine hydroxylase (TH), glial fibrilary acidic protein (GFAP), and iron content in ventral midbrain were quantified. All dopamine measures, as well as TH and GFAP, demonstrated wide, genotype-dependent differences in response to MPTP. Serotonin was largely unaffected. Principal components analysis (PC) on difference values, saline minus MPTP, for DA, DOPAC, HVA, and TH, yielded a dominant principal component. The PC trait residuals for each genotype were compared against complementary expression data for striatum of the same strains. Three transcripts representing Mtap2, Lancl 1, and Kansl1l were highly correlated with the PC, as was the difference score, MPTP minus saline for GFAP. This systems approach to the study of environmental neurotoxicants holds promise to define individual genetic differences that contribute to variability in susceptibility to risk factors for diseases such as Parkinson's disease.