The present study was designed to test the hypothesis that chronic very mild prenatal carbon monoxide (CO) exposure (25 parts per million) subverts the normal development of the rat cerebellar cortex. Studies at this chronic low CO exposure over the earliest periods of mammalian development have not been performed to date. Pregnant rats were exposed chronically to CO from gestational day E5 to E20. In the postnatal period, rat pups were grouped as follows: Group A: prenatal exposure to CO only; group B: prenatal exposure to CO then exposed to CO from postnatal day 5 (P5) to P20; group C: postnatal exposure only, from P5 to P20, and group D, controls (air without CO). At P20, immunocytochemical analyses of oxidative stress markers, and structural and functional proteins were assessed in the cerebellar cortex of the four groups. Quantitative real time PCR assays were performed for inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) nitric oxide synthases.
Superoxide dismutase-1 (SOD1), SOD2, and hemeoxygenase-1 (HO-1) immunoreactivity increased in cells of the cerebellar cortex of CO-exposed pups. INOS and nitrotyrosine immunoreactivity also increased in blood vessels and Purkinje cells (PCs) of pups from group-A, B and C. By contrast, nNOS immunoreactivity decreased in PCs from group-B. Endothelial NOS immunoreactivity showed no changes in any CO-exposed group. The mRNA levels for iNOS were significantly up-regulated in the cerebellum of rats from group B; however, mRNA levels for nNOS and eNOS remained relatively unchanged in groups A, B and C. Ferritin-H immunoreactivity increased in group-B. Immunocytochemistry for neurofilaments (structural protein), synapsin-1 (functional protein), and glutamic acid decarboxylase (the enzyme responsible for the synthesis of the inhibitory neurotransmitter GABA), were decreased in groups A and B. Immunoreactivity for two calcium binding proteins, parvalbumin and calbindin, remained unchanged. The immunoreactivity of the astrocytic marker GFAP increased after prenatal exposure.
We conclude that exogenously supplied CO during the prenatal period promotes oxidative stress as indicated by the up-regulation of SOD-1, SOD-2, HO-1, Ferritin-H, and iNOS with increased nitrotyrosine in the rat cerebella suggesting that deleterious and protective mechanisms were activated. These changes correlate with reductions of proteins important to cerebellar function: pre-synaptic terminals proteins (synapsin-1), proteins for the maintenance of neuronal size, shape and axonal quality (neurofilaments) and protein involved in GABAergic neurotransmission (GAD). Increased GFAP immunoreactivity after prenatal CO-exposure suggests a glial mediated response to the constant presence of CO. There were differential responses to prenatal vs. postnatal CO exposure: Prenatal exposure seems to be more damaging; a feature exemplified by the persistence of markers indicating oxidative stress in pups at P20, following prenatal only CO-exposure. The continuation of this cellular environment up to day 20 after CO exposure suggests the condition is chronic. Postnatal exposure without prenatal exposure shows the least impact, whereas prenatal followed by postnatal exposure exhibits the most pronounced outcome among the groups.
Higher aluminum (Al) content in infant formula and its effects on neonatal brain development are a cause for concern. This study aimed to evaluate the distribution and concentration of Al in neonatal rat brain following Al treatment, and oxidative stress in brain tissues induced by Al overload.
Postnatal day 3 (PND 3) rat pups (n =46) received intraperitoneal injection of aluminum chloride (AlCl3), at dosages of 0, 7, and 35 mg/kg body wt (control, low Al (LA), and high Al (HA), respectively), over 14 d.
Aluminum concentrations were significantly higher in the hippocampus (751.0 ± 225.8 ng/g v.s. 294.9 ± 180.8 ng/g; p < 0.05), diencephalon (79.6 ± 20.7 ng/g v.s. 20.4 ± 9.6 ng/g; p < 0.05), and cerebellum (144.8 ± 36.2 ng/g v.s. 83.1 ± 15.2 ng/g; p < 0.05) in the HA group compared to the control. The hippocampus, diencephalon, cerebellum, and brain stem of HA animals displayed significantly higher levels of lipid peroxidative products (TBARS) than the same regions in the controls. However, the average superoxide dismutase (SOD) activities in the cerebral cortex, hippocampus, cerebellum, and brain stem were lower in the HA group compared to the control. The HA animals demonstrated increased catalase activity in the diencephalon, and increased glutathione peroxidase (GPx) activity in the cerebral cortex, hippocampus, cerebellum, and brain stem, compared to controls.
Aluminum overload increases oxidative stress (H2O2) in the hippocampus, diencephalon, cerebellum, and brain stem in neonatal rats.
Aluminum; Neonatal rats; Functional brain tissues; Intraperitoneal injection
Prenatal exposure to environmental contaminants, such as Benzo(a)pyrene [B(a)P] has been shown to impair brain development. The overarching hypothesis of our work is that glutamate receptor subunit expression is crucial for cortical evoked responses and that prenatal B(a)P exposure modulates the temporal developmental expression of glutamatergic receptor subunits in the somatosensory cortex. To characterize prenatal B(a)P exposure on the development of cortical function, pregnant Long Evans rats were exposed to low-level B(a)P (300μg/kg BW) by oral gavage on gestational days 14 to 17. At this exposure dose, there was no significant effect of B(a)P on 1) the number of pups born per litter, 2) the pre-weaning growth curves and 3) initial and final brain to body weight ratios. Control and B(a)P-exposed offspring were profiled for B(a)P metabolites in plasma and whole brain during the pre-weaning period. No detectable levels of metabolites were found in the control offspring. However, a time-dependent decrease in total metabolite concentration was observed in B(a)P-exposed offspring. On PND100-120, cerebrocortical mRNA expression was determined for the glutamatergic NMDA receptor subunit (NR2B) in control and B(a)P-exposed offspring. Neural activity was also recorded from neurons in primary somatic sensory (barrel) cortex. Semiquantitative PCR from B(a)P-exposed offspring revealed a significant 50% reduction in NR2B mRNA expression in B(a)P-exposed offspring relative to controls. Recordings from B(a)P-exposed offspring revealed that N-methyl-D-aspartate (NMDA) receptor -dependent neuronal activity in barrel cortex evoked by whisker stimulation was also significantly reduced (70%) as compared to controls. Analysis showed that the greatest deficit in cortical neuronal responses occurred in the shorter latency epochs from 5-20ms post-stimulus. The results suggest that in utero exposure to benzo(a)pyrene results in diminished mRNA expression of the NMDA NR2B receptor subunit to result in late life deficits in cortical neuronal activity in the offspring. The findings from this study lead to a strong prediction that in utero exposure to benzo(a)pyrene at a time when synapses are first formed and adjusted in strength by activity in the sensory pathways will produce a strong negative effect on brain function in offspring progeny.
polycyclic aromatic hydrocarbon-(PAH); benzo(a)pyrene-B(a)P; World Trade Center (WTC); small for gestational age (SGA); intrauterine growth restriction (IUGR); Bailey Scales of Infant Development (BSID-II); susceptibility-exposure paradigm; somatosensory cortex-S1 cortex; cortical neuronal activity and behavior; alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA); N-methyl-D-aspartate (NMDA); environmental aryl hydrocarbon receptor agonists-eAhR agonist; developmental neurotoxicity
Inorganic lead produces cerebral dysfunction and clinically definable encephalopathies in man. To date there have been few studies on the biochemical changes in brain following exposure to inorganic lead. Studies correlating toxicity with behavioral and brain neurochemical changes following lead exposure have been hindered because adult laboratory animals are resistant to the central nervous system effects of lead poisoning. Such studies have been impeded by lack of suitable experimental models until Pentschew and Garro showed that brain lesions develop in neonatal rats when a pregnant rat newly delivered of her litter is placed on a 4% lead carbonate containing diet. Lead passes into the developing sucklings via maternal milk. Lead-poisoned new-borns have pronounced retardation of growth and during the fourth week of ilfe develop the severe signs of lead encephalopathy, namely, extensive histological lesions of the cerebellum, brain edema, and paraplegia. There is an approximate 85-fold increase in the lead concentration of both the cerebellum and cerebral cortex relative to controls, but edema and gross vascular changes are confined to the cerebellum. Ingested lead had little effect on RNA, DNA, and protein concentrations of developing rat cerebellum and cerebral cortex. However, there was a reduction of between 10 and 20% in the DNA content of the cerebellum around 3 weeks of age in the lead-exposed sucklings. This suggests a failure of cell multiplication in this part of the brain.
A critical evaluation of this experimental approach indicated that under similar dietary conditions experimental lactating rats eat 30% less food than controls resulting in: (a) sustained loss in body weight of nursing mothers and that (b) offsprings who develop paraplegia and cerebellar damage do so after gaining access to lead containing diet.
We have studied mothers' food consumption and body weight changes and blood, milk, and brain lead content; and newborns' body and brain weight changes, blood and brain lead content, and brain serotonin (5HT), norepinephrine (NE), dopamine (DA), and γ-aminobutyric acid (GABA). We have found that a lactating mother rat eating 5% lead acetate (2.73% Pb) produced milk containing 25 ppm lead. When the mothers' diet is changed at day 16 from 5% PbAc to one containing 25 ppm Pb, and neonates allowed free access to the solid diet, the sucklings still have retarded body growth but do not develop paraplegia or grossly apparent vascular damage of the cerebellum. However, during the fourth week these animals exhibit a less severe form of “encephalopathy” consisting of hyperactivity, tremors, and stereotype behavior. Pair-fed controls coetaneous to experimental groups do not display such activities. There was no change in brain 5HT, GABA, or NE, but a 15–20% decrease in brain DA. Change in DA relative to other monoamines suggests a relationship between CNS dysfunction due to lead and DA metabolism in the brain.
The experimental design as discribed provides a model of CNS dysfunction due to lead exposure without debilitating histopathologies. It is possible that our findings on increased motor activity and changes in brain dopamine may correspond to early responses to lead exposure before recognized overt signs of toxicity.
Nicotine, the major psychoactive ingredient in tobacco interacting with nicotinic acetylcholine receptors (nAChR), is believed to have neuroprotective and neurotoxic effects on the developing brain. Neurotoxicity has been attributed to activation of homomeric α7 nAChRs, neuroprotection to heteromeric α4β2 nAChRs. Thus, developmental nicotine could have opposite effects in different brain regions, depending on nAChR subtype expression. Here, we determined if chronic neonatal nicotine exposure (CNN), during a period of brain growth corresponding to the third human trimester, differentially regulates nAChR expression, cell death, and morphological properties in hippocampus and cerebellum, two structures maturing postnatally. Rat pups were orally treated with 6 mg/kg/day nicotine from postnatal day (P)1 to P7. On P8, expression for α4, α7 and β2 mRNA was determined by in situ hybridization; nAChR binding sites in by receptor autoradiography, dying neurons by TUNEL and Fluoro-Jade staining and morphological properties by analysis of Cresyl-Violet stained sections. In control cerebellum, strong expression of α4, β2 mRNA and heteromeric nAChRs labeled with [125I]-Epibatidine were found in granule cells, and α7 mRNA and homomeric nAChRs labeled with [125I]-αBungarotoxin in the external germinal layer. In control hippocampus, low expression of α4 mRNA and heteromeric nAChRs and high expression of α7 mRNA and homomeric nAChRs were detected. CNN increased heteromeric nAChR binding in hippocampus but not cerebellum and significantly decreased neuronal soma size and increased packing density in hippocampal principal cells but not in cerebellum. CNN did not increase the number of dying cells in any area, but significantly fewer TUNEL-labeled cells were found in CA3 strata oriens and radiatum and cerebellar granule layer. Thus, the hippocampus seems to be more sensitive than the cerebellum to CNN which could result from different nAChR subtype expression and might explain long-lasting altered cognitive functions correlated with gestational nicotine exposure due to changes in hippocampal cell morphology.
apoptosis; development; nicotinic; receptor; neuronal density
Previous reports have recently shown the prototypic neurotoxicant, lead, to induce apoptosis in the brains of developing organisms. In the current study, timed-pregnant rats were exposed to lead acetate (0.2% in the drinking water) 24 hrs following birth at postnatal day 1 (PND 1). Dams and pups were continuously exposed to lead through the drinking water of the dam until PND 20. Postnatal exposure in the pups resulted in altered mRNA levels of the following apoptotic and neurotrophic factors: caspase 2 and 3, bax, bcl-x, and brain-derived neurotrophic factor (BDNF). Ribonuclease protection assays were conducted to measure the factors simultaneously at the following postnatal time points: 9, 12, 15, 20, and 25 days. Our results suggest a brain region- and time-specific response following lead acetate exposure. The region most vulnerable to alterations occurs in the hippocampus with alterations beginning at PND 12 in which caspase 3, bcl-x, and BDNF increase with lead exposure. Significant treatment effects were not observed for both the cortex and cerebellum.
lead acetate; apoptosis; neurotrophic factor; cortex; hippocampus; cerebellum; caspase 2; caspase 3; bax; bcl-x; bdnf
The adolescent brain is particularly vulnerable to the effects of alcohol, with intoxications at this developmental age often producing long-lasting effects. The present study addresses the effects of a single acute ethanol exposure on GAP-43 and BDNF gene expression in neurons in the cerebellum and hippocampus of adolescent rats. Male postnatal day 23 (P23) Sprague-Dawley rats were exposed to ethanol vapors for two hours and after a recovery period of two hours, the cerebellum and hippocampus were harvested and samples were taken for blood alcohol concentration (BAC) determinations. We found that this exposure resulted in a mean BAC of 174 mg/dl, which resembles levels in human adolescents after binge drinking. Analyses of total RNA and protein by qRT-PCR and western blotting, respectively, revealed that this single ethanol exposure significantly decreased the levels of GAP-43 mRNA and protein in the cerebellum but increased the levels of mRNA and protein in the hippocampus. BDNF mRNA and protein levels were also increased in the hippocampus but not in the cerebellum of these animals. In situ hybridizations revealed that GAP-43 and BDNF mRNA levels were primarily increased by alcohol exposure in hippocampal dentate granule cells and CA3 neurons. Overall, the reported alterations in the expression of the plasticity-associated genes GAP-43 and BDNF in juvenile rats are consistent with the known deleterious effects of binge drinking on motor coordination and cognitive function.
GAP-43; BDNF; ethanol; hippocampus; cerebellum; juvenile rats
CYP2D6 levels are higher in many brain regions of human smokers in comparison with nonsmokers. We have shown that CYP2D is expressed in rat brain regions and that enzyme activities correlate with protein and messenger ribonucleic acid (mRNA) levels. The aims of this study were to investigate whether nicotine can induce rat brain CYP2D, to determine the recovery time course of the induction and to investigate the mechanism of induction through measuring mRNA levels over time.
Rats were either treated once with either saline or nicotine (1 mg base/kg, subcutaneous and sacrificed 8 hours after the treatment or treated daily for 7 days and sacrificed 0.5–24 hours after the last injection. The CYP2D protein and mRNA levels were assessed by immunoblotting, immunocytochemistry and slot blotting.
There were no changes in brain CYP2D levels after a single nicotine injection. Following chronic nicotine treatment, levels were maximal at 8 hours and returned to control levels by 12 hours after nicotine treatment in all 3 regions assessed. At 8 hours after nicotine treatment, CYP2D levels were significantly (p < 0.05) higher than levels in saline-treated control animals in the cerebellum (1.4-fold), hippocampus (1.3-fold) and striatum (3.2-fold); they tended to be higher in the frontal cortex, brainstem and thalamus. Induction was specific to brain region and cell, for example, in some striatal neurons and in neurons in the cerebellar granular layer and white matter. At no time was there any increase in brain CYP2D mRNA levels. Hepatic CYP2D levels were unchanged at all times tested.
Chronic nicotine treatment induced CYP2D enzymes in rat brain but not rat liver. The induction was maximal 8 hours after the last injection and did not involve alterations in mRNA, indicating a posttranscriptional mechanism. These findings suggest that, in humans exposed to nicotine, response to centrally acting drugs metabolized by CYP2D, susceptibility to neurotoxins either activated or inactivated by CYP2D and the general homeostasis of endogenous neurochemicals metabolized by CYP2D may be affected, owing to increased CYP2D in the brain.
animals; cytochrome P450 enzyme system; nicotine; brain; central nervous system; metabolism; smoking; Parkinson disease
Human preterm neonates are subjected to repetitive pain during neonatal intensive care. We hypothesized that exposure to repetitive neonatal pain may cause permanent or long-term changes because of the developmental plasticity of the immature brain. Neonatal rat pups were stimulated one, two, or four times each day from P0 to P7 with either needle prick (noxious groups N1, N2, N4) or cotton tip rub (tactile groups T1, T2, T4). In groups N2, N4, T2, T4 stimuli were applied to separate paws at hourly intervals; each paw was stimulated only once a day. Identical rearing occurred from P7 to P22 days. Pain thresholds were measured on P16, P22, and P65 (hot-plate test), and testing for defensive withdrawal, alcohol preference, air-puff startle, and social discrimination tests occurred during adulthood. Adult rats were exposed to a hot plate at 62°C for 20 s, then sacrificed and perfused at 0 and 30 min after exposure. Fos expression in the somatosensory cortex was measured by immunocytochemistry. Weight gain in the N2 group was greater than the T2 group on P16 (p < 0.05) and P22 (p < 0.005); no differences occurred in the other groups. Decreased pain latencies were noted in the N4 group [5.0 ± 1.0 s vs. 6.2 ± 1.4 s on P16 (p < 0.05); 3.9 ± 0.5 s vs. 5.5 ±1.6 s on P22 (p < 0.005)], indicating effects of repetitive neonatal pain on subsequent development of the pain system. As adults, N4 group rats showed an increased preference for alcohol (55 ± 18% vs. 32 ± 21%; p < 0.004); increased latency in exploratory and defensive withdrawal behavior (p < 0.05); and a prolonged chemosensory memory in the social discrimination test (p < 0.05). No significant differences occurred in corticosterone and ACTH levels following air-puff startle or in pain thresholds at P65 between N4 and T4 groups. Fos expression at 30 min after hot-plate exposure was significantly greater in all areas of the somatosensory cortex in the T4 group compared with the N4 group (p < 0.05), whereas no differences occurred just after exposure. These data suggest that repetitive pain in neonatal rat pups may lead to an altered development of the pain system associated with decreased pain thresholds during development. Increased plasticity of the neonatal brain may allow these and other changes in brain development to increase their vulnerability to stress disorders and anxiety-mediated adult behavior. Similar behavioral changes have been observed during the later childhood of expreterm neonates who were exposed to prolonged periods of neonatal intensive care.
Behavioral effects; Repetitive pain; Rat pups
Lead is a male reproductive toxicant. Data suggest that rats dosed with relatively high levels of lead acetate for short periods of time induced changes in the hypothalamic gonadotropin-releasing hormone (GnRH) at the molecular level, but these changes were attenuated with increased concentration of exposure. The current study evaluated whether exposure to low levels of lead acetate over longer periods of time would produce a similar pattern of adaptation to toxicity at the molecular and biologic levels. Adult 100-day-old Sprague-Dawley male rats were dosed with 0, 0.025, 0.05, 0.1, and 0.3% lead acetate in water. Animals were killed after 1, 4, 8, and 16 weeks of treatment. Luteinzing hormone (LH) and GnRH levels were measured in serum, and lead levels were quantified in whole blood. Hypothalamic GnRH mRNA levels were also quantified. We found no significant differences in serum LH and GnRH among the groups of animals treated within each time period. A significant dose-related increase of GnRH mRNA concentrations with lead dosing occurred in animals treated for 1 week. Animals treated for more than 1 week also exhibited a significant increase in GnRH mRNA, but with an attenuation of the increase at the higher concentrations of lead with increased duration of exposure. We conclude that the signals within and between the hypothalamus and pituitary gland appear to be disrupted by long-term, low-dose lead exposure.
Sulfotransferase catalyzed sulfation regulates the biological activities of various neurotransmitters/hormones and detoxifies xenobiotics. Rat sulfotransferase rSULT1A1 catalyzes the sulfation of neurotransmitters and xenobiotic phenolic compounds. rSULT2A1 catalyzes the sulfation of hydroxysteroids and xenobiotic alcoholic compounds. In this work, Western blot and real-time RT-PCR were used to investigate the effect of methamphetamine on rSULT1A1 and rSULT2A1 protein and mRNA expression in rat cerebellum, frontal cortex, hippocampus, and striatum. After 1-day treatment, significant induction of rSULT1A1 was observed only in the cerebellum; rSULT2A1 was induced significantly in the cerebellum, frontal cortex, and hippocampus. After 7-days of exposure, rSULT1A1 was induced in the cerebellum, frontal cortex, and hippocampus, while rSULT2A1 was induced significantly in all four regions. Western blot results agreed with the real-time RT-PCR results, suggesting that the induction occurred at the gene transcriptional level. Results indicate that rSULT1A1 and rSULT2A1 are expressed in rat frontal cortex, cerebellum, striatum, and hippocampus. rSULT1A1 and rSULT2A1are inducible by methamphetamine in rat brain sections in a time dependable manner. rSULT2A1 is more inducible than rSULT1A1 by methamphetamine in rat brain sections. Induction activity of methamphetamine is in the order of cerebellum > frontal cortex, hippocampus > striatum. These results suggest that the physiological functions of rSULT1A1 and rSULT2A1 in different brain regions can be affected by methamphetamine.
sulfotransferase; methamphetamine; gene regulation; rat brain sections; rSULT1A1; rSULT2A1
Chronic exposure to nicotine during the first postnatal week in rats, a developmental period that corresponds to the third trimester of human gestation, results in sexually dimorphic long-term functional defects in the adult hippocampus. One potential cause could be the sex-specific differences in the maturation of GABAA receptor-mediated responses from excitatory to inhibitory, which depends on the expression of the Na2+/K+/Cl−-co-transporter NKCC1 and the K+/Cl− co-transporter KCC2. In the rat hippocampus, this switch occurs during the first and second postnatal week in females and males, respectively, and is regulated by nicotinic receptor activation. Excitatory GABAergic signaling can increase BDNF expression, which might exacerbate sex differences by impacting synaptogenesis. We hypothesized that chronic neonatal nicotine (CNN) exposure differentially regulates the expression of these co-transporters and BDNF in males and females. We use quantitative isotopic in situ hybridization to examine the expression of mRNAs for NKCC1, KCC2, BDNF, and NMDA receptor subunits NR2A and NR2B in the postnatal day (P) 5 and 8 rat hippocampus in both sexes that were either control-treated or with 6 mg/kg/day nicotine in milk formula (CNN) via gastric intubation starting at P1. In line with prolonged GABAergic excitation, we found that at P5 males had significantly higher mRNA expression of NKCC1 and BDNF than females. CNN treatment resulted in a significant increase in KCC2 and BDNF mRNA expression in male but not female hippocampus (p<0.05). Males also had higher expression of NR2A and lower expression of NR2B at P5 compared to females (p<0.05). At P8, there were neither sex nor treatment effects on mRNA expression, indicating the end of a critical period for sensitivity to nicotine. These results suggest that differential maturation of GABAAR-mediated responses result in sex-specific sensitivity to nicotine during early postnatal development, potentially explaining the differential long-term effects of CNN on hippocampal function.
nicotinic; nAChR; GABAergic; NMDA; development; in situ hybridization
Fetal alcohol exposure is a leading cause of preventable birth defects, yet drinking during pregnancy remains prevalent worldwide. Studies suggest that activation of the neuroimmune system plays a role in the effects of alcohol exposure during the rodent equivalent to the third trimester of human pregnancy (i.e., first week of neonatal life), particularly by contributing to neuronal loss. Here, we performed a comprehensive study investigating differences in the neuroimmune response in the cerebellum and hippocampus, which are important targets of third trimester-equivalent alcohol exposure.
To model heavy, binge-like alcohol exposure during this period, we exposed rats to alcohol vapor inhalation during postnatal days (P)3–5 (blood alcohol concentration = 0.5 g/dL). The cerebellar vermis and hippocampus of rat pups were analyzed for signs of glial cell activation and neuronal loss by immunohistochemistry at different developmental stages. Cytokine production was measured by reverse transcriptase polymerase chain reaction during peak blood alcohol concentration and withdrawal periods. Additionally, adolescent offspring were assessed for alterations in gait and spatial memory.
We found that this paradigm causes Purkinje cell degeneration in the cerebellar vermis at P6 and P45; however, no signs of neuronal loss were found in the hippocampus. Significant increases in pro-inflammatory cytokines were observed in both brain regions during alcohol withdrawal periods. Although astrocyte activation occurred in both the hippocampus and cerebellar vermis, microglial activation was observed primarily in the latter.
These findings suggest that heavy, binge-like third trimester-equivalent alcohol exposure has time- and brain region-dependent effects on cytokine levels, morphological activation of microglia and astrocytes, and neuronal survival.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-015-0382-9) contains supplementary material, which is available to authorized users.
Microglia; Cytokines; Astrocytes; Alcohol; Neurodegeneration; Fetal; Cerebellum; Hippocampus; Development
Fluoride and arsenic are two common inorganic contaminants in drinking water that are associated with impairment in child development and retarded intelligence. The present study was conducted to explore the effects on spatial learning, memory, glutamate levels, and group I metabotropic glutamate receptors (mGluRs) expression in the hippocampus and cortex after subchronic exposure to fluoride, arsenic, and a fluoride and arsenic combination in rats. Weaned male Sprague-Dawley rats were assigned to four groups. The control rats drank tap water. Rats in the three exposure groups drank water with sodium fluoride (120 mg/L), sodium arsenite (70 mg/L), and a sodium fluoride (120 mg/L) and sodium arsenite (70 mg/L) combination for 3 months. Spatial learning and memory was measured in Morris water maze. mGluR1 and mGluR5 mRNA and protein expression in the hippocampus and cortex was detected using RT-PCR and Western blot, respectively. Compared with controls, learning and memory ability declined in rats that were exposed to fluoride and arsenic both alone and combined. Combined fluoride and arsenic exposure did not have a more pronounced effect on spatial learning and memory compared with arsenic and fluoride exposure alone. Compared with controls, glutamate levels decreased in the hippocampus and cortex of rats exposed to fluoride and combined fluoride and arsenic, and in cortex of arsenic-exposed rats. mGluR5 mRNA and protein expressions in the hippocampus and mGluR5 protein expression in the cortex decreased in rats exposed to arsenic alone. Interestingly, compared with fluoride and arsenic exposure alone, fluoride and arsenic combination decreased mGluR5 mRNA expression in the cortex and protein expression in the hippocampus, suggesting a synergistic effect of fluoride and arsenic. These data indicate that fluoride and arsenic, either alone or combined, can decrease learning and memory ability in rats. The mechanism may be associated with changes of glutamate level and mGluR5 expression in cortex and hippocampus.
AIM: To investigate the effect and possible mechanisms of antiangiogenesis therapy for HCC in rats.
METHODS: Adult male LEW/SsN rats were divided into 3 groups, 25 animals each. Group A was the control group. Groups B and C were given diethylnitrosamine, 5 mg/kg/d. In addition, group C rats received an intraperitoneal injection of fumagillin, 30 mg/(kg·d). Five animals in each group were killed at 6th, 12th, 18th, 20th and 24th wk to evaluate the development of HCC and metastasis. Weight of the rats, liver tumors, and number of organs involved by HCC were measured at each stage. We compared methionine aminopeptidase-2 (MetAP-2) mRNA, Bcl-2 mRNA, telomerase mRNA, and telomerase activity at 24th wk in the liver tissue of group A rats and tumor tissue of HCC from group B and C rats.
RESULTS: No HCC developed in group A, but tumors were present in group B and C rats by the 18th wk. At wk 20 and 24, the median liver weight in group B was 0.64 g (range: 0.58-0.70 g) and 0.79 g (range: 0.70-0.90 g) (P = 0.04), and that in group C was 0.37 g (range: 0.35-0.42 g) and 0.39 g (range: 0.35-0.47 g) (P = 0.67). The liver weight in group C rats was significantly lower than that in group B rats (P = 0.009). At the same time, the median metastasis score (number of organ systems involved) was 3 (range 2-3) in group B, and 1 (range 1-2) in group C, a significant difference between the groups (P = 0.007, 0.004). The levels of MetAP-2 mRNA were significantly higher in groups B and C than in group A (P = 0.025), and significantly higher in group C than in group B (P = 0.047). The level of Bcl-2 mRNA was significantly higher in group B than in group A (P = 0.024), but lower in group C than in group B, although not significantly (P = 0.072). Telomerase mRNA was significantly higher in group B than in group A (P = 0.025), but significantly lower in group C than in group B (P = 0.016). The same inter-group relationship was also true for telomerase activity (P = 0.025 and 0.046).
CONCLUSION: Fumagillin effectively inhibits both liver tumor growth and metastasis in rats in vivo. A possible mechanism is fumagillin-induced inhibition of MetAP-2, which plays an essential role in endothelial cell proliferation. Inhibition of MetAP-2 also results in inhibition of Bcl-2 and telomerase activity.
Hepatocellular carcinoma; Antiangiogenesis therapy; Fumagillin; MetAP-2
The focus of this study was to characterize the impact of gestational exposure to benzo(a)pyrene, [B(a)P] on modulation of glutamate receptor subunit expression that is critical for the maintenance of synaptic plasticity mechanisms during hippocampal or cortical development in offspring. Previous studies have demonstrated that hippocampal and/or cortical synaptic plasticity (as measured by long-term potentiation and S1-cortex spontaneous/evoked neuronal activity) and learning behavior (as measured by fixed-ratio performance operant testing) is significantly impaired in polycyclic aromatic or halogenated aromatic hydrocarbon-exposed offspring as compared to controls. These previous studies have also revealed that brain to body weight ratios are greater in exposed offspring relative to controls indicative of intrauterine growth retardation which has been shown to manifest as low birth weight in offspring. Recent epidemiological studies have identified an effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 Years of life among inner-city children (Perera et al., 2006). The present study utilizes a well-characterized animal model to test the hypothesis that gestational exposure to B(a)P causes dysregulation of developmental ionotropic glutamate receptor subunit expression, namely the N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR) both critical to the expression of synaptic plasticity mechanisms. To mechanistically ascertain the basis of B(a)P-induced plasticity perturbations, timed pregnant Long-Evans rats were exposed in an oral subacute exposure regimen to 0, 25 and 150µg/kg BW B(a)P on gestation days 14–17. The first sub-hypothesis tested whether gestational exposure to B(a)P would result in significant disposition in offspring. The second sub-hypothesis tested whether gestational exposure to B(a)P would result in downregulation of early developmental expression of NMDA and AMPA receptor subunits in the hippocampus of offspring as well as in primary neuronal cultures. The results of these studies revealed significant: 1) disposition to the hippocampus and cortex, 2) down-regulation of developmental glutamate receptor mRNA and protein subunit expression and 3) voltage-dependent decreases in the amplitude of inward currents at negative potentials in B(a)P-treated cortical neuronal membranes.
These results suggest that plasticity and behavioral deficits produced as a result of gestational B(a)P exposure are at least, in part, a result of down-regulation of early developmental glutamate receptor subunit expression and function at a time when excitatory synapses are being formed for the first time in the developing central nervous system. The results also predict that in B(a)P-exposed offspring with reduced early glutamate receptor subunit expression, a parallel deficit in behaviors that depend on normal hippocampal or cortical functioning will be observed and that these deficits will be present throughout life.
While both epidemiological and experimental animal studies have demonstrated that perinatal exposure to polychlorinated biphenyls (PCBs) negatively impacts cognitive and psychomotor function, there remains considerable uncertainty regarding mechanisms by which PCBs cause these functional deficits. In vitro studies have shown that PCBs can trigger apoptosis in cultured neurons and suggest this effect is mediated in part by increased levels of reactive oxygen species (ROS). However, whether PCBs cause similar effects in vivo in the developing brain has yet to be reported. In this study, rat pups were exposed to the commercial PCB mixture Aroclor 1254 (A1254) at 0.1 or 1.0 mg/kg/d in the maternal diet throughout gestation and lactation. Apoptosis and oxidative damage were quantified in three brain regions within several days after birth and at weaning. Caspase-3 activity was significantly increased in the cortex, hippocampus and cerebellum of newborn but not weanling rats exposed to A1254 at 1.0 mg/kg/d in the maternal diet. The most prominent effect was observed in the cerebellum, and PCB-induced apoptosis in this brain region was confirmed by TUNEL. Western blotting revealed that developmental A1254 exposure also increased levels of 3-nitrotyrosine and 4-hydroxynonenal levels in the cerebellum of new-born rats, indicating increased oxidative damage of proteins and lipids, respectively. These findings provide the first in vivo data in support of the hypothesis that PCB-induced oxidative stress alters spatiotemporal profiles of apoptosis, and suggest that this is an important mechanism contributing to the developmental neurotoxicity of PCBs.
Apoptosis; developmental neurotoxicity; neurodevelopment; oxidative stress; polychlorinated biphenyls
1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.
The cerebellum is involved in the control of motor functions with Purkinje cells serving as the only output from the cerebellum. Purkinje cells are important targets for toxic substances and are vulnerable to prenatal insults. Intrauterine infection (IUI) has been shown to selectively target the developing cerebral white matter through lesioning, necrosis and inflammatory cytokine activation. Developmental and cognitive delays have been associated with animal models of IUI. The aim of this study was to determine if IUI leads to damage to Purkinje cells in the developing cerebellum and if any damage is associated with decreases in calbindin and motor behaviors in surviving pups. Pregnant rats were injected with Escherichia coli (1 × 105 colony-forming units) or sterile saline at gestational day 17. Beginning at postnatal day (PND) 2, the pups were subjected to a series of developmental tests to examine developmental milestones. At PND 16, some pups were sacrificed and their brains extracted and processed for histology or protein studies. Hematoxylin and eosin (HE) staining was done to examine the general morphology of the Purkinje cells and to examine Purkinje cell density, area and volume. Calbindin expression was examined in the cerebellum via immunohistochemistry and Western blot techniques. The remaining rat pups were used to examine motor coordination and balance on a rotating rotarod at the prepubertal and adult ages. Prenatal E. coli injection did not significantly change birth weight or delivery time, but did delay surface righting and negative geotaxis in pups. Pups in the E. coli group also had a decrease in the number of Purkinje cells, as well as a decrease in Purkinje cell density and volume. HE staining demonstrated a change in Purkinje cell morphology. Calbindin expression was decreased in rats from the E. coli group as well. Locomotor tests indicated that while there were no significant changes in gross motor activity, motor coordination and balance was impaired in both prepubertal and adult rats from the E. coli group. In this model of IUI, we observed changes in Purkinje cell development which were associated with alterations in cerebellum-dependent motor behaviors. The decreases in calbindin and Purkinje cells were associated with developmental delays. These data further support the importance of IUI in brain development.
Calbindin; Brain development, fetal; Motor behavior; Purkinje neurons; Escherichia coli
Although developmental lead exposure is known to have detrimental effects on a variety of cognitive functions that depend on the integrity of the hippocampus and frontal cortex, little is known about how low levels of lead exposure affect expression of key families of genes in these structures. The present study examined the effects of exposure to environmentally-relevant levels of lead during the sensitive early post-weaning period in the rat on the expression profiles of a select number of neurobiologically relevant genes (i.e., genes for neurotrophic factors, NMDA receptors, metabotropic glutamate receptors, synaptic function/plasticity, cell signaling, and transcription/regulation) in the rat hippocampus and frontal cortex. Exposure to lead (180 and 375 ppm lead acetate in food for 30 days) significantly increased blood lead levels (5.8 to 10.3 μg/dl) and significantly affected expression of many of the genes examined. In many instances, lead exposure had different effects on the same gene depending on the brain region in which the expression of that gene was examined. Gene expression in the frontal cortex was often more sensitive to modification than gene expression in the hippocampus. These results suggest that even past infancy, exposures to low levels of lead can have significant effects on gene expression in frontal cortex and the hippocampus with the potential to exert long-term effects on behavior and cognition.
Lead; gene expression; mRNA; hippocampus; frontal cortex
Phencyclidine (PCP), used to mimic certain aspects of schizophrenia, induces sexually dimorphic, cognitive deficits in rats. In this study, the effects of sub-chronic PCP on expression of brain-derived neurotrophic factor (BDNF), a neurotrophic factor implicated in the pathogenesis of schizophrenia, have been evaluated in male and female rats. Male and female hooded-Lister rats received vehicle or PCP (n = 8 per group; 2 mg/kg i.p. twice daily for 7 days) and were tested in the attentional set shifting task prior to being sacrificed (6 weeks post-treatment). Levels of BDNF mRNA were measured in specific brain regions using in situ hybridisation. Male rats were less sensitive to PCP-induced deficits in the extra-dimensional shift stage of the attentional set shifting task compared to female rats. Quantitative analysis of brain regions demonstrated reduced BDNF levels in the medial prefrontal cortex (p < 0.05), motor cortex (p < 0.01), orbital cortex (p < 0.01), olfactory bulb (p < 0.05), retrosplenial cortex (p < 0.001), frontal cortex (p < 0.01), parietal cortex (p < 0.01), CA1 (p < 0.05) and polymorphic layer of dentate gyrus (p < 0.05) of the hippocampus and the central (p < 0.01), lateral (p < 0.05) and basolateral (p < 0.05) regions of the amygdaloid nucleus in female PCP-treated rats compared with controls. In contrast, BDNF was significantly reduced only in the orbital cortex and central amygdaloid region of male rats (p < 0.05). Results suggest that blockade of NMDA receptors by sub-chronic PCP administration has a long-lasting down-regulatory effect on BDNF mRNA expression in the female rat brain which may underlie some of the behavioural deficits observed post PCP administration.
PCP; BDNF; Gender; Set-shifting; Rats
Perinatal exposure to polychlorinated biphenyls (PCBs) is associated with decreased IQ scores, impaired learning and memory, psychomotor difficulties, and attentional deficits in children. It is postulated that these neuropsychological deficits reflect altered patterns of neuronal connectivity. To test this hypothesis, we examined the effects of developmental PCB exposure on dendritic growth.
Rat dams were gavaged from gestational day 6 through postnatal day (PND) 21 with vehicle (corn oil) or the commercial PCB mixture Aroclor 1254 (6 mg/kg/day). Dendritic growth and molecular markers were examined in pups during development.
Golgi analyses of CA1 hippocampal pyramidal neurons and cerebellar Purkinge cells indicated that developmental exposure to PCBs caused a pronounced age-related increase in dendritic growth. Thus, even though dendritic lengths were significantly attenuated in PCB-treated animals at PND22, the rate of growth was accelerated at later ages such that by PND60, dendritic growth was comparable to or even exceeded that observed in vehicle controls. Quantitative reverse transcriptase polymerase chain reaction analyses demonstrated that from PND4 through PND21, PCBs generally increased expression of both spinophilin and RC3/neurogranin mRNA in the hippocampus, cerebellum, and cortex with the most significant increases observed in the cortex.
This study demonstrates that developmental PCB exposure alters the ontogenetic profile of dendritogenesis in critical brain regions, supporting the hypothesis that disruption of neuronal connectivity contributes to neuropsychological deficits seen in exposed children.
dendritogenesis; developmental neurotoxicology; learning and memory; molecular markers; polychlorinated biphenyls
Previous study has demonstrated that dietary taurine supplement protected rats from impairments of synaptic plasticity induced by postnatal lead exposure. However, little is known about the role of taurine in the presence of prenatal and perinatal lead exposure. We investigated the possible effect of taurine supplement on prenatal and perinatal lead-induced synaptic plasticity deficit and determined developmental periods critical for the effect of taurine.
In the present study, taurine was administrated to prenatal and perinatal lead-exposed rats in different developmental periods: from prenatal to weaning (Lead+PW-Tau), from weaning to life (Lead+WL-Tau), and from prenatal to life (Lead+PL-Tau). We examined the input-output (I/O) function, paired-pulse facilitation (PPF) and the long-term potentiation (LTP) of field excitatory postsynaptic potential (fEPSP) in the hippocampal CA1 area of rats on postnatal days 18–25 (P18–25) or days 60–75 (P60–75). We found that (1) on P18–25, taurine had no evident effect on I/O functions and PPF ratios of lead-exposed rats but caused a 12.0% increase in the LTP amplitudes of these animals; (2) on P60–75, taurine significantly elevated lead depressed I/O functions and PPF ratios in Lead+PW-Tau and Lead+PL-Tau rats, but failed in Lead+WL-Tau rats. The amplitudes of LTP of lead-exposed rats were all significantly increased by additional taurine supplement in any developmental period compared with untreated rats. Thus, taurine appeared to have the most effect during the prenatal and lactation periods and its effects on younger rats would not be manifest until the adult life; and (3) the level of lead deposition in hippocampus was evidently reduced by additional treatment of taurine in lead-exposed rats, compared with untreated rats.
Taurine supplement can protect the adult rats from synaptic plasticity deficits following prenatal and perinatal lead exposure, and the protective effects are critical for the prenatal and lactation periods of lead-exposed rats.
Catecholamine is a group of neurotransmitters that is believed to be responsible for the normal function of animal brain. Physiological and behavioral changes of human body have been reported due to the damage of the brain function following lead exposure. Due to the assumption of lead disposal in brain tissue with two year for its half-life, which results in alteration of brain function, we investigated the ability of lead to change the brain catecholamines during short and long-term studies. Rats were exposed daily with varying amounts of lead and catecholamine contents of cerebellum, mid-brain and brain cortex were determined. Acute peritoneal administration of single dose of lead as lead acetate (260 μmol/Kg) after 2 h reduced (p < 0.05) the catecholamine levels of cerebellum, mid-brain and cortex part by 34.9%, 35.44% and 23.8%, respectively. The extension of experiment time to 5 h, significant (p < 0.05) reductions in catecholamine levels of mentioned regions of brain by 32.35%, 12.35% and 19.3% were seen respectively. Daily intraperitoneal administration of 10 μmol/Kg lead for 30 and 60 days reduced catecholamines levels of cerebellum (22.22% and 30.44%), midbrain (12.48% and 26.27%) and brain cortex (11.58% and 26.7%) respectively. It might be concluded that brain dysfunction in lead intoxicated rat occurred through the reduction in the catecholamine levels of different parts of brain. Lead might be therefore considered as a probable factor in causing neurological disease in lead exposed man.
Lead; Catecholamine; Brain region; Pb+2
Infantile spasms are seizures manifesting in infantile epileptic encephalopathies that are associated with poor epilepsy and cognitive outcomes. The current therapies are not always effective or are associated with serious side effects. Early cessation of spasms has been proposed to improve long-term outcomes. To identify new therapies for infantile spasms with rapid suppression of spasms, we are using the multiple-hit rat model of infantile spasms, which is a model of refractory infantile spasms. Here, we are testing the efficacy and tolerability of a single dose of the galanin receptor 1 preferring analog, NAX 5055, in the multiple-hit model of spasms. To induce the model, postnatal day 3 (PN3) male Sprague-Dawley rats underwent right intracerebral infusions of doxorubicin and lipopolysaccharide; p-chlorophenylalanine was then injected intraperitoneally (i.p.) at PN5. After the onset of spasms at PN4, 11–14 rats/group were injected i.p. with either NAX 5055 (0.5, 1, 2, or 4 mg/kg) or vehicle. Video monitoring for spasms included a 1hour pre-injection period, followed by 5 hours of recording post-injection, and two 2 hour sessions on PN5. The study was conducted in a randomized, blinded manner. Neurodevelopmental reflexes were assessed daily as well as at 2 hours after injection. Respiratory function, heart rate, pulse distension, oximetry and blood glucose were measured 4 hours after injection. The relative expression of GalR1 and GalR2 mRNA over β-actin in the cerebral cortex and hippocampus was determined with real time reverse transcription polymerase chain reaction. There was no acute effect of NAX 5055 on spasm frequency after the single dose of NAX 5055 (n=11–13 rats/group, following exclusions). Neurodevelopmental reflexes, vital signs, blood glucose measured 4 hours post-injection, and survival were not affected. A reduction in pulse and breath distention of unclear clinical significance was observed with the 7mg/kg NAX 5055 dose. GalR1 mRNA was present in the cerebral cortex and hippocampus of PN4 and adult rats. The hippocampal –but not the cortical- GalR1 mRNA expression was significantly lower in PN4 pups than in adults. GalR1 mRNA was also at least 20 times less abundant in the PN4 cortex than GalR2 mRNA. In conclusion, a single dose of NAX 5055 has no acute efficacy on spasms or toxicity in the multiple hit rat model of medically refractory infantile spasms. Our findings cannot exclude the possibility that repetitive NAX 5055 administration may show efficacy on spasms. The higher expression of GalR2 in the PN4 cortex suggests that GalR2-preferring analogs may be of interest to test for efficacy on spasms.
Antiepileptic; Galanin receptor; glucose; antibody; neurodevelopmental reflexes; cerebral cortex