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Mammals have a marked period of rapid brain growth and development (BGS), which is postnatal in mice and rats, spanning the first 3-4 weeks of life and reaching its peak around postnatal day 10. CaMKII, GAP-43 and BDNF play important roles during the BGS in mammals. One class of flame retardants, polybrominated diphenyl ethers (PBDEs), are present and increasing in the environment and in human milk, which is also true for the only congener still in use, decabrominated diphenyl ether (PBDE 209). In the present study, the brains from 1, 3, 7, 10, 14 and 28 days old mice, were analysed for CaMKII and GAP-43. The level of CaMKII increases continuously during the neonatal period, while GAP-43 has a bell-shaped ontogeny curve, which peaks around postnatal day 10, in mouse brain. Furthermore, the effects of PBDE 209 on the developmental expression of CaMKII, GAP-43 and BDNF were examined in mice. Neonatal NMRI-male mice were orally exposed on days 3-20.1mgPBDE 209/kg body weight. The animals were euthanized 7 days after exposure to PBDE 209 and levels of CaMKII, GAP-43 and BDNF were analysed in different brain regions. The protein analysis showed that CaMKII increased significantly in hippocampus, but not in cortex, in animals 7 days after exposure to PBDE 209. GAP-43 showed a significant increase in hippocampus and a significant decrease in cortex of animals 7 days after exposure to PBDE 209. BDNF decreased significantly in hippocampus, but not in cortex, in mice 7 days after exposure to PBDE 209. This shows that PBDE 209 affects important proteins involved in normal maturation of the brain and further strengthen our findings concerning PBDE 209 as a developmental neurotoxicological agent.

It is widely recognized that prolonged increases in reactive oxygen species (ROS) and intracellular free calcium ([Ca2+]i) are part of a signaling pathway leading to cell death. ROS production resulting in oxidative stress and disruption of calcium homeostasis leading to increases in [Ca2+]i have been described as early events following exposure to a number of neurotoxicants. In order to determine the intrinsic sensitivity of developing neurons to toxicant-induced oxidative stress and disruption of calcium homeostasis, we exposed immature neurons to iron (Fe2+) or methylmercury (MeHg). Primary cultures of cortical cells (prepared from 1 day old rats) or cerebellar granule cells (prepared from 7 day old rats) were exposed to the toxicants on day in vitro (DIV) 1 (immature response to receptor agonists) or DIV 7 (mature response to receptor agonists). ROS was measured using the fluorescent probe 2',7'-dichlorodihydrofluorescin. In both cerebellar granule cells and cortical cells, Fe2+ or MeHg exposure (0.1-30 microM) produced time- and concentration-dependent increases in ROS. In general, the increase in ROS induced by both metals was greater in immature cells compared to mature cells, except for cerebellar granule cells in which the effects of Fe2+ were similar at DIV1 and 7. Changes in intracellular cation concentrations (including Ca2+) were measured using the fluorescent probe fluo-3. MeHg exposure produced a time- and concentration-dependent increase in fluo-3 fluorescence in both cerebellar granule cells and cortical cells. In cortical cultures, the fluorescence increase after MeHg exposure was greater in immature cells. In contrast, mature and immature cells were equally sensitive to the effects of MeHg in cerebellar granule cell cultures. These results suggest that there may be inherent differences in the sensitivity of neurons to toxicant-induced increases in ROS and [Ca2+] depending upon age and cell type.

Recent evidence indicates that the neurotoxic metal aluminum interferes with the phosphoinositide second messenger system in adult rats both in vitro and in vivo. We have examined the age-related effects of aluminum chloride (AlCl3) on receptor-stimulated inositol phosphate (IP) accumulation in brain slices from neonatal and adult rats in vitro. Carbachol-stimulated (1 mM) IP accumulation was greatest in frontal cortex slices from 7 day old rats, decreased in 14 day old and 21 day old rats, and was lowest in adults (120 days old). AlCl3 (500 microM) inhibited both basal and carbachol-stimulated IP accumulation in neonatal and adult rats. The effects of AlCl3 were concentration-related and produced significant decreases (15-25%) in IP accumulation at 500 and 1000 microM. The concentration-response curve for AlCl3 was similar in 7 day old and adult rats. AlCl3 reduced carbachol-, norepinephrine- and quisqualate-stimulated IP accumulation in both 7 day old and adult rats. The effects of 500 microM AlCl3 were examined on carbachol-stimulated IP accumulation in slices prepared from frontal cortex, hippocampus, striatum, and cerebellum. Although IP accumulation was greater in slices from the 7 day old rats compared to adults in each tissue, AlCl3 (500 microM) decreased IP accumulation by approximately 20% in all regions at both ages. Aluminum produced concentration-dependent inhibition of phospholipase C in cortical homogenates which was similar in 7 day old and adult rats. These results show that in vitro exposure to aluminum decreases IP accumulation through a mechanism which is not age-dependent.

Direct infusion of colchicine into the dentate gyrus of the hippocampus kills granule cells and elicits behavioral, neurochemical and neuroanatomical changes. Colchicine-treated rats are less sensitive to the behavioral effects of cholinergic muscarinic receptor antagonists and more sensitive to cholinergic agonists. These behavioral changes are associated with time- and dose-dependent alterations in the cholinergic signal transduction mechanism. Carbachol-stimulated turnover of phosphoinositides is increased in the hippocampus of colchicine-treated rats; similar changes are not observed in the cortex or striatum of colchicine-treated animals. Intradentate colchicine produces a significant increase in choline- acetyltransferase activity and staining for acetylcholinesterase activity in the hippocampus, suggesting reactive synaptogenesis of cholinergic fibers. Other studies have shown that the integrity of the septohippocampal pathway is necessary for these colchicine-induced compensatory changes to occur. It is suggested that the mechanism for these neurochemical changes in colchicine-treated animals may be occurring via alterations in negative feedback control of receptor-G-protein-mediated phosphoinositide hydrolysis.

On postnatal day 21 (PND21), Long-Evans rat pups received a single subcutaneous injection of either 0 (corn oil vehicle), 90, 120, or 240 mg/kg chlorpyrifos and were then tested for T-maze delayed alternation on PND23 or 26. Acetylcholinesterase (AChE) activity and muscaranic receptor density [i.e., quinuclidinyl benzilate (QNB) binding] were determined in hippocampus and cortex of brains taken from pups 15 hours after the end of behavioral testing (i.e., the morning of PND24, and 27). Pups exposed to the 240 mg/kg dose of chlorpyrifos showed signs of overt toxicity that precluded behavioral testing. Exposure to the 120 mg/kg dose produced a selective memory impairment (ie., a deficit in delayed alternation but not position discrimination) relative to the 90 mg/kg and vehicle groups. This impairment was transient, however, as it appeared on PND23 and was absent by PND26. PND21 exposure to chlorpyrifos produced dose-related inhibition and recovery of brain AChE over the PND24-27 age range. A similar pattern was observed in hippocampus. Binding of [3H]QNB was reduced in frontal cortex on PND27 only at the 240 mg/kg dose. No significant effects were observed in the hippocampus. These results suggest that the neurochemical effects of acute chlorpyrifos administration are more transient, and the behavioral effects are smaller and shorter-lived than what has been reported in adult rats.

Neurotoxicants have found increasing use as tools to study the structure and function of the central nervous system. One class of compounds which block mitosis and disrupt axoplasmic transport includes colchicine, a chemical which is highly toxic to certain neuronal populations. Colchicine administered directly into the hippocampus of rats results in the preferential destruction of dentate gyrus granule cells without affecting the surrounding pyramidal cells. Injection of colchicine into other brain areas also destroys neurons but with less selectivity than is observed in the hippocampus. The neurotoxicity of colchicine appears to be related to the ability to bind to tubulin, although its exact mechanism remains to be elucidated.

Rats were given bilateral injections of colchicine into the nucleus basalis (NBM; 1.0 or 2.0 micrograms/site), hippocampus (HPC; 1.25 or 2.5 micrograms/site), or both areas (COM; 1.0 NBM, 1.25 HPC) and examined for changes in locomotor activity, passive avoidance behavior, and spatial navigation in a water maze task. Colchicine injected into the HPC caused a dose-related increase in locomotor activity 7 days after treatment which declined with repeated testing. Motor activity in NBM-lesioned rats was not significantly different from control. Rats with the COM lesion were more active than controls 7 days after treatment and remained hyperactive over the 3 week testing period. Retention of a step-through passive avoidance task was examined 18 days after surgery. HPC lesions had no apparent effect on passive avoidance behavior. NBM lesions causes a dose-dependent decrease in step-through latencies, while latencies in the combined group were comparable to the low dose NBM group. In the spatial navigation task, HPC and COM lesions impaired acquisition, with little indication of learning in the combined group. NBM lesions had no effect in the water maze. These data suggest that combined lesions of the NBM and HPC cause lasting behavioral impairments and may be useful as a model for neurodegenerative disorders such as Alzheimer's disease.

Rats were given bilateral injections of colchicine into the dorsal and ventral hippocampus to study the role of the dentate gyrus granule cells in the acquisition and performance of a spatial, working memory task in the radial arm maze. Three weeks after intradentate injections, rats were trained in a task in which all eight arms were baited prior to each daily trail. For up to 20 days of training, colchicine-treated rats were significantly impaired in the performance of the task. In another study, rats received 20 days of training and then were given intradentate colchicine. Three weeks later, the performance of the colchicine-treated rats was impaired for up to 20 days of testing. A third experiment tested the ability of colchicine-treated rats to learn a task in which the same four arms of the maze were baited, while the remaining arms were never baited. Colchicine-treated rats were significantly impaired in their ability to perform this version of the task. Histological verification indicated that colchicine resulted in a relatively select loss of granule cells, while sparing pyramidal cells in the hippocampus. These data suggest that the hippocampus plays an integral role in the performance of the place tasks used in these experiments.

Evidence is accumulating which suggests that brain damage associated with certain neurodegenerative conditions may be at least partially produced by the overactivation of N-methyl-D-aspartate receptors (N-MDA). To systematically examine the overactivation of N-MDA receptors, N-MDA was administered directly to the frontal cortex and to the hippocampus in rats. It was found that cortical application (1, 2, or 4 mg) had no effect on motor activity 1, 2, or 3 wk after surgery. Four mg N-MDA failed to affect acquisition of a water maze task despite large decreases in cortical width at the site of application. In addition, no alterations in striatal, hippocampal, or cortical cholineacetyltransferase (CHAT) activity were detected after cortical application. Intrahippocampal N-MDA (0, 2.5, 5.0, 10.0, 20.0 micrograms/site) increased motor activity in a dose dependent manner 1, 2, and 3 wk post-surgery. Furthermore, 10 micrograms/site significantly impaired water maze acquisition. Intrahippocampal N-MDA also increased hippocampal CHAT activity and resulted in a loss of pyramidal and dentate granule cells. These results suggest that N-MDA may serve as a useful tool in studying the effects of glutaminergic hyperfunction and its role in neurodegenerative disorders which involve the overactivation of N-MDA receptors.