Perinatal manganese exposure and hydroxyl radical formation in rat brain.

The present study was designed to investigate the role of pre- and postnatal manganese (Mn) exposure on hydroxyl radical (HO(•)) formation in the brains of dopamine (DA) partially denervated rats (Parkinsonian rats). Wistar rats were given tap water containing 10,000 ppm manganese chloride during the duration of pregnancy and until the time of weaning. Control rat dams consumed tap water without added Mn. Three days after birth, rats of both groups were treated with 6-hydroxydopamine at one of three doses (15, 30, or 67 µg, intraventricular on each side), or saline vehicle. We found that Mn content in the brain, kidney, liver, and bone was significantly elevated in dams exposed to Mn during pregnancy. In neonates, the major organs that accumulated Mn were the femoral bone and liver. However, Mn was not elevated in tissues in adulthood. To determine the possible effect on generation of the reactive species, HO(•) in Mn-induced neurotoxicity, we analyzed the contents of 2.3- and 2.5-dihydroxybenzoic acid (spin trap products of salicylate; HO(•) being an index of in vivo HO(•) generation), as well as antioxidant enzyme activities of superoxide dismutase (SOD) isoenzymes and glutathione S-transferase (GST). 6-OHDA-depletion of DA produced enhanced HO(•) formation in the brain tissue of newborn and adulthood rats that had been exposed to Mn, and the latter effect did not depend on the extent of DA denervation. Additionally, the extraneuronal, microdialysate, content of HO(•) in neostriatum was likewise elevated in 6-OHDA-lesioned rats. Interestingly, there was no difference in extraneuronal HO(•) formation in the neostriatum of Mn-exposed versus control rats. In summary, findings in this study indicate that Mn crosses the placenta but in contrast to other heavy metals, Mn is not deposited long term in tissues. Also, damage to the dopaminergic system acts as a "trigger mechanism," initiating a cascade of adverse events leading to a protracted increase in HO(•) generation, and the effects of Mn and 6-OHDA are compounded. Moreover, HO(•) generation parallels the suppression of SOD isoenzymes and GST in the brains of rats lesioned with 6-OHDA and/or intoxicated with Mn-the most prominent impairments being in frontal cortex, striatum, and brain stem. In conclusion, ontogenetic Mn exposure, resulting in reactive oxygen species, HO(•) formation, represents a risk factor for dopaminergic neurotoxicity and development of neurodegenerative disorders.

A small-molecule therapeutic lead for Huntington's disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse.

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a glutamine expansion within huntingtin protein. The exact pathological mechanisms determining disease onset and progression remain unclear. However, aggregates of insoluble mutant huntingtin (mhtt), a hallmark of HD, are readily detected within neurons in HD brain. Although aggregated polyglutamines may not be inherently toxic, they constitute a biomarker for mutant huntingtin useful for developing therapeutics. We previously reported that the small molecule, C2-8, inhibits polyglutamine aggregation in cell culture and brain slices and rescues degeneration of photoreceptors in a Drosophila model of HD. In this study, we assessed the therapeutic potential of C2-8 in the R6/2 mouse model of HD, which has been used to provide proof-of-concept data in considering whether to advance therapies to human HD. We show that, at nontoxic doses, C2-8 penetrates the blood-brain barrier and is present in brain at a high concentration. C2-8-treated mice showed improved motor performance and reduced neuronal atrophy and had smaller huntingtin aggregates. There have been no prior drug-like, non-toxic, brain-penetrable aggregation inhibitors to arise from cell-based high-throughput screens for reducing huntingtin aggregation that is efficacious in preclinical in vivo models. C2-8 provides an essential tool to help elucidate mechanisms of neurodegeneration in HD and a therapeutic lead for further optimization and development.

Cholinergic nicotinic receptor involvement in movement disorders associated with Lewy body diseases. An autoradiography study using [(125)I]alpha-conotoxinMII in the striatum and thalamus.

The presence of alpha6 subunit containing nicotinic acetylcholine receptors on nigrostriatal dopaminergic neurons has been demonstrated in rodents and monkeys. [(125)I]alpha-conotoxinMII is a radioligand that binds to alpha6, and also alpha3 subunits of nicotinic acetylcholine receptors (nAChRs). In the present study, we have compared the distribution of [(125)I]alpha-conotoxinMII binding in post mortem human tissue from four groups of patients: individuals with dementia with Lewy bodies displaying extra-pyramidal features (DLB + EPF), DLB without extra-pyramidal features (DLB - EPF) Parkinson's disease without dementia (PD) and age-matched controls. Reduced binding was observed in the putamen and caudate in PD and both DLB groups. In DLB patients, the decline was greater in DLB + EPF compared to DLB - EPF group. The declines in nicotinic receptor binding in the striatum were in part paralleled by reductions in the striatal dopamine transporter. In the thalamus, [(125)I]alpha-conotoxinMII binding was significantly reduced in the centromedian nucleus in both DLB groups, and also in the parafascicular nucleus in the DLB - EPF group. In DLB + EPF and PD patients, there was decreased binding in the ventral lateral nucleus. This study demonstrates alterations of alpha6 and/or alpha3 nAChRs binding in DLB and PD, which are likely to relate to extra-pyramidal symptoms.

Synthesis and biological evaluation of pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane derivatives as potential therapeutic agents in Parkinson's disease.

In previous studies, the polycyclic cage amine 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane (NGP1-01) and a number of its derivatives showed positive effects in neuroprotection studies with MPTP, in vivo. In view of these findings, we examined these compounds for their effects on [(3)H]dopamine ([(3)H]DA) release and uptake inhibition in murine striatal synaptosomes, as well as for inhibition of baboon liver monoamine oxidase (MAO) B. In order to assess specificity, initial experiments focused on compounds that blocked dopamine uptake without causing appreciable release (<40% at 100 microM) of the transmitter. NGP1-01 blocked the uptake of [(3)H]DA with an IC(50) of 57 microM, while another compound, 8-phenylethyl-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane, blocked uptake at an IC(50) value of 23 microM. These values were comparable to that of another polycyclic cage amine, amantadine (IC(50); 82 micro), that is used in parkinsonian therapy. Structure-activity relationships of this series of compounds support the importance of geometric and steric, rather than electronic effects, in determining biological activity. MAO-B inhibition for this group was weak, with less than 50% inhibition at 300 microM for any of the compounds in the series. The present study suggests that blockage of the dopamine transporter may underlie, at least in part, their neuroprotective effects against MPTP-induced parkinsonism. These compounds may be considered as potential lead compounds for Parkinson's Disease therapy.

Group I metabotropic glutamate receptors in the monkey striatum: subsynaptic association with glutamatergic and dopaminergic afferents.

Group I metabotropic glutamate receptors (mGluRs) are involved in long-term synaptic plasticity and neuroprotection in the striatum, but the specific role(s) of mGluR1 and mGluR5 remain poorly understood. In this study, we used electron-microscopic immunocytochemistry to compare the pattern of subsynaptic and subcellular distribution of mGluR1a and mGluR5 in relation to putative glutamatergic and dopaminergic inputs to the monkey striatum. At the light-microscopic level, both group I mGluRs are expressed in the striatal neuropil. In addition, numerous perikarya of striatal output neurons are immunostained for mGluR5, but much less frequently for mGluR1a. At the electron-microscopic level, immunoreactivity for both receptor subtypes is primarily expressed postsynaptically in dendrites and spines, although presynaptic mGluR1a labeling of glutamatergic thalamostriatal boutons and, less frequently, dopaminergic and corticostriatal terminals is also seen. In contrast to mGluR1a, mGluR5 immunoreactivity is rarely encountered presynaptically. In postsynaptic elements, 40-70% of immunoreactivity for both receptor subtypes is expressed intracellularly, whereas 30-60% is apposed to the plasma membrane. More than 80% of the labeling apposed to the plasma membrane is extrasynaptic. The remaining 20% is located at the edges of putative glutamatergic synapses or in the active zone of symmetric synapses. In mGluR5-, but not mGluR1a-immunostained sections, approximately 70% of dopaminergic symmetric synapses are labeled perisynaptically. These data emphasize the differential pattern of subsynaptic localization of the two group I mGluRs and provide various presynaptic and postsynaptic sites whereby mGluR1 and mGluR5 could mediate different, but complementary, effects on glutamatergic and dopaminergic transmission in the primate striatum.

Effect of prenatal sound stimulation on medio-rostral neostriatum/hyperstriatum ventrale region of chick forebrain: a morphometric and immunohistochemical study.

The higher auditory association area in chick forebrain, i.e. medio-rostral neostriatum/hyperstriatum ventrale region (MNH), is involved in juvenile auditory filial imprinting. Studies show that neuronal size as well as expression of calcium-binding proteins, parvalbumin (PV) and calbindin D28K (CaBP) are regulated by neuronal activation. In the present study, we have determined the effect of extra auditory stimulation, given as a prenatal sound enrichment protocol, on MNH neurons of posthatch day 1 chicks. Patterned species-specific or musical (sitar) sounds were provided in a graded manner from embryonic day 10 through hatching. Thionin and immunohistochemically stained (PV and CaBP) neurons were evaluated by morphometric methods. The thionin-stained MNH neurons of both the auditory stimulated groups showed a significant increase in nuclear area compared to controls. The change in nuclear dimension was greater in the music-stimulated than in the species-specific sounds-stimulated group. These observations indicate a positive influence of prenatal sound stimulation on MNH neurons. The auditory stimulated groups also demonstrated an increase in the proportion of PV- and CaBP-neurons compared to controls, with the species-specific sounds-stimulated group showing a significantly higher percentage of immunostained cells than the music-stimulated group. However, immunostained cells of both the auditory stimulated groups did not show a significant change in size. These cytoplasmic proteins, by acting as intracellular buffers, enable neurons to display high electrical activity without calcium overload. The influx of Ca(2+) ions is essential for long-term potentiation, a phenomenon important for learning and memory. The increase in percentage of the neurons containing calcium-binding proteins may provide a morphological basis for enhancement of auditory imprinting and learning.

Distribution of serotonin 5-HT(2A) receptors in afferents of the rat striatum.

Treatment with conventional antipsychotic drugs (APDs) is accompanied by extrapyramidal side effects (EPS), which are thought to be due to striatal dopamine D(2) receptor blockade. In contrast, treatment with atypical APDs is marked by a low incidence or absence of EPS. The reduced motor side effect liability of atypical APDs has been attributed to a high serotonin 5-HT(2A) receptor affinity coupled with a relatively low D(2) affinity. Despite the high density of 5-HT(2A) binding sites in the striatum, there are few detectable 5-HT(2A) mRNA-expressing neurons in the striatum. This suggests that most striatal 5-HT(2A) receptors are heteroceptors located on afferent axons. A combined retrograde tracer-immunohistochemistry method was used to determine the sites of origin of striatal 5-HT(2A)-like immunoreactive axons. 5-HT(2A)-like immunoreactive neurons in both the cortex and globus pallidus were retrogradely labeled from the striatum; very few nigrostriatal or thalamostriatal neurons expressed 5-HT(2A)-like immunoreactivity. Within the striatum, parvalbumin-containing interneurons displayed 5-HT(2A) immunolabeling; these neurons are the targets of cortical and pallidal projections. Our data indicate that cortico- and pallido-striatal neurons are the major source of 5-HT(2A) receptor binding in the striatum, and suggest that cortico- and pallido-striatal neurons are strategically positioned to reduce the motor side effects that accompany striatal D(2) receptor blockade or are seen in parkinsonism.

Sexual dimorphism in the response to N-methyl-D-aspartate receptor antagonists and morphine on behavior and c-Fos induction in the rat brain.

It has been suggested that there are sex differences in the neural response to drugs of abuse. Previous studies have shown that, upon administration of morphine, the immediate early gene c-Fos is induced in the striatum, nucleus accumbens and cortex of the rat brain. This induction of c-Fos is reduced by administration of the N-methyl-D-aspartate receptor antagonist dizocilpine maleate. However, in studies using immunocytochemistry, we found that the pattern of this expression differed markedly between the sexes. In male rats treated with morphine (10 mg/kg, s.c.) and killed 2 h later, there was an induction of c-Fos in the dorsomedial caudate-putamen, the nucleus accumbens and in the intralaminar nuclei of the thalamus. Administration of dizocilpine maleate (0.2 mg/kg, i.p.; 30 min before morphine) partially blocked the response in the caudate-putamen, but not in the thalamus. In females, morphine induced c-Fos in the caudate-putamen, but with more inter-animal variability than in males. In the midline intralaminar thalamic nuclei, female rats showed less induction than males. In male rats, dizocilpine maleate alone caused negligible induction of c-Fos, whereas in female rats, it caused a large induction in the rhomboid, reuniens and central medial nuclei of the thalamus, and in the cortex. Whereas dizocilpine maleate partially blocked the morphine-induced c-Fos expression in the caudate-putamen of males, it completely blocked this response in females. With dizocilpine maleate alone, there was little or no effect on behavior in male rats, whereas in female rats, it caused head bobbing, thrashing, hyperactivity and uncoordinated movements. These behavioral sex differences were not seen on treatment of rats with the competitive N-methyl-D-aspartate receptor antagonist 2R,4R,5S-2-amino-4,5-(1,2-cyclohexyl)-7-phosphoheptanoic acid (NPC-17742; 10 mg/kg, i.p.) and this drug did not induce c-Fos expression in either sex. In the caudate-putamen, morphine-induced c-Fos expression was significantly reduced by NPC-17742 (30 min before morphine) in males and completely blocked in females. These results suggest that the responses to both morphine and N-methyl-D-aspartate receptor antagonists differ between the sexes and emphasize that glutamate is involved in morphine-induced immediate early gene expression in the brain. These studies thus have important implications for gender differences in drug addiction.

Effects of repeated amphetamine treatment on regional GABAA receptor binding.

The present study examined the effects of repeated exposure to amphetamine on GABAA receptor binding in cortical and subcortical areas. The goal of the study was to determine whether changes in specific binding were related to behavioral sensitization. Animals were exposed to either saline (0.3 ml, s.c.; n=12) or d-amphetamine (2.5 mg/kg, s.c.; n=12) for 6 consecutive days and sacrificed after a 14-day withdrawal period. Differences in GABAA receptor binding in these two groups of animals were assessed using the GABAA receptor antagonist [3H]SR 95531. To verify that the preceding treatment regimen led to the development of behavioral sensitization, a separate set of animals (n=8/group) was exposed to the same regimen and challenged with d-amphetamine (2.5 mg/kg, s.c.) after the 14-day withdrawal period. As expected, preexposure to amphetamine led to the development of amphetamine sensitization. There were no differences in GABAA receptor binding in animals preexposed to saline and amphetamine in the prefrontal cortex, caudate-putamen, hypothalamus, or cerebellum. These findings do not provide support for the idea that changes in GABAA receptor binding in the medial prefrontal cortex or various subcortical areas are related to the development of behavioral sensitization.

Neuroprotective effects of creatine and cyclocreatine in animal models of Huntington's disease.

The gene defect in Huntington's disease (HD) may result in an impairment of energy metabolism. Malonate and 3-nitropropionic acid (3-NP) are inhibitors of succinate dehydrogenase that produce energy depletion and lesions that closely resemble those of HD. Oral supplementation with creatine or cyclocreatine, which are substrates for the enzyme creatine kinase, may increase phosphocreatine (PCr) or phosphocyclocreatine (PCCr) levels and ATP generation and thereby may exert neuroprotective effects. We found that oral supplementation with either creatine or cyclocreatine produced significant protection against malonate lesions, and that creatine but not cyclocreatine supplementation significantly protected against 3-NP neurotoxicity. Creatine and cyclocreatine increased brain concentrations of PCr and PCCr, respectively, and creatine protected against depletions of PCr and ATP produced by 3-NP. Creatine supplementation protected against 3-NP induced increases in striatal lactate concentrations in vivo as assessed by 1H magnetic resonance spectroscopy. Creatine and cyclocreatine protected against malonate-induced increases in the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid, biochemical markers of hydroxyl radical generation. Creatine administration protected against 3-NP-induced increases in 3-nitrotyrosine concentrations, a marker of peroxynitrite-mediated oxidative injury. Oral supplementation with creatine or cyclocreatine results in neuroprotective effects in vivo, which may represent a novel therapeutic strategy for HD and other neurodegenerative diseases.

Decreased TrkA gene expression in cholinergic neurons of the striatum and basal forebrain of patients with Alzheimer's disease.

In addition to cortical pathology, Alzheimer's disease is characterized by a loss of cholinergic neurons in the basal forebrain and the ventral striatum. Since cholinergic neurons which degenerate in Alzheimer's disease are sensitive to nerve growth factor, a link between nerve growth factor sensitivity and the vulnerability of cholinergic neurons has been suspected. The purpose of this study was to determine, in cholinergic neurons, the level of expression of TrkA, the high affinity receptor for nerve growth factor, in control subjects and Alzheimer patients. The study was performed by in situ hybridization using a 35S-labeled RNA probe complementary to human TrkA mRNA on immunohistochemically identified cholinergic neurons of the nucleus basalis of Meynert, the ventral striatum, and the putamen in postmortem brains of patients with clinically and neuropathologically confirmed Alzheimer's disease and control subjects. In patients with Alzheimer's disease, a decrease in TrkA mRNA expression was observed in the nucleus basalis of Meynert (-75%, P < 0.001) and the ventral striatum (-41%, P < 0.01), where the cholinergic neurons degenerate, and also in the anterior (-43%, P < 0.01) and posterior (-51%, P < 0.01) parts of the putamen, where they are spared but display precocious signs of cell alterations. These results, taken in conjunction with the reduced choline acetyltransferase activity and our previously published data showing a loss of high affinity nerve growth factor binding in both the dorsal and the ventral striatum of patients with Alzheimer's disease, indicate that receptor loss and the consequent decrease in trophic support may be associated with the degeneration of cholinergic neurons during Alzheimer's disease.

Habrec1, a novel serine/threonine kinase TGF-beta type I-like receptor, has a specific cellular expression suggesting function in the developing organism and adult brain.

Members of the TGF-beta superfamily signal through a dual receptor system consisting of a type II receptor protein kinase that binds the ligand, after which this complex associates with a type I receptor to mediate intracellular signaling. In mammals, six type I and five type II receptors mediating responses to different TGF-beta family members have been identified to date. Using primers from conserved regions of the protein kinase domain of the serine/threonine kinase receptors in a low-stringency polymerase chain reaction-based screening procedure, and deselecting known receptors with colony hybridization, we now report cloning a novel receptor member. The novel receptor was found in a cDNA library prepared from the habenular nucleus area and was designated Habrec1. Although only a partial sequence is available, it fits the criteria for a TGF-beta type I serine/threonine kinase receptor. In situ hybridization of Habrec1 reveals mRNA expression in several distinct areas of the developing central nervous system, including cortex cerebri, cerebellum, hippocampus, striatum, and thalamic nuclei. Expression is also seen in the anterior pituitary. In the periphery, strong expression prenatally includes brown fat, the gastrointestinal tract, liver, pancreas, thymus, and nasal cavity epithelium. In the adult brain Habrec1 mRNA is prominently found in cerebellum, cortex cerebri, and striatum, but at lower levels in several additional areas. We conclude that Habrec1 is a member of the TGF-beta type I receptor family with expression patterns in the developing animal, suggesting specific functions in and outside the nervous system, and in the adult CNS, suggesting roles in both cortical and subcortical brain circuitry.

The metabotropic glutamate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynaptic and glial localizations.

Glutamate neurotransmission involves numerous ionotropic and metabotropic glutamate receptor types in postsynaptic, presynaptic and glial locations. Distribution of the metabotropic glutamate receptors mGluR2 and mGluR3 was studied with an affinity-purified, characterized polyclonal antibody made from a C-terminus peptide. This antibody, mGluR2/3, recognized both mGluR2 and mGluR3, but did not cross-react with any other type of metabotropic glutamate receptor except for a very slight recognition of mGluR5. Light microscope distribution of the antibody binding sites in the nervous system matched the combined distributions of messenger RNA for mGluR2 and mGluR3. For example, dense staining seen in the accessory olfactory bulb and cerebellar Golgi cells matched high levels of mGluR2 messenger RNA in these structures, while moderately dense staining in the reticular nucleus of the thalamus and light to moderate staining in glia throughout the brain matched significant levels of mGluR3 messenger RNA in these structures. In the rostral olfactory structures, the densest stained neurons belonged to presumptive "necklace olfactory glomeruli." In the hippocampus, staining was densest in the neuropil of the stratum lucidum/pyramidale, stratum lacunosum/moleculare, hilus and middle third of the molecular layer of the dentate gyrus. Ultrastructural studies of the cerebral cortex, hippocampus and caudate-putamen revealed significant staining in postsynaptic and presynaptic structures and glial wrappings of presumptive excitatory synapses; frequently, this staining was concentrated in discrete patches at or near active zones. In the hippocampus, presynaptic staining appeared to be concentrated in terminals of two populations of presumptive glutamatergic axons: mossy fibers originating from granule cells and perforant path fibers originating from the entorhinal cortex. These data suggest that populations of mGluR2 and/or mGluR3 receptors are localized differentially in synapses, i.e. those in and near the postsynaptic and presynaptic membranes and in glial wrappings of synapses, in several regions of the brain. In addition, we provide immunocytochemical evidence of mGluR2 or mGluR3 receptors in presynaptic terminals of glutamatergic synapses. Thus, mGluR2 and mGluR3 are found in various combinations of presynaptic, postsynaptic and glial localizations that may reflect differential modulation of excitatory amino acid transmission.

alpha-Linolenic acid dietary deficiency alters age-related changes of dopaminergic and serotoninergic neurotransmission in the rat frontal cortex.

The effects of alpha-linolenic acid diet deficiency on rat dopaminergic and serotoninergic neurotransmission systems were investigated in the frontal cortex, striatum, and cerebellum of male rats 2,6,12, and 24 months of age. The diet deficiency induced severe decrease in the 22:6n-3 fatty acid levels in all regions and a compensatory increase in n-6 fatty acid levels. A recovery in the levels of 22:6n-3 was observed in deficient rats between 2 and 12 months of age; however, this recovery was lower in frontal cortex than in striatum and cerebellum. In the striatum and cerebellum, dopaminergic and serotoninergic receptor densities and endogenous dopamine and serotonin levels were affected by aging regardless of the diet. In contrast, a 40-75% lower level of endogenous dopamine in the frontal cortex occurred in deficient rats according to age. The deficiency also induced an 18-46% increase in serotonin 5-HT2 receptor density in the frontal cortex during aging, without variation in endogenous serotonin level, and a 10% reduction in density of dopaminergic D2 receptors. Monoamine oxidase-A and -B activities showed specific age-related variations but regardless of the diet. Our results suggest that a chronically alpha-linolenic-deficient diet specifically affects the monoaminergic systems in the frontal cortex.