We conducted a genome-wide association study of essential tremor, a common movement disorder characterized mainly by a postural and kinetic tremor of the upper extremities. Twin and family history studies show a high heritability for essential tremor. The molecular genetic determinants of essential tremor are unknown. We included 2807 patients and 6441 controls of European descent in our two-stage genome-wide association study. The 59 most significantly disease-associated markers of the discovery stage were genotyped in the replication stage. After Bonferroni correction two markers, one (rs10937625) located in the serine/threonine kinase STK32B and one (rs17590046) in the transcriptional coactivator PPARGC1A were associated with essential tremor. Three markers (rs12764057, rs10822974, rs7903491) in the cell-adhesion molecule CTNNA3 were significant in the combined analysis of both stages. The expression of STK32B was increased in the cerebellar cortex of patients and expression quantitative trait loci database mining showed association between the protective minor allele of rs10937625 and reduced expression in cerebellar cortex. We found no expression differences related to disease status or marker genotype for the other two genes. Replication of two lead single nucleotide polymorphisms of previous small genome-wide association studies (rs3794087 in SLC1A2, rs9652490 in LINGO1) did not confirm the association with essential tremor.

Rotenone, a pesticide and complex I inhibitor, causes nigrostriatal degeneration similar to Parkinson disease pathology in a chronic, systemic, in vivo rodent model [M. Alam, W.J. Schmidt, Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats, Behav. Brain Res. 136 (2002) 317–324; R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson's disease, Nat. Neurosci. 3 (2000) 1301–1306; S.M. Fleming, C. Zhu, P.O. Fernagut, A. Mehta, C.D. DiCarlo, R.L. Seaman, M.F. Chesselet, Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone, Exp. Neurol. 187 (2004) 418–429; T.B. Sherer, J.H. Kim, R. Betarbet, J.T. Greenamyre, Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation, Exp. Neurol. 179 (2003) 9–16.]. To better investigate the role of mitochondria and complex I inhibition in chronic, progressive neurodegenerative disease, we developed methods for long-term culture of rodent postnatal midbrain organotypic slices. Chronic complex I inhibition over weeks by low dose (10–50 nM) rotenone in this system lead to dose- and time-dependent destruction of substantia nigra pars compacta neuron processes, morphologic changes, some neuronal loss, and decreased tyrosine hydroxylase (TH) protein levels. Chronic complex I inhibition also caused oxidative damage to proteins, measured by protein carbonyl levels. This oxidative damage was blocked by the antioxidant α-tocopherol (vitamin E). At the same time, α-tocopherol also blocked rotenone-induced reductions in TH protein and TH immunohistochemical changes. Thus, oxidative damage is a primary mechanism of mitochondrial toxicity in intact dopaminergic neurons. The organotypic culture system allows close study of this and other interacting mechanisms over a prolonged time period in mature dopaminergic neurons with intact processes, surrounding glia, and synaptic connections.

Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinson's disease, including selective nigrostriatal dopaminergic degeneration and alpha-synuclein-positive cytoplasmic inclusions (Betarbet et al., 2000; Sherer et al., 2003). Here, we examined mechanisms of rotenone toxicity using three model systems. In SK-N-MC human neuroblastoma cells, rotenone (10 nm to 1 microm) caused dose-dependent ATP depletion, oxidative damage, and death. To determine the molecular site of action of rotenone, cells were transfected with the rotenone-insensitive single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (NDI1), which incorporates into the mammalian ETC and acts as a "replacement" for endogenous complex I. In response to rotenone, NDI1-transfected cells did not show mitochondrial impairment, oxidative damage, or death, demonstrating that these effects of rotenone were caused by specific interactions at complex I. Although rotenone caused modest ATP depletion, equivalent ATP loss induced by 2-deoxyglucose was without toxicity, arguing that bioenergetic defects were not responsible for cell death. In contrast, reducing oxidative damage with antioxidants, or by NDI1 transfection, blocked cell death. To determine the relevance of rotenone-induced oxidative damage to dopaminergic neuronal death, we used a chronic midbrain slice culture model. In this system, rotenone caused oxidative damage and dopaminergic neuronal loss, effects blocked by alpha-tocopherol. Finally, brains from rotenone-treated animals demonstrated oxidative damage, most notably in midbrain and olfactory bulb, dopaminergic regions affected by Parkinson's disease. These results, using three models of increasing complexity, demonstrate the involvement of oxidative damage in rotenone toxicity and support the evaluation of antioxidant therapies for Parkinson's disease.

Recent studies suggest that metabotropic glutamate receptors (mGluRs) may play a significant role in regulating basal ganglia functions. In this study, we investigated the localization of mGluR4a protein in the mouse and rat basal ganglia. Polyclonal antibodies that specifically react with the metabotropic glutamate receptor subtype mGluR4a were produced and characterized by Western blot analysis. These antibodies recognized a native protein in wild-type mouse brain with a molecular weight similar to the molecular weight of the band from a mGluR4a-transfected cell line. The immunoreactivity was absent in brains of knockout mice deficient in mGluR4. mGluR4a immunoreactivity was most intense in the molecular layer of the cerebellum. We also found a striking mGluR4a immunoreactivity in globus pallidus, and moderate staining in substantia nigra pars reticulata and entopeduncular nucleus. Moderate to low mGluR4a immunoreactivity was present in striatum and other brain regions, including hippocampus, neocortex, and thalamus. Double labeling with mGluR4a antibodies and antibodies to either a dendritic marker or a marker of presynaptic terminals suggest a localization of mGluR4a on presynaptic terminals. Immunocytochemistry at electron microscopy level confirmed these results, revealing that in the globus pallidus, mGluR4a is mainly localized in presynaptic sites in axonal elements. Finally, quinolinic acid lesion of striatal projection neurons decreased mGluR4a immunoreactivity in globus pallidus, suggesting a localization of mGluR4a on striatopallidal terminals. These data support the hypothesis that mGluR4a serves as a presynaptic heteroreceptor in the globus pallidus, where it may play an important role in regulating g-amino-n-butyric acid (GABA) release from striatopallidal terminals.

Metabotropic glutamate receptors (mGluRs), which couple glutamate to second messengers, have important roles in the regulation of movement by the basal ganglia. We used two polyclonal antisera to mGluR1a and mGluR2/3 and confocal laser microscopy to investigate the localization of these receptors in the basal ganglia of the rat. The mGluRs were visualized in combination with an antibody to tyrosine hydroxylase (TH), an antibody to microtubule-associated protein 2 (MAP2, a dendritic marker), or SV2 (an antibody to a protein associated with presynaptic terminals). In the neostriatum, punctate mGluR1a immunoreactivity (ir) was present in the neuropil. This staining did not colocalize with MAP2-ir or SV2-ir and was not altered by decortication or unilateral 6-hydroxydopamine (6-OHDA) lesions. In the globus pallidus and substantia nigra pars reticulata, however, mGluR1a-ir was tightly clustered along large MAP2-ir dendrites. In contrast to the variations in mGluR1a-ir staining, similar punctate neuropil mGluR2/3-ir staining was observed within all basal ganglia structures. In the neostriatum, these puncta were abundant; unlike mGluR1a, many mGluR2/3-ir puncta colocalized with SV2-ir, and striatal mGluR2/3-ir puncta were markedly reduced in number after decortication. Neither mGluR1a-ir nor mGluR2/3-ir could be detected in TH-ir soma within substantia nigra pars compacta, or in TH-ir striatal terminals. Overall, our observations suggest that mGluR1a and mGluR2/3 receptors have distinct cellular localizations in different components of the basal ganglia circuitry and are likely to subserve distinct functions. Our data support the presence of mGluR2/3 on the terminals of corticostriatal afferents, where they may regulate glutamate release. In contrast, mGluR1a appears to be a postsynaptic receptor of neurons in the neostriatum, globus pallidus, and substantia nigra pars reticulata.

Metabotropic glutamate receptors, which are linked via G-proteins to second messenger systems, have been implicated in the physiological regulation of dopaminergic neurons of the substantia nigra pars compacta as well as in neurodegeneration. Of the eight known metabotropic glutamate receptors, metabotropic glutamate receptor 1 is the most abundant subtype in the substantia nigra pars compacta. Metabotropic glutamate receptor 1 is alternatively spliced at the carboxy terminal region to yield five variants: 1a, 1b, 1c, 1d and a form recently identified in human brain, 1g. We used an antibody recognizing metabotropic glutamate receptor 1, and another recognizing the splice form 1a only, to study the localization of these receptors in dopaminergic neurons identified by the presence of tyrosine hydroxylase. Metabotropic glutamate receptor immunoreactivity was present within the somata, axons, and dendrites of substantia nigra pars compacta neurons. The 1a splice form specific antibody, however, did not label these cells, suggesting that they express a metabotropic glutamate receptor 1 splice form different from 1a. In situ hybridization with splice form-specific oligonucleotide probes was used to determine which of the other known metabotropic glutamate receptor 1 splice forms might be present in the substantia nigra pars compacta. Each probe produced a very distinct labelling pattern in the rat brain with the exception of the 1g specific probe which produced only background signal. Substantia nigra pars compacta neurons were most intensely labelled by the metabotropic glutamate receptor 1d splice form specific probe. Metabotropic glutamate receptor 1a was expressed weakly whereas there was no detectable 1b, c, or g signal in the substantia nigra pars compacta. These data demonstrate thar metabotropic glutamate receptor 1 protein is present within the perikarya and processes of dopaminergic neurons in the substantia nigra pars compacta. The majority of this protein is not the la splice form. which is abundant in other brain regions, and may be the 1d isoform. Since splicing alters the carboxy terminus of the receptor, it is likely to affect the interaction of the receptor with intracellular signalling systems. (C) 1998 IBRO. Published by Elsevier Science Ltd.

Glutamate is the principal excitatory transmitter of the mammalian brain and plays a particularly important role in the physiology of the basal ganglia structures responsible for movement regulation. Using in situ hybridization with oligonucleotide probes, we examined the expression patterns of the five known kainate type glutamate receptor subunit genes, KA1, KA2 and GluR5-7, in the basal ganglia of adult and developing rat brain. In the adult rat, a highly organized and selective pattern of expression of the kainate subunits was observed in the basal ganglia and associated structures as well as in other regions of the brain. KA2 mRNA was abundant in the striatum, nucleus accumbens, subthalamic nucleus and substantia nigra pars compacta, and was present at lower levels in the globus pallidus and substantia nigra pars reticulata. Neither KA1 nor GluR5 expression was observed in the basal ganglia of adult rats, although these messages were present in other regions. GluR6 was highly expressed in the striatum and subthalamic nucleus and to a lesser extent in the substantia nigra pars reticulata, while no hybridization signal was detectable in the large, presumably dopaminergic neurons of the substantia nigra pars compacta. In contrast, GluR7 was strongly expressed in the substantia nigra pars compacta, was present at lower levels in the striatum, globus pallidus and substantia nigra pars reticulata, and was not detectable in the subthalamic nucleus. During postnatal development, expression of the kainate receptor subunits was characteristically highest on postnatal day 1 and declined to adult levels by day 20; however, in the globus pallidus we did observe the transient expression of KA1 and GluR5 between day 1 and day 10. These results demonstrate that the neuronal structures comprising the basal ganglia express a distinct combination of kainate receptor subunit genes, suggesting that the pharmacological properties of the resultant glutamate receptors are likely to be regionally specific. The organization of expression of these genes is established early in life, which is consistent with the important role they may play in establishing the functions of the motor system.

Metabotropic glutamate receptors (mGluRs) are thought to mediate diverse processes in brain including synaptic plasticity and excitotoxicity. These receptors are often divided into three groups by their pharmacological profiles. [3H]Glutamate binding in the presence of compounds selective for ionotropic glutamate receptors can be used as a general assay for these receptors; subtypes of this non-ionotropic [3H]glutamate binding differ in both pharmacology and anatomical distribution, and are differentially sensitive to quisqualate. The characteristics of these binding sites are consistent with those of group 1 (high-affinity quisqualate) and group 2 (low-affinity quisqualate) mGluRs. Under our assay conditions, no [3H]glutamate binding to group 3-like (L-AP4 sensitive) sites could be demonstrated. We have attempted to characterize particular agents which may selectively measure [3H]glutamate binding to mGluR subtypes. We used two isomers of 2-(carboxycyclopropyl)glycine, L-CCG-I and L-CCG-II, and the (2S,1'R,2'R,3'R) isomer of 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) as competitors of non-ionotropic [3H]glutamate binding sites. DCG-IV clearly distinguishes two binding sites. Quantitative levels of DCG-IV binding by anatomic region correlate with quisqualate-defined binding subtypes: high-affinity DCG-IV binding correlates with low-affinity quisqualate binding, whereas low-affinity DCG-IV binding correlates with high-affinity quisqualate binding. L-CCG-II displaces only one type of non-ionotropic [3H]glutamate binding, corresponding to high-affinity quisqualate binding. Therefore DCG-IV and L-CCG-II at appropriate concentrations appear to distinguish binding to putative group 2 vs. group 1 mGluRs. L-CCG-I displaces both high- and low-affinity quisqualate binding sites, but unlike the other two compounds, does not clearly distinguish between them.

N-methyl-D-aspartate (NMDA) glutamate receptors have an established role in the regulation of motor behavior by the basal ganglia. Recent studies have revealed that NMDA receptors are heteromeric assemblies of structurally related subunits from two families: NMDAR1, which is required for channel activity, and NMDAR2A-D, which modulate the properties of the channels. In the rat, the NMDA receptor subunits exhibit anatomically restricted patterns of expression, so that each component of the basal ganglia has a distinct NMDA receptor subunit mRNA phenotype. We have used in vivo intrastriatal injection of synthetic antisense oligodeoxynucleotides (ODNs) to examine the roles of particular NMDA receptor subunits in the regulation of motor behavior in rats. Injection of 15 nmol of a 20-mer ODN targeted to the NMDAR1 subunit induced spontaneous ipsilateral rotation. Smaller doses of NMDAR1 antisense ODN did not lead to spontaneous rotation, but prominent ipsilateral rotation was observed after systemic administration of D-amphetamine. An antisense ODN to NMDAR2A was also effective in eliciting amphetamine-inducible rotation, although the magnitude of the effect was less than that seen with NMDAR1, whereas ODNs targeted to NMDAR2B, NMDAR2C and an NMDAR1 sense strand ODN had no effect on behavior. In situ hybridization demonstrated that injection of the NMDAR1, NMDAR2A or NMDAR2B antisense ODNs produced specific reductions in target mRNA signal intensity in the injected striatum. After NMDAR1 antisense ODN injection, striatal binding of 3H-glutamate target mRNA signal intensity in the injected striatum. After NMDAR1 antisense ODN injection, striatal binding of 3H-glutamate to NMDA sites was not altered, although strychnine-insensitive 3H-glycine binding sites exhibited a small but significant reduction. These observations suggest that NMDA receptor complexes containing NMDAR1 and, to a lesser extent, NMDAR2A subunits play particularly important roles in the regulation of motor behavior by neostriatal neurons.