NMDA receptor losses in putamen from patients with Huntington's disease.

N-Methyl-D-aspartate (NMDA), phencyclidine (PCP), and quisqualate receptor binding were compared to benzodiazepine, gamma-aminobutyric acid (GABA), and muscarinic cholinergic receptor binding in the putamen and cerebral cortex of individuals with Huntington's disease (HD). NMDA receptor binding was reduced by 93 percent in putamen from HD brains compared to binding in normal brains. Quisqualate and PCP receptor binding were reduced by 67 percent, and the binding to other receptors was reduced by 55 percent or less. Binding to these receptors in the cerebral cortex was unchanged in HD brains. The results support the hypothesis that NMDA receptor-mediated neurotoxicity plays a role in the pathophysiology of Huntington's disease.

Localization of dopaminergic markers in the human subthalamic nucleus.

The potential role for dopamine in the subthalamic nucleus was investigated in human postmortem tissue sections by examining; (1) immunostaining for tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis; (2) binding of [(3)H]-SCH23390 (D1-like), [(3)H]-YM-09151-2 (D2-like), and [(3)H]-mazindol (dopamine uptake); and (3) expression of dopamine D1 and D2 receptor mRNAs. Immunostaining for tyrosine hydroxylase was visualized in Bouin's-fixed tissue by using a monoclonal antibody and the avidin-biotin-complex method. The cellular localization of the dopamine D1 and D2 receptor mRNAs was visualized by using a cocktail of human specific oligonucleotide probes radiolabeled with (35)S-dATP. Inspection of immunostained tissue revealed a fine network of tyrosine hydroxylase-immunostained fibers traversing the nucleus; no immunopositive cells were detected. Examination of emulsion-coated tissue sections processed for D1 and D2 receptor mRNA revealed, as expected, an abundance of D1 and D2 mRNA-positive cells in the caudate nucleus and putamen. However, no D1 or D2 receptor mRNA-expressing cells were detected in the subthalamic nucleus. Further, semiquantitative analysis of D1-like, D2-like and dopamine uptake ligand binding similarly revealed an enrichment of specific binding in the caudate nucleus and putamen but not within the subthalamic nucleus. However, a weak, albeit specific, signal for [(3)H]-SCH23390 and [(3)H]-mazindol was detected in the subthalamic nucleus, suggesting that the human subthalamic nucleus may receive a weak dopaminergic input. As weak D1-like binding is detected in the subthalamic nucleus, and subthalamic neurons do not express dopamine D1 or D2 receptor mRNAs, together these data suggest that the effects of dopaminergic agents on the activity of human subthalamic neurons may be indirect and mediated via interaction with dopamine D1-like receptors.

Longitudinal neuropsychological and genetic linkage analysis of persons at risk for Huntington's disease.

OBJECTIVE: To determine (1) whether the neuropsychological profiles of healthy individuals at risk (AR) for Huntington's disease who were positive (AR/+) or negative (AR/-) for the Huntington's disease genetic marker differed from those of symptomatic patients with Huntington's disease and normal control individuals and (2) whether the neuropsychological performance of the two AR groups differed from each other on three assessments during a 4-year span. DESIGN: Case-control, double-blind study, with AR status determined by genetic linkage analysis (G8 probe), in addition to examination of trinucleotide repeats for most AR subjects. SETTING: The Neuropsychology Program in the Department of Psychiatry and the Department of Neurology at the University of Michigan Medical Center, Ann Arbor, a tertiary care center. PARTICIPANTS: Eight subjects matched as closely as possible for age, gender, and education in each of the following groups: AR/+, AR/-, normal control, and Huntington's disease. MEASURES: A battery of neuropsychological tasks, including measures of intelligence, memory, problem solving, and motor ability. RESULTS: Although both AR groups demonstrated variability on select intellectual subtests relative to normal subjects, they did not differ from each other on the three assessments during a 4-year span. Patients with Huntington's disease performed more poorly than the other groups across a range of neuropsychological measures. CONCLUSIONS: These results do not support previous evaluations concluding that AR/+ individuals demonstrate cognitive impairments as compared with AR/- individuals. Findings in earlier studies without genetic linkage analysis of lower performance of AR individuals, including children, as compared with normal controls may relate to extraneous environmental and familial issues that interfere with intellectual development.

Dopamine-receptor autoradiography of human narcoleptic brain.

Although the pathology of human narcolepsy is unknown, studies of human and canine narcolepsy have suggested that dopamine metabolism may be disturbed. We used quantitative autoradiography to assess dopamine D1- and D2-receptor binding in basal ganglia and amygdala of five narcoleptic and 17 control human brains. In caudate, narcoleptic brains had a statistically significant increase of 57% in D1-receptor binding, and large but not significant increases of 54% in medial globus pallidus D1 binding, 63% in caudate D2-receptor binding, 95% in lateral globus pallidus D2 binding, and 93% in lateral amygdala D2 binding. We found no major changes in the putamen or in the basal or accessory basal nuclei of the amygdala. These results suggest that narcolepsy is associated with upregulation of dopamine receptors in specific areas of the brain, although medications used prior to death may have contributed to the findings.

Glutamate receptors in the substantia nigra of Parkinson's disease brains.

Glutamic acid and its analogs are excitotoxins that might contribute to the pathogenesis of Parkinson's disease (PD). We measured four subtypes of glutamate binding sites autoradiographically in 20-microns sections from control and PD midbrains. N-Methyl-D-aspartate (NMDA) binding sites (eight control, eight PD) were very low in control (20 +/- 7 [SEM] fmol/mg protein) and were reduced in the PD pars compacta (2.6 +/- 1.1 fmol/mg protein; p less than 0.02). NMDA binding was also reduced in the red nucleus but not in periaqueductal gray (PAG). We measured alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), metabotropic, and non-NMDA, nonkainate, non-quisqualate (NNKQ) sites in 10 PD and 12 control midbrains. AMPA binding sites were reduced from 175 +/- 20 to 99 +/- 16 (p less than 0.05) fmol/mg protein in PD pars compacta, NNKQ sites from 86 +/- 10 to 50 +/- 12 (p less than 0.05) fmol/mg protein in total nigra, and metabotropic sites (15 +/- 5 fmol/mg protein) were unchanged. AMPA, metabotropic, and NNKQ binding were unchanged in red nucleus and PAG. The very low number of NMDA binding sites suggests that factors other than excitotoxicity mediated via NMDA receptors on nigral cell bodies play roles in the pathogenesis of PD. There may be a generalized loss of NMDA receptors in PD brains. AMPA and NNKQ binding sites appear to be located on dopamine neurons, although the role of NNKQ sites in normal nervous system function and human disease is unknown.

Dopamine transmission in DYT1 dystonia: a biochemical and autoradiographical study.

Indices of dopamine transmission were measured in the postmortem striatum of DYT1 dystonia brains. A significant increase in the striatal 3,4-dihydroxyphenylacetic acid/dopamine ratio was found. Quantitative autoradiography revealed no differences in the density of dopamine transporter or vesicular monoamine transporter-2 binding; however, there was a trend toward a reduction in D(1) receptor and D(2) receptor binding. One brain with DYT1 parkinsonism was similarly evaluated and marked reductions in striatal dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid content as well as the density of binding of all four dopaminergic ligands were measured.

Excitatory amino acid binding sites in the basal ganglia of the rat: a quantitative autoradiographic study.

Quantitative receptor autoradiography was used to determine the distribution of excitatory amino acid binding sites in the basal ganglia of rat brain. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-D-aspartate, kainate, quisqualate-sensitive metabotropic and non-N-methyl-D-aspartate, non-kainate, non-quisqualate glutamate binding sites had their highest density in striatum, nucleus accumbens, and olfactory tubercle. Kainate binding was higher in the lateral striatum but there was no medial-lateral striatal gradient for other binding sites. N-Methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding sites were most dense in the nucleus accumbens and olfactory tubercle. There was no dorsal-ventral gradient within the striatal complex for the other binding sites. Other regions of the basal ganglia had lower densities of ligand binding. To compare binding site density within non-striatal regions, binding for each ligand was normalized to the striatal binding density. When compared to the striatal complex, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and metabotropic binding sites had higher relative density in the globus pallidus, ventral pallidum, and subthalamic nucleus than other binding sites. Metabotropic binding also had a high relative density in the substantia nigra. Non-N-methyl-D-aspartate, non-kainate, non-quisqualate glutamate binding sites had a high relative density in globus pallidus, ventral pallidum, and substantia nigra. N-Methyl-D-aspartate binding sites had a low relative density in pallidum, subthalamic nucleus, substantia nigra and ventral tegmental area. Our data indicate heterogeneous distribution of excitatory amino acid binding sites within rat basal ganglia and suggest that the character of excitatory amino acid-mediated neurotransmission within the basal ganglia is also heterogeneous.

Neurochemical studies of human narcolepsy: alpha-adrenergic receptor autoradiography of human narcoleptic brain and brainstem.

Studies of human and canine narcolepsy-cataplexy syndrome suggest that noradrenergic function may be abnormal. We used quantitative autoradiography to assess noradrenergic alpha-1 and alpha-2 receptors in several regions of seven human narcoleptic and 18 control brains using [3H]prazosin to evaluate alpha-1 receptors, and [3H]UK14304 and [3H]rauwolscine to evaluate alpha-2 receptors. Specific blocking agents were used in combination with the tritiated ligands to assess alpha-1 and alpha-2 receptor subtypes. Although we found few statistically significant differences between narcoleptic and control brains, there were a number of trends. [3H]Prazosin binding to sites in the amygdala, globus pallidus and putamen was reduced by 22-68%, whereas binding was increased by 40% to the inferior olive and by 84% to portions of the dorsal pons. Binding was similar to control values in other regions. In all seven brainstem regions that were evaluated, the ratio of alpha-1b receptor binding to alpha-1a receptor binding was increased. Binding of [3H]UK14304 was increased by 35-74% in the caudate nucleus, putamen and portions of the amygdala and pons. [3H]rauwolscine binding data suggested that increase of alpha-2 receptor binding in the dorsal pons were not due to effects at the imidazole receptor. findings suggest that noradrenergic function may be altered in specific regions of the brain and brainstem in human narcolepsy, although the absence of statistical significance indicates that these trends should be considered preliminary. The trend toward a relative increase of alpha-1b receptor binding in narcoleptic brainstem is consistent with data from studies of canine narcolepsy and suggests that altered activity at this receptor may contribute to the pathogenesis of human narcolepsy. Studies of additional brains will be required to confirm these findings.

Quisqualate resolves two distinct metabotropic [3H]glutamate binding sites.

Metabotropic receptor subtypes have been proposed based on pharmacological, signal transduction and cDNA sequence data. We assessed potential metabotropic binding site subtypes with in vitro quantitative [3H]glutamate autoradiography in adult rat brains in the presence of saturating concentrations of N-methyl-D-aspartate (NMDA) and (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA). Quisqualate (QUIS) competition curves resolved two differentially distributed binding sites (KIhigh = 17 nM; KIlow = 62 microM). Trans-1-amino-cyclopentane- 1,3-dicarboxylic acid (t-ACPD) and 1S,3R-ACPD displaced [3H]glutamate binding both in the absence and presence of a quisqualate concentration (2.5 microM) that saturates the high affinity sites, suggesting that both sites are linked to metabotropic receptors. We conclude that two metabotropic binding sites with different distributions and pharmacological profiles can be detected with selective [3H]glutamate binding assays.

Contamination of commercially available quisqualic acid by glutamate-like and aspartate-like substances.

Six different batches of the glutamic acid analogue quisqualic acid were analyzed with high-pressure liquid chromatography (HPLC). All batches examined showed contaminant peaks. Different batches had different contaminant peaks and differing amounts of each contaminant. Every batch of quisqualic acid tested demonstrated a contaminant peak which co-eluted with exogenously added glutamic acid. Certain batches possessed a contaminant which co-eluted with aspartic acid. The levels of glutamate-like contamination ranged from 0.08 to 0.60%, and the levels of aspartate-like contamination ranged from undetectable amounts to 0.80%. The amount of combined glutamate- and aspartate-like contamination of each batch of quisqualate correlated very highly with the ability of that batch to interact with non-quisqualate receptors in an autoradiographic binding assay. These non-quisqualate receptors are likely N-methyl-D-aspartate (NMDA) receptors. Thus, when high concentrations of quisqualate are used experimentally, contamination is likely to produce spurious effects at non-quisqualate glutamate receptors. Quisqualate itself may be a more specific agonist than assumed previously.

Excitatory amino acid receptor regulation after subthalamic nucleus lesions in the rat.

The subthalamic nucleus plays a pivotal role in the regulation of basal ganglia output. Recent electrophysiologic, lesion and immunocytochemical studies suggest that the subthalamic nucleus uses an excitatory amino acid as a neurotransmitter. After complete ablation of the subthalamic nucleus, we have examined the NMDA, AMPA, kainate and metabotropic subtypes of excitatory amino acid receptors in two major subthalamic projection areas (globus pallidus and substantia nigra pars reticulata) with quantitative autoradiography. Two weeks after ablation, binding sites for [3H]AMPA and [3H]kainate increased in substantia nigra pars reticulata ipsilateral to the lesion. In globus pallidus on the lesioned side, [3H]glutamate binding to the NMDA recognition site decreased. The results suggest that glutamate receptors regulate after interruption of subthalamic nucleus output.

Inhibitory and excitatory amino acid neurotransmitter binding sites in cynomolgus monkey (Macaca fascicularis) cervical spinal cord.

Autoradiography of inhibitory and excitatory amino acid neurotransmitter binding sites in the cervical spinal cord of M. fascicularis spinal cord revealed inhomogeneous distribution of all binding sites in spinal gray matter. Quisqualate-sensitive [3H]glutamate binding, [3H]MK-801 binding, benzodiazepine binding, kainate binding, and GABAB binding had highest levels in the superficial layers of the dorsal horn (laminae 1 and 2) and substantially lower levels in other laminae. [3H]Strychnine binding was more uniformly distributed throughout all laminae with highest levels in the superficial layers of the dorsal horn. These results are similar to those found in other mammals.

Selective metabotropic receptor agonists distinguish non-ionotropic glutamate binding sites.

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.

Autoradiographic evidence for increased dopamine uptake sites in striatum of hypoxic mice.

Ligand binding to dopamine uptake sites, D1 and D2 dopamine receptors was measured autoradiographically in brain sections of mice exposed to intermittent hypobaric hypoxia (450 Torr; 4,300 m) for 14 days and compared to sea level controls. Desipramine-insensitive [3H]mazindol, [3H]SCH23390 and [3H]YM-09151-2 were used respectively for the labeling of the three binding sites. After 14 days, the striatum of hypoxic mice showed a significant 21% increase in dopamine uptake sites, one of the loci of action of cocaine. A similar (28%) but non-significant increase was found in the ventral tegmental area. No changes were seen in the activities of D1 or D2 receptors in several areas examined including the substantia nigra and the striatum.

Excitatory amino acid binding sites in the periaqueductal gray of the rat.

We used receptor autoradiography to determine the distribution of excitatory amino acid (EAA) binding site subtypes in the periaqueductal gray (PAG) of the rat. N-Methyl-D-aspartate (NMDA), kainate, quisqualate-ionotropic, and quisqualate-metabotropic binding sites were all present in the PAG. Distribution was inhomogeneous with greatest density of all binding site subtypes in the dorsolateral subdivision and lowest density in the ventrolateral subdivision. Relative to regions of brain with high densities of EAA binding site subtypes, quisqualate-metabotropic binding sites had the highest relative density and NMDA binding sites the least. The presence of all subtypes of EAA binding sites in the PAG suggests that EAA action within the PAG is likely to be complex.

Autoradiographic studies of post-mortem human narcoleptic brain.

Although the pathological basis for narcolepsy is unknown, studies of human and canine narcolepsy have suggested that monoamine and cholinergic metabolism may be altered. We used quantitative autoradiography to assess binding of dopaminergic, noradrenergic, and cholinergic ligands to basal ganglia and amygdala of five narcoleptic and 17 control human brains. Dopamine receptor studies revealed significant increases in D-1 and D-2 receptor binding in the caudate nucleus, as well as large but not significant increases of D-1 binding in the medial globus pallidus, and D-2 binding in the lateral globus pallidus and the lateral nucleus of the amygdala. Alpha-adrenergic receptor studies revealed a significant increase in alpha-2 receptor binding in the putamen and large but not significant increases of alpha-2 binding in the caudate nucleus, and basal and lateral nuclei of the amygdala. Alpha-1 receptor binding was decreased in several areas but the changes were not statistically significant. Studies of two narcoleptic brains revealed small but not statistically significant increases in muscarinic receptor binding in the caudate nucleus, putamen, and amygdala. Although we cannot exclude the possibility that stimulant medications used before death may be partly responsible for these findings, the results suggest that human narcolepsy is associated with upregulation of dopamine and alpha-2 adrenergic receptors in specific brain regions.

Absence of alpha-synuclein mRNA expression in normal and multiple system atrophy oligodendroglia.

alpha-Synuclein is a major constituent of glial cytoplasmic inclusions (GCIs), which are pathognomic for multiple system atrophy (MSA). We have previously demonstrated that in normal human brain, alpha-synuclein mRNA has a restricted pattern of neuronal expression and no apparent glial expression. The current study used double-label in situ hybridization to determine if alpha-synuclein mRNA is expressed by oligodendroglia of MSA cases. Analysis of MSA brain tissue revealed depletion of regional signal for this transcript in many brain areas due to extensive neurodegeneration. Cellular analysis of oligodendroglia in crus cerebri, a GCI-rich region ventral to substantia nigra, revealed an absence of alpha-synuclein mRNA signal in control and MSA cases. However, an abundance of this transcript was detected in melanin-containing neurons of substantia nigra. Therefore, oligodendroglia do not express alpha-synuclein mRNA in control and MSA cases suggesting that involvement of alpha-synuclein in GCI pathology of MSA is due to its ectopic presence in oligodendroglia.

Phospholipase A2 modulates different subtypes of excitatory amino acid receptors: autoradiographic evidence.

Exogenous phospholipases have been used extensively as tools to study the role of membrane lipids in receptor mechanisms. We used in vitro quantitative autoradiography to evaluate the effect of phospholipase A2 (PLA2) on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat brain. PLA2 pretreatment induced a significant increase in alpha-[3H]amino-3-hydroxy-5-methyl-isoxazole-4-propionate ([3H]AMPA) binding in the stratum radiatum of the CA1 region of the hippocampus and in the stratum moleculare of the cerebellum. No modification of [3H]AMPA binding was found in the stratum pyramidale of the hippocampus at different ligand concentrations. [3H]-Glutamate binding to the metabotropic glutamate receptor and the non-NMDA-, non-kainate-, non-quisqualate-sensitive [3H]glutamate binding site were also increased by PLA2 pretreatment. [3H]Kainate binding and NMDA-sensitive [3H]glutamate binding were minimally affected by the enzyme pretreatment. The PLA2 effect was reversed by EGTA, the PLA2 inhibitor p-bromophenacyl bromide, and prolonged pretreatment with heat. Bovine serum albumin (1%) prevented the increase in metabotropic binding by PLA2. Arachidonic acid failed to mimic the PLA2 effect on metabotropic binding. These results indicate that PLA2 can selectively modulate certain subtypes of excitatory amino acid receptors. This effect is due to the enzymatic activity but is probably not correlated with the formation of arachidonic acid metabolites. Independent of their possible physiological implications, our results provide the first autoradiographic evidence that an enzymatic treatment can selectively affect the binding properties of excitatory amino acid receptors in different regions of the CNS.

Inhibition of N-methyl-D-aspartate glutamate receptor subunit expression by antisense oligonucleotides reveals their role in striatal motor regulation.

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.