Kynurenine pathway measurements in Huntington's disease striatum: evidence for reduced formation of kynurenic acid.

Recent evidence suggests that there may be overactivation of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors in Huntington's disease (HD). Tryptophan metabolism by the kynurenine pathway produces both quinolinic acid, an NMDA receptor agonist, and kynurenic acid, an NMDA receptor antagonist. In the present study, multiple components of the tyrosine and tryptophan metabolic pathways were quantified in postmortem putamen of 35 control and 30 HD patients, using HPLC with 16-sensor electrochemical detection. Consistent with previous reports in HD putamen, there were significant increases in 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, and serotonin concentrations. Within the kynurenine pathway, the ratio of kynurenine to kynurenic acid was significantly (p less than 0.01) increased twofold in HD patients as compared with controls, consistent with reduced formation of kynurenic acid in HD. CSF concentrations of kynurenic acid were significantly reduced in HD patients as compared with controls and patients with other neurologic diseases. Because kynurenic acid is an endogenous inhibitor of excitatory neurotransmission and can block excitotoxic degeneration in vivo, a relative deficiency of this compound could directly contribute to neuronal degeneration in HD.

Distribution of DARPP-32 in the basal ganglia: an electron microscopic study.

DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein, has been studied by light and electron microscopical immunocytochemistry in the rat caudatoputamen, globus pallidus and substantia nigra. In the caudatoputamen, DARPP-32 was present in neurons of the medium-sized spiny type. Immunoreactivity for DARPP-32 was present in dendritic spines, dendrites, perikaryal cytoplasm, most but not all nuclei, axons and a small number of axon terminals. Immunoreactive axon terminals in the caudatoputamen formed symmetrical synapses with immunolabeled dendritic shafts or somata. Neurons having indented nuclei were never immunoreactive. In the globus pallidus and substantia nigra pars reticulata, DARPP-32 was present in myelinated and unmyelinated axons and in axon terminals. The labelled axon terminals in these regions formed symmetrical synaptic contacts on unlabelled dendritic shafts or on unlabelled somata. These data suggest that DARPP-32 is present in striatal neurons of the medium-sized spiny type and that these DARPP-32-immunoreactive neurons form symmetrical synapses on target neurons in the globus pallidus and substantia nigra. The presence of DARPP-32 in these striatal neurons and in their axon terminals suggests that DARPP-32 mediates part of the response of medium-size spiny neurons in the striatum to dopamine D-1 receptor activation.

What excitotoxin kills striatal neurons in Huntington's disease? Clues from neurochemical studies.

Amino acid analyses of both caudate nucleus and putamen obtained at autopsy from patients dying with Huntington's disease (HD), and from control subjects, showed significantly decreased mean glutamate contents in the HD patients. In addition, the mean glutamate concentration was significantly increased in the CSF of living HD patients as compared with controls. Neurochemical studies also showed that neither aspartic acid, proline, 5-oxoproline, nor homocysteic acid is likely to act as a causative excitotoxin in HD. Excessive striatal glycine content, or deficient glutathione content, is unlikely to contribute to the effects of a causative excitotoxin in HD. We suggest that glutamic acid may be the proximate causative neurotoxin in the striatum in HD, as a result of an unexplained failure in the reuptake mechanism for glutamate released there as an excitatory neurotransmitter.

Distribution of monoamines and metabolites in rabbit neostriatum, hippocampus and cortex.

The monoamines noradrenaline (NA), dopamine (DA), adrenaline (AD) and 5-hydroxytryptamine (5-HT) were assayed in the putamen (PUT), the lateral (lCAU) and medial (mCAU) portions of the caudate, the dorsal (dHIP) and ventral (vHIP) hippocampus, as well as in four cortical areas, i.e., anterior cingulate (CIN), entorhinal-piriform (EnPi), sensorimotor (SSC; somatosensory) and primary visual (VIS). The use of an HPLC procedure enabled us to perform these measurements in microdissected samples and to assay as well monoamine metabolites. The DA levels were highest in the neostriatum, moderate in the EnPi and CIN and very low in the SSC, VIS and hippocampus. The distribution of NA was more uniform, although higher concentrations were measured in the neostriatum, hippocampus and EnPi. The largest amounts of 5-HT were in the EnPi, while moderate concentrations were found in the other regions. The ratios between the neurotransmitters and their metabolites were used as an index of turnover and indicate that the terminal fields of the monoamine systems are heterogenous within the neostriatal, hippocampal and cortical subdivisions.

Analysis of serotonin, dopamine and their metabolites in the caudate putamen, the suprachiasmatic nucleus and the median raphe nucleus of euthermic and torpid deermice, Peromyscus maniculatus.

Deermice, subjected to food rationing and low ambient temperature, were sacrificed in normothermia or during daily torpor. Levels of monoamines and their metabolites in the caudate putamen (CPN), the suprachiasmatic nuclear area (SCN), and the median raphe nucleus (MRN) were quantified through the use of HPLC with electrochemical detection. Significant elevations in levels (pg/mg protein) of the serotonin (5-HT) metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA) were noted in torpid individuals in all nuclei examined. The dopamine (DA) metabolite, homovanillic acid (HVA) was significantly elevated in the CPN and MRN of torpid individuals. Moreover, a significant increase in the HVA to DA ratio was also noted in the CPN and the MRN. In the SCN, the concentrations of 5-hydroxytryptophan (5-HTP), 5-HT, DA and 3,4-dihydroxyphenylacetic acid (DOPAC) were also increased significantly during torpor. These significant elevations suggest that an increase in the activity of the serotonergic and dopaminergic systems occurs in these nuclei during daily torpor in the deermouse.

Striatal phosphoproteins in Parkinson disease and progressive supranuclear palsy.

This study was undertaken to evaluate the levels of cAMP-regulated phosphoproteins in the striatum of patients with neurodegenerative diseases of the dopaminergic system. Postmortem samples of caudate nucleus and putamen from 24 control subjects, 23 patients with Parkinson disease, and 13 patients with progressive supranuclear palsy were studied with immunoblotting techniques. The levels of tyrosine hydroxylase were reduced in patients with Parkinson disease (levels were 24% and 10% of controls in caudate nucleus and putamen, respectively) and with progressive supranuclear palsy (levels were 11% and 6% of controls in caudate nucleus and putamen, respectively). Five phosphoproteins, which are present in striatal neurons and are likely to play a role in the postsynaptic actions of dopamine, were measured. These included ARPP-16, ARPP-19, ARPP-21 (cAMP-regulated phosphoproteins of Mr 16,000, 19,000, and 21,000, respectively), DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32,000), and phosphatase inhibitor I. The levels of these phosphoproteins were inversely correlated with postmortem delay. In brains of patients with Parkinson disease or progressive supranuclear palsy with postmortem delays comparable to those of controls, the levels of these proteins as well as those of synaptic (synapsin I and synaptophysin) and glial (glial fibrillary acidic protein and myelin basic protein) markers were not significantly modified. We conclude that the levels of several phosphoproteins involved in signal transduction in striatal neurons are not altered in Parkinson disease and progressive supranuclear palsy. This observation supports the view that the striatal output neurons are intact in both diseases.

Quantitative radioimmunohistochemical method using [125I]-protein A to measure the content of methionine enkephalin in discrete rat brain areas.

We report a quantitative radioimmunohistochemical method, using [125I]-protein A in combination with a specific antibody to methionine enkephalin (Met-enk), for determination of the content of this peptide in discrete areas of rat brain. After paraformaldehyde fixation, rat brain sections were incubated with a Met-enk polyclonal antibody, followed by incubation with [125I]-protein A. After autoradiography with 3H-sensitive Ultrofilm, optical densities (OD) were quantified by computerized microdensitometry. The OD obtained were compared to a standard curve, constructed after determination by radioimmunoassay of the Met-enk content in corresponding brain areas from adjacent tissue sections. After comparing 15 different brain areas over a ninetyfold range of concentrations, we found a linear relationship between the content of Met-enk, as determined by radioimmunoassay, and the OD generated by autoradiography. The content of Met-enk in other discrete brain areas can be quantified by interpolation of the OD determined by autoradiography in the standard curve. The method allows, for the first time, precise quantification of peptide concentrations in discrete areas and nuclei from thin sections of rat brain. This technique has a more than 100-fold higher sensitivity than classical radioimmunoassays, with the additional advantage of neuroanatomical localization. It also has the potential for application to the quantification of many other antigens present in brain and other tissues.

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.

Subcellular distribution of mammalian tachykinins in rat basal ganglia.

A combined differential and density gradient centrifugation procedure was used to study the subcellular localisation of the mammalian tachykinins in rat caudateputamen and substantia nigra. Substance P, neurokinin A, neuropeptide K, and neurokinin B were found to be concentrated in the synaptosomal fractions and in fractions containing heavy synaptic vesicles in both regions studied. In contrast, the catecholamines dopamine and noradrenaline had a more widespread distribution throughout the gradient. HPLC analysis of the immunoreactivity recovered showed that the tachykinin immunoreactivity coeluted with the relevant synthetic tachykinins, except in the soluble gradient fraction where neurokinin A immunoreactivity eluted in position consistent with neurokinin A3-10. These results suggest that, in the basal ganglia, the mammalian tachykinins are localised in fractions containing large dense cored synaptic vesicles. This vesicular localisation would be consistent with the proposed role of the tachykinins as neurotransmitters and neuromodulators.

Polyamine changes in reversible cerebral ischemia.

Putrescine, spermidine, and spermine levels were measured in the cortex, caudoputamen, and hippocampus of rats during 30 min of severe forebrain ischemia (induced by occlusion of both carotid and vertebral arteries) and subsequent recirculation. During ischemia, polyamine levels did not change significantly. During postischemic recirculation, however, putrescine levels dramatically increased whereas those of spermine and spermidine did not change, with the exception of the severely damaged caudoputamen, where the concentration declined after 24 h. The increase of putrescine is explained by postischemic activation of ornithine decarboxylase and inhibition of S-adenosylmethionine decarboxylase. It is suggested that the accumulation of putrescine during postischemic recirculation may be responsible for the delayed neuronal death occurring after ischemia.

Decreased glutamic acid and increased 5-hydroxytryptamine in Huntington's disease brain.

gamma-Aminobutyric acid (GABA), glutamate and serotonin (5-HT), along with its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), have been measured in 4 brain regions taken post-mortem from patients with Huntington's disease and from control subjects. GABA was found to be significantly reduced, not only in the basal ganglia, but also in the cortex and hippocampus. Glutamate was also reduced in all regions except the pallidum, and thus was unrelated to the GABA deficit which was greatest in this area. 5-HT and 5-HIAA were significantly increased in all areas except the hippocampus.

Divergent changes in D-1 and D-2 dopamine binding sites in human brain during aging.

The density of D-1 and D-2 dopamine receptors in human caudate nucleus and putamen, obtained postmortem, were studied throughout the adult lifespan using [3H]fluphenazine as the dopamine receptor ligand. The D-1 subtype increased progressively with age in both regions, while the D-2 subtype declined in caudate nucleus. The ratio of D-1/D-2 Bmax in both regions increased from approximately 1 at age 20 to 2 by age 75. The dopamine content in putamen declined with age and was inversely correlated with D-1 receptor density. We suggest that D-1 receptor density is up-regulated by loss of dopamine during aging. The D-2 receptor density in caudate nucleus was positively correlated with choline acetyltransferase activity, suggesting that loss of intrastriatal neurons with age may contribute to the decrease in D-2 sites. These divergent changes in dopamine receptor subtypes with age result in an altered complement of dopamine receptors in older humans and may provide a basis for selective pharmacotherapy in disorders of the basal ganglia.

Haloperidol increases proenkephalin mRNA levels in the caudate-putamen of the rat: a quantitative study at the cellular level using in situ hybridization.

Previous immunocytochemical studies have shown that the opioid peptides, Met-enkephalin and Leu-enkephalin, are present in medium-sized, spiny projection neurons of the caudate-putamen. It has also been demonstrated that chronic treatment of rats with the dopamine receptor blocker, haloperidol, results in an increase in the levels of enkephalin peptides and proenkephalin mRNA in this brain region. To determine whether this increase in proenkephalin mRNA content is exhibited by all enkephalinergic neurons of the caudate-putamen or by only a subpopulation, we have used in situ nucleic acid hybridization to examine the haloperidol-induced increase in proenkephalin mRNA levels at the cellular level. Results of in situ hybridization suggest that all enkephalinergic neurons in the caudate-putamen can respond to haloperidol treatment with an increase in steady state levels of proenkephalin mRNA, and that the mean induction is an approximate 3-fold increase in the message levels. This suggests that dopamine exerts a tonic inhibitory effect on the expression of the proenkephalin gene in all of the enkephalinergic neurons of the caudate-putamen. Dot blot analysis indicated a 2.4-fold increase in the tissue levels of this mRNA. The agreement between the in situ hybridization results and dot blot analysis supports in situ hybridization as a reliable method for quantitative studies of alterations in neuropeptide precursor mRNAs in the brain.

Quantitative autoradiography of dopamine D2 sites in rat caudate-putamen: localization to intrinsic neurons and not to neocortical afferents.

Dopamine D2 receptors, labeled with [3H]spiroperidol or [3H]sulpiride, show a lateral-to-medial gradient in the caudate-putamen, with a more than two-fold greater density laterally than medially. It has been thought that D2 receptors are located on at least two neuronal elements of the caudate-putamen, neurons intrinsic to this structure and axons whose cell bodies reside in the cortex. As a first step in establishing what neuronal elements underlie this heterogeneous organization of D2 receptors, we took advantage of quantitative autoradiography to examine the association of these receptors with those elements. The present findings show that the D2 sites are almost exclusively located on neurons whose somata reside in the caudate-putamen and are not located on terminals of corticostriatal axons. A detailed comparison of the distribution of histochemically identified acetylcholinesterase neurons with that of D2 receptors in serially adjoining sections suggests a common organizational pattern. The density of [3H]spiroperidol sites in rat caudate-putamen was determined after unilateral injection of the neurotoxin quinolinic acid into this structure or after ablation of neocortical regions. Quantification of the tissue damage was achieved by acetylcholinesterase histochemistry (following diisopropylfluorophosphate treatment), as well as by thionin and luxol fast staining of sections adjacent to those used for [3H]spiroperidol autoradiography. In identically treated animals, biochemical determination of the extent of tissue damage was made utilizing assays for high-affinity [3H]choline and [3H]glutamate uptake in the caudate-putamen. In quinolinic acid-injected rats, the density of D2 sites was decreased by 90-95% at the site of complete loss of large acetylcholinesterase-positive neurons. Other animals, given ablations of specific neocortical fields (medial prefrontal, motor, somatosensory) or of the entire parietal-frontal cortex of one hemisphere, showed no loss of caudate-putamen D2 sites unless the cortical ablation caused accompanying damage of the caudate-putamen. In the caudate-putamen of all animals there was a close correspondence between the D2 sites and the striatal neurons (and processes) that show strong acetylcholinesterase reactivity. We suggest that the caudate-putamen topography of D2 sites is based largely on the internal organization of this structure and may preferentially involve acetylcholine-containing intrinsic neurons.

In situ analysis of neostriatal RNA and chromatin in Alzheimer's disease.

Scanning-integrating microdensitometry of azure B-RNA- and Feulgen-Schiff-stained tissue sections was used to measure neostriatal neuronal RNA levels and susceptibility of neuronal and oligodendrocyte chromatin to acid hydrolysis in Alzheimer's disease (AD) patients and controls. AD was associated with neuronal RNA depletion (17 to 23%) in both caudate nucleus and putamen. While neuronal chromatin was found to be more acid-labile than that of the oligodendrocytes, there were no differences in either cell type between AD and controls. These data support the existence of a macromolecular disturbance (RNA loss) occurring within neostriatal neurons, perhaps related to the extrapyramidal dysfunction of AD, but fail to demonstrate that an alteration in chromatin is responsible for this effect.

Age-related decreases in dopamine receptors in the caudate nucleus and putamen of the rhesus monkey (Macaca mulatta).

Specific binding of the dopamine receptor ligand 3H-spiroperidol to cell membranes prepared from the caudate nuclei and putamens of 29 rhesus monkeys (M. mulatta), ranging in age from 2 to 22 years, was investigated. Receptor concentration (Bmax) decreased in the caudate nucleus and putamen with age at mean rates of 2.1 and 1.7% per year, respectively, whereas binding affinity (Kd) did not change significantly with age. The rate of decline in Bmax appeared to be more rapid before adulthood and in old age than during young adulthood and middle age. These data are compared with the results from similar studies of other animal species including human, rabbit, rat, and mouse. The rate of decline in striatal dopamine receptors is closely related to the rate of aging and maximal life span of the species. It may reflect both maturational and senescent processes modifying the behavior of animals as they age.

HPLC analysis of somatostatin related peptides in putamen of Huntington's disease patients.

Size exclusion high performance liquid chromatography of post mortem human putamen samples revealed three somatostatin-immunoreactive peaks. The two major peaks cochromatographed with synthetic somatostatin-14 and somatostatin-28, respectively whereas the presumed somatostatin precursor preceded these peaks. In 11 samples obtained post mortem from patients suffering from Huntington's Disease somatostatin-like immunoreactivity was increased by about 55% in the mean. This increase was mainly due to increases in somatostatin-14 and--to a minor extent--of somatostatin-28, whereas the presumed precursor was not significantly changed.

3-Methoxytyramine in the putamen as a gauge of the postmortem interval.

The armamentarium of the forensic thanatologist includes the examiner's subjective assessment of bodily changes together with comprehensive evaluation of environmental and associated factors to estimate the postmortem interval (PMI). Of the various objective means, postmortem accumulation of potassium in vitreous humor is a widely used gauge. In view of the considerable variability inherent in these techniques, an additional marker with greater accuracy would be a welcome supplement to these indices. We have developed a method of estimating PMI from the level of 3-methoxytyramine (3-MT) in the dorsal putamen of the brain. The supernatant of a sample of putamen, sonicated in 6 volumes (weight/volume) of 0.1 M perchloric acid and centrifuged at 51 000 times gravity for 10 min, was analyzed for 3-MT using high-pressure liquid chromatography with electrochemical detection. The change of 3-MT in the putamen is linearly related to the PMI (range: 5.5 to 60 h), irrespective of patient age (1 to 84 years). This observation is attributable to several factors, including high substrate (dopamine) concentration, preservation of catechol-O-methyl transferase activity, and an inactivation of monoamine oxidase activity, as a result of a decrease in tissue pO2.

[CRF-like immunoreactivities in mammalian brains examined with a radioimmunoassay for CRF].

A sensitive and useful radioimmunoassay for CRF was established using synthetic ovine CRF. CRF-like immunoreactivities of hypothalami in the bovine, human, rat, guinea pig, and mouse could be detected with this radioimmunoassay. A variety of CRF-like immunoreactivities of hypothalami in the mammalian brains led us to discuss a putative species difference of amino-acid structure in CRF. In the bovine, CRF-like immunoreactivities of the frontal cortex, caudate nucleus, putamen, and hippocampus could be detected in addition to CRF-like immunoreactivity of hypothalamus. These observations suggested a wide distribution of CRF in the central nervous system, and gave room for discussion about a physiological role of extrahypothalamic CRF. Gel chromatographic studies revealed a high-molecular-weight form of CRF-like immunoreactivity in the bovine frontal cortical profile that was not found in the hypothalamic profile. The results show a multiplicity of CRF-like immunoreactivity in the bovine brain.