Huntingtin inclusions do not down-regulate specific genes in the R6/2 Huntington's disease mouse.

Transcriptional dysregulation is a central pathogenic mechanism in Huntington's disease (HD); HD and transgenic mouse models of HD demonstrate down-regulation of specific genes at the level of mRNA expression. Furthermore, neuronal intranuclear inclusions (NIIs) have been identified in the brains of R6/2 mice and HD patients. One possibility is that NIIs contribute to transcriptional dysregulation by sequestering transcription factors. We therefore assessed the relationship between NIIs and transcriptional dysregulation in the R6/2 mouse, using double-label in situ hybridization combined with immunohistochemistry, and laser capture microdissection combined with quantitative real-time PCR. There was no difference in transcript levels of specific genes between NII-positive and NII-negative neurons. These results demonstrate that NIIs do not cause decreases in D2, PPE and PSS mRNA levels in R6/2 striatum and therefore are not involved in the down-regulation of these specific genes in this HD model. In addition, these observations argue against the notion that NIIs protect against transcriptional dysregulation in HD.

Gene expression profiling in the post-mortem human brain--no cause for dismay.

Global expression profiling techniques such as microarray technology promise to revolutionize biology. Soon it will be possible to investigate alterations at the transcript level of the entire human genome. There is great hope that these techniques will at last shed light on the pathological processes involved in complex neuropsychiatric disorders such as schizophrenia. These scientific advances in turn have re-kindled a great interest and demand for post-mortem brain tissue. Good quality post-mortem tissue undoubtedly is the fundamental prerequisite to investigate complex brain disorders with molecular profiling techniques. In this review we show that post-mortem brain tissue can yield good quality mRNA and intact protein antigens which allow the successful application of traditional molecular biology methods as well as novel profiling techniques. We also consider the use of laser-capture microdissection on post-mortem tissue. This recently developed technique allows the experimenter to explore the molecular basis of cellular function at the single cell level. The combination of laser-capture microdissection with high throughput profiling techniques offers opportunities to obtain precise genetic fingerprints of individual neurons allowing comparisons of normal and pathological states.

New insights into progressive supranuclear palsy.

Increased oxidative damage and mitochondrial dysfunction have been suggested to play crucial roles in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. In this review, we will focus on progressive supranuclear palsy (PSP), a rare parkinsonian disorder with tau pathology. Particular emphasis is placed on the genetic and biochemical data that has emerged, offering new perspectives into the pathogenesis of this devastating disease, especially the contributory roles of oxidative damage and mitochondrial dysfunction.

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.

Expression of alpha-synuclein, parkin, and ubiquitin carboxy-terminal hydrolase L1 mRNA in human brain: genes associated with familial Parkinson's disease.

Mutations in the alpha-synuclein, parkin, and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) genes have been linked to some cases of familial Parkinson's disease. To provide insight into how these genes may relate to each other and contribute to the pathology of Parkinson's disease, their expression was examined in normal human brain. Tissue sections from multiple regions of 11 normal human brains were hybridized with radiolabeled and digoxygenin-labeled cRNA probes for alpha-synuclein, parkin, and UCH-L1 mRNA. Expression of each of these three genes was predominantly neuronal. Alpha-synuclein and parkin mRNAs were expressed in a restricted number of brain regions, whereas UCH-L1 mRNA was more uniformly expressed throughout brain. The melanin-containing dopamine neurons of the substantia nigra had particularly robust expression. The expression patterns of alpha-synuclein and parkin mRNAs were similar, suggesting that these two proteins may be involved in common pathways contributing to the pathophysiology of Parkinson's disease.

Frontal lobe dysfunction in progressive supranuclear palsy: evidence for oxidative stress and mitochondrial impairment.

Recent data from our laboratory have shown a regionally specific increase in lipid peroxidation in postmortem progressive supranuclear palsy (PSP) brain. To extend this finding, we measured activities of mitochondrial enzymes as well as tissue malondialdehyde (MDA) levels in postmortem superior frontal cortex (Brodmann's area 9; SFC) from 14 pathologically confirmed cases of PSP and 13 age-matched control brains. Significant decreases (-39%) in alpha-ketoglutarate dehydrogenase complex/glutamate dehydrogenase ratio and significant increases (+36%) in tissue MDA levels were observed in the SFC in PSP; no differences in complex I or complex IV activities were detected. Together, these results suggest that mitochondrial dysfunction and lipid peroxidation may underlie the frontal metabolic and functional deficits observed in PSP.

Distribution of the mRNAs encoding torsinA and torsinB in the normal adult human brain.

To gain insight into the neural pathways involved in the pathogenesis of DYT1 dystonia, we have mapped the cellular expression of the mRNA encoding torsinA and the closely related family member, torsinB, in normal adult human brain. Here, we report an intense expression of torsinA mRNA in the substantia nigra pars compacta dopamine neurons, the locus ceruleus, the cerebellar dentate nucleus, Purkinje cells, the basis pontis, numerous thalamic nuclei, the pedunculopontine nucleus, the oculomotor nucleus, the hippocampal formation, and the frontal cortex. Within the caudateputamen, the cellular expression of torsinA mRNA was heterogeneous; a moderate signal was found overlying large cholinergic neurons, and most striatal neurons exhibited only a very weak signal. A moderate signal was detected in numerous midbrain and hindbrain nuclei. A weak cellular signal was detected in neurons of the globus pallidus and subthalamic nucleus. In marked contrast to torsinA, no specific mRNA signal was detected for torsinB. That torsinA mRNA is enriched in several basal ganglia nuclei, including the dopamine neurons in the substantia nigra, is intriguing since it suggests that DYT1 dystonia may be associated with a dysfunction in dopamine transmission.

Measurement of GABAergic parameters in the prefrontal cortex in schizophrenia: focus on GABA content, GABA(A) receptor alpha-1 subunit messenger RNA and human GABA transporter-1 (HGAT-1) messenger RNA expression.

The hypothesis that the pathophysiology of schizophrenia may be associated with a dysfunction in GABA transmission in the human prefrontal cortex was investigated. Human post mortem brain tissue from 10 control cases and six cases of schizophrenia were processed for amino acid analysis and for radioactive in situ hybridization. Laminae III and V of three prefrontal cortical areas were examined in detail, namely Brodmann areas 9, 10 and 11. Of these three areas significant changes in GABAergic markers were found only in areas 9 and 10. Of note, a significant decrease in the tissue content of GABA was observed and this was accompanied by a marked increase in the cellular expression of the GABA(A) receptor alpha-1 subunit messenger RNA and a marked decrease in the expression of human GABA transporter-1, the messenger RNA encoding the neuronal GABA transporter protein. The amino acid analysis data provided in this study coupled with the detailed cellular study of several GABAergic markers in the human prefrontal cortex provide direct evidence in support of a disturbance in GABA transmission in the prefrontal cortex, which may be loosely termed "hypofrontality".

Evidence for oxidative stress in the subthalamic nucleus in progressive supranuclear palsy.

Increased free radical production and oxidative stress have been proposed as pathogenic mechanisms in several neurodegenerative disorders. Free radicals interact with biological macromolecules, such as lipids, which can lead to lipid peroxidation. A well-established marker of oxidative damage to lipids is malondialdehyde (MDA). We measured tissue MDA levels in the subthalamic nucleus (STN) and cerebellum from 11 progressive supranuclear palsy (PSP) cases and 11 age-matched control cases using sensitive HPLC techniques. In PSP, a significant increase in tissue MDA levels was observed in the STN when compared with the age-matched control group. By contrast, no significant difference between tissue MDA content was observed in cerebellar tissue from the same PSP and age-matched control cases. These results indicate that lipid peroxidation may play a role in the pathogenesis of PSP.

Localization of calcium-binding proteins and GABA transporter (GAT-1) messenger RNA in the human subthalamic nucleus.

The distribution of messenger RNA encoding the human GAT-1 (a high-affinity GABA transporter) was investigated in the subthalamic nucleus of 10 neurologically normal human post mortem cases. Further, the distribution of messenger RNA and protein encoding the three neuronally expressed calcium-binding proteins (calbindin D28k, parvalbumin and calretinin) was similarly investigated using in situ hybridization and immunohistochemical techniques. Cellular sites of calbindin D28k, parvalbumin, calretinin and GAT-1 messenger RNA expression were localized using human-specific oligonucleotide probes radiolabelled with [35S]dATP. Sites of protein localization were visualized using specific anti-calbindin D28k, anti-parvalbumin and anti-calretinin antisera. Examination of emulsion-coated tissue sections processed for in situ hybridization revealed an intense signal for GAT-1 messenger RNA within the human subthalamic nucleus, indeed the majority of Methylene Blue-counterstained cells were enriched in this transcript. Further, a marked heterogeneity was noted with regard to the expression of the messenger RNA's encoding the three calcium-binding proteins; this elliptical nucleus was highly enriched in parvalbumin messenger RNA-positive neurons and calretinin mRNA-positive cells but not calbindin messenger RNA-positive cells. Indeed, only an occasional calbindin messenger RNA-positive cell was detected within the mediolateral extent of the nucleus. In marked contrast, numerous parvalbumin messenger RNA-positive cells and calretinin messenger RNA-positive cells were detected and they were topographically distributed; parvalbumin messenger RNA-positive cells were highly enriched in the dorsal subthalamic nucleus extending mediolaterally; calretinin messenger RNA-positive cells were more enriched ventrally although some degree of overlap was apparent. Computer-assisted analysis of the average cross-sectional somatic area of parvalbumin, calretinin and GAT-1 messenger RNA-positive neurons revealed them all to be in the range of 300 microm2. The unique patterns of calcium-binding protein gene expression were similarly reflected at the protein level; an abundance of parvalbumin- and calretinin-immunopositive neurons were observed whereas only occasional intensely-labelled calbindin-immunopositive fibres were seen, no calbindin-immunopositive cells were detected. Single and double labelling studies show that parvalbumin-immunopositive neurons were mainly localized in the dorsal region of the nucleus, and calretinin-immunopositive neurons were mainly localized in the ventral region although there was overlap with double-labelled neurons located in the middle and dorsal regions. The significance of these findings, in particular the expression of GAT-1, a high-affinity GABA uptake protein, for basal ganglia signalling is discussed.

Expression of the human excitatory amino acid transporter 2 and metabotropic glutamate receptors 3 and 5 in the prefrontal cortex from normal individuals and patients with schizophrenia.

A disturbance of glutamatergic transmission has been suggested to contribute to the development of schizophrenic pathophysiology based primarily on the ability of glutamate receptor antagonists to induce schizophrenic-like symptoms, and recent studies suggesting reduced glutamatergic function in the prefrontal cortex (PFC) of individuals with a diagnosis of schizophrenia. In order to investigate this hypothesis further, the expression of several 'glutamatergic' markers, the metabotropic glutamate receptors (mGluRs; mGluR3, 5) and the human excitatory amino acid transporter (EAAT2) were compared in the PFC of normal individuals and schizophrenics. The present results showed that glial cells in the pyramidal layers of the PFC from schizophrenics had decreased EAAT2 mRNA content relative to controls in Brodmann areas 9 and 10. The cellular levels of expression of the two mGluR signals investigated (mGluR3, and 5) were not significantly changed relative to controls except for an increase in the neuronal mGluR5 in the pyramidal cell layers of area 11. Comparing the ratio of cellular mGluR expression to that of EAAT2, the mGluR/EAAT2 ratio showed that schizophrenics had a significantly increased mGluR/EAAT2 ratios in the pyramidal cell layers of all three PFC regions examined. The glutamate content of consecutive sections analyzed by high pressure liquid chromatography (HPLC), although decreased in schizophrenics did not reach significance and did not correlate with either EAAT2 or mGluR mRNA content. These results are discussed in the light of current results on the neurochemistry and pharmacology of schizophrenia.

Expression of the early-onset torsion dystonia gene (DYT1) in human brain.

Early-onset torsion dystonia, an autosomal dominant disease associated with the DYT1 locus on 9q34, is the most frequent genetic form of dystonia. Recent work has revealed that the causative mutation in most cases is deletion of a glutamate residue from the carboxy terminal of torsinA, a 332 amino acid protein encoded by the DYT1 gene. To gain insight into how deletion of a single amino acid can produce such a profound movement disorder, we have mapped the expression of the DYT1 gene in normal human postmortem brain. DYT1 mRNA is highly enriched in the dopamine neurons of the substantia nigra pars compacta. Intense expression was also found in the cerebellum and hippocampal subfields. The prominent expression of the DYT1 gene within the substantia nigra pars compacta, which provides dopaminergic innervation to the basal ganglia, implicates a disturbance of dopaminergic function in the pathophysiology of early-onset torsion dystonia.

Contrasting effects of excitotoxic lesions of the prefrontal cortex on the behavioural response to D-amphetamine and presynaptic and postsynaptic measures of striatal dopamine function in monkeys.

The effects of excitotoxic lesions of the prefrontal cortex on behavioural, neurochemical and molecular indices of dopamine function in the caudate nucleus were studied in the marmoset. The lesion, which encompassed both the lateral and orbital regions of prefrontal cortex, made the animals more sensitive to the performance disrupting effects of the dopamine releasing drug, D-amphetamine, in a variation of the object retrieval task. Specifically, following drug administration, the lesioned marmosets were less able to gain access to food reward in the minimum number of responses. Analysis of the nature of the errors suggested that the deficit was not due to inhibition of a prepotent response as the lesioned monkeys were just as likely to make a detour reach to the unopened side of the box as a direct "line-of-sight" reach into the unopened front of the box. Rather, the data indicated a general disorganization of behaviour. The enhanced behavioural responsiveness to manipulations increasing presynaptic dopamine function was accompanied by neurochemical changes indicating a reduced responsiveness, as revealed by in vivo microdialysis. Thus, in lesioned animals, whilst there were no effects on baseline levels of extracellular dopamine in dorsolateral caudate, evoked release, both to systemic D-amphetamine and to a local depolarizing pulse of potassium ions, was attenuated. These opposite effects of the prefrontal cortex lesion on behavioural and neurochemical indices of striatal dopamine function occurred in the absence of any changes in striatal dopamine receptors of the D1 and D2 subtype, as determined both by radioligand binding assays and measurements of messenger RNA using in situ hydridization techniques. These data provide further insight into the interactions between prefrontal cortex and striatal dopamine function in the non-human primate. In particular, when taken in the light of our previous studies they indicate that following prefrontal manipulations, concurrence between behavioural and neurochemical indices of striatal dopamine function depends, critically, on the behavioural task. These findings are discussed with respect to the growing body of evidence implicating abnormalities in frontostriatal neurotransmission in complex disorders such as schizophrenia.

Dopamine D1 and D2 receptor gene expression in the striatum in Huntington's disease.

The cellular expression of dopamine D1 and D2 receptor mRNAs was investigated in the postmortem human caudate nucleus of control cases and genetically and pathologically confirmed cases of Huntington's disease (HD) by using quantitative in situ hybridization. The HD cases were categorized (0-4) by severity of striatal neuropathology according to the Vonsattel scale. For the HD grade 0 case, a pronounced reduction in the number of D1 and D2 mRNA-positive cells was observed compared with controls; however, the abundance of both receptor mRNAs per remaining cell was within the control range. For D2 receptor mRNA, the number of detectable D2-positive medium-sized cells decreased with increasing pathology; this decrease was accompanied by a gradual reduction in the intensity of D2 signal per cell. By contrast, for D1 receptor mRNA, despite a decrease in the number of D1 mRNA-positive cells detected, the average cellular expression of D1 mRNA was markedly reduced in the HD grade 1 case and then increased (relative to the grade 1 case) with increasing pathology, presumably reflecting the relative survival of D1-expressing striatal interneurons. The implications of these findings for providing further information on the neurodegenerative process in HD are discussed.

Monoamine transporter gene expression in the central nervous system in diabetes mellitus.

Diabetic animals exhibit altered neurotransmission in brain monoaminergic systems. By means of in situ hybridization, we have investigated the expression of dopamine, noradrenaline, and serotonin transporter (DA-T, NA-T, and 5-HT-T, respectively) mRNAs in the brains of alloxan- and streptozotocin-diabetic rats. The expression of DA-T mRNA is decreased in 1- (-11%) and 4- (-17%) week alloxan-diabetic and 4- (-9%) and 8- (-20%) week streptozotocin-diabetic rats in the ventral medial bundle. The expression of NA-T mRNA is decreased in the locus coeruleus of 8- (-26%) week streptozotocin-diabetic rats, in the noradrenergic A1 cell group of 4- (-27%) week alloxan- and 8- (-25%) week streptozotocin-diabetic rats, and in the noradrenergic A2 cell group of 1- (-21%) and 4- (-28%) week alloxan-diabetic and 4- (-27%) and 8- (-25%) week streptozotocin-diabetic animals. The expression of 5-HT-T mRNA in the dorsal raphe nucleus is increased in 1- (+14%) and 4- (+44%) week alloxan- and 4- (+28%) and 8- (+44%) week streptozotocin-diabetic rats. The expression of each of the three monoamine transporter genes may be differentially regulated in diabetes and dependent on the duration of diabetes. Altered monoamine transporter gene expression may possibly contribute to the observed dysfunctions in brain monoamine transmission in chronic diabetes.

Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study.

The chemical phenotype of proneurotensin messenger RNA-expressing cells was determined in the acute haloperidol-treated rat striatum using a combination of (35S)-labelled and alkaline phosphatase-labelled oligonucleotides. Cellular sites of proneurotensin messenger RNA expression were visualized simultaneously on tissue sections processed to reveal cellular sites of preproenkephalin A messenger RNA or the dopamine and adenylate cyclase phosphoprotein-32, messenger RNA. The cellular co-expression of preproenkepahlin A (enkephalin) and preprotachykinin (substance P) messenger RNA was also examined within forebrain structures. Cellular sites of enkephalin (substance P) and dopamine and adenylate cyclase phosphoprotein-32 messenger RNAs were visualized using alkaline phosphatase-labelled oligonucleotides whilst sites of substance P and proneurotensin messenger RNA expression were detected using (35S)-labelled oligos. Cellular sites of enkephalin and dopamine and adenylate cyclase phosphoprotein-32 gene expression were identified microscopically by the concentration of purple alkaline phosphatase reaction product within the cell cytoplasm, whereas sites of substance P and proneurotensin gene expression were identified by the dense clustering of silver grains overlying cells. An intense hybridization signal was detected for all three neuropeptide messenger RNAs in the striatum, the nucleus accumbens and septum. Dopamine and adenylate cyclase phosphoprotein-32 messenger RNA was detected within the neostriatum but not within the septum. In all forebrain regions examined, with the exception of the islands of Calleja, the cellular expression of enkephalin messenger RNA and substance P messenger RNA was discordant; the two neuropeptide messenger RNAs were detected essentially in different cells, although in the striatum and nucleus accumbens occasional isolated cells were detected which contained both hybridization signals; dense clusters of silver grains overlay alkaline phosphatase-positive cells, demonstrating clearly that these dual-labelled cells expressed both messenger RNAs. By contrast, the hybridization signals for proneurotensin and enkephalin, and proneurotensin and dopamine and adenylate cyclase phosphoprotein-32 were generally coincident, at least within the neostriatum; most proneurotensin messenger RNA-positive cells expressed enkephalin messenger RNA and were also positive for dopamine and adenylate cyclase phosphoprotein-32 messenger RNA. However, occasional proneurotensin messenger RNA-positive striatal cells were identified that were single-labelled and did not express enkephalin messenger RNA. Within the septal nucleus, enkephalin messenger RNA and substance P messenger RNA were expressed essentially within segregated cell populations. These studies illustrate further the utility of co-expression techniques for investigating the chemical phenotype of cells within the CNS and demonstrate that the distribution of neuropeptide co-expressing cells is different within different brain regions. That several populations of proneurotensin messenger RNA-positive striatal cells may exist, of which one population is sensitive to haloperidol, co-expresses enkephalin messenger RNA and is positive for dopamine and adenylate cyclase phosphoprotein-32 messenger RNA may be of some significance in neuropsychiatric/neurological disorders given that the translated peptide, neurotensin, is known to influence and interact closely with the dopamine systems.

Effects of L-DOPA-therapy on dopamine D2 receptor mRNA expression in the striatum of MPTP-intoxicated parkinsonian monkeys.

The cellular expression of dopamine D2 receptor mRNA was examined in striatal (caudate nucleus and putamen) neurones of 9 Macaca fascicularis monkeys rendered parkinsonian by systemic injection of MPTP. Messenger RNA abundance was determined by quantitative in situ hybridization using human-specific 35S-labelled oligonucleotides. Control monkeys were untreated and received neither MPTP nor L-DOPA while the rest were rendered parkinsonian and received chronic levodopa therapy to induce dyskinesia. In the control brains a strong dopamine D2 receptor hybridization signal was detected overlying medium-sized and some large neurons in both the caudate nucleus and putamen. Neurons from the lateral and medial regions of the caudate nucleus, and from the dorsal and ventral regions of the putamen were analysed separately. A significant increase in the cellular abundance of dopamine D2 receptor mRNA was seen in the striatum of MPTP-treated monkeys; this increase being restricted to the population of medium-sized striatal cells. No such increase in dopamine D2 receptor mRNA was observed in (dyskinetic) L-DOPA-treated monkeys suggesting that levodopa-therapy normalises D2 receptor expression in post-synaptic striatal cells. The cellular abundance of dopamine D2 receptor mRNA expressed by large striatal neurons (putative cholinergic cells) was unaffected by either MPTP treatment or levodopa therapy. The implications of these findings for the development of levodopa-induced dyskinesias is discussed.

Reduction in enkephalin and substance P messenger RNA in the striatum of early grade Huntington's disease: a detailed cellular in situ hybridization study.

The expression of enkephalin and substance P messenger RNAs was examined in the caudate-putamen of human post mortem tissue from control and Huntington's disease tissue using in situ hybridization techniques and human specific enkephalin and substance P [35S] oligonucleotides. Macroscopic and microscopic quantification of enkephalin and substance P gene expression was carried out using computer-assisted image analysis. Tissue was collected from six control cases with no sign of neurological disease and six Huntington's disease cases ranging from grades 0 to 3 as determined by neuropathological evaluation. The clinical and pathological diagnosis of Huntington's disease was confirmed unequivocally by genetic analysis of the CAG repeat length in both copies of IT15, the Huntington's disease gene. A marked reduction in both enkephalin and substance P messenger RNAs was detected in all regions of the caudate nucleus and putamen in Huntington's disease grades 2/3 when compared to controls; in the dorsal caudate few enkephalin or substance P messenger RNA-positive cells were detected. For the early grade (0/1) Huntington's disease cases, a heterogeneous reduction in both enkephalin and substance P messenger RNAs were noted; for enkephalin messenger RNA the striatal autoradiograms displayed a conspicuous patchy appearance. Detailed cellular analysis of the dorsal caudate revealed a striking reduction in the number of enkephalin and substance P messenger RNA-positive cells detected and in the intensity of hybridization signal/cell. These data suggest that both the "indirect" GABA/enkephalin and "direct" GABA/substance P pathways are perturbed very early in the course of the disease and that these early changes in chemical signalling may possibly underlie the onset of clinical symptoms.