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To examine the consequences of nigrostriatal denervation and chronic levodopa (L-DOPA) treatment on functional activity of the basal ganglia, we analyzed, using in situ hybridization, the cellular expression of the mRNA encoding for cytochrome oxidase subunit I (COI mRNA), a molecular marker for functional neuronal activity, in the basal ganglia. This analysis was performed in monkeys rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Intoxication, some of which had been receiving L-DOPA, and in patients with Parkinson's disease (PD). In MPTP-intoxicated monkeys compared with control animals, COI mRNA expression was increased in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia, i.e., the internal segment of the globus pallidus and the substantia nigra pars reticulata. This increase was partially reversed by L-DOPA treatment. COI mRNA expression remained unchanged in the external segment of the globus pallidus (GPe). In PD patients, all of whom had been treated chronically by L-DOPA, COI mRNA expression in the analyzed basal ganglia structures was similar to that in control subjects. These results are in agreement with the accepted model of basal ganglia organization, to the extent that the output nuclei of the basal ganglia are considered to be overactive after nigrostriatal denervation, partly because of increased activity of excitatory afferents from the STN. Yet, our results would also seem to contradict this model, because the overactivity of the STN does not seem to be attributable to a hypoactivation of the GPe.

The mRNA levels encoding for the enzyme glutamate decarboxylase (GAD67) were measured by computerized image analysis after in situ hybridization histochemistry and radioautography in the striatum and pallidum of normal squirrel monkeys (Saimiri sciureus), or after treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). All MPTP-injected monkeys exhibited profound motor deficits including akinesia. The dopaminergic innervation, as visualized and quantified on x-ray films after 3H-mazindol binding on tissue sections, was uniformly lost throughout the striatum of MPTP-treated monkeys. Brain sections processed with a probe synthesized from a feline or human GAD67 cDNA exhibited intense radioautographic labeling throughout the striatum. When measured on x-ray films, the intensity of GAD67 mRNA labeling was increased in the striatum of MPTP-treated versus control monkeys. Increased labeling reached statistical significance in the dorsolateral sector of the rostral putamen and throughout the putamen and the caudate at the caudal, postcommissural, level. Analysis of emulsion radioautographs demonstrated that the increase in GAD67 mRNA labeling in MPTP-treated monkeys occurred in individual neurons of the striatum. In the external and internal segments of the pallidum, numerous neurons labeled with the GAD67 cRNA probe were visualized on emulsion radioautographs. The intensity of GAD67 mRNA labeling in single neurons of both pallidal segments was increased in MPTP-treated versus control monkeys. Construction of the histograms of frequency distribution of labeling indicated that this increase occurred in a majority of labeled neurons. The present study demonstrates that GAD67 mRNA levels are significantly altered in the striatum and pallidum of parkinsonian monkeys. The preferential increase of GAD67 mRNA labeling in the dorsolateral putamen, which receives afferents from the sensorimotor cortex, provides further evidence of the involvement of GABAergic transmission in the expression of the motor deficits elicited after MPTP. In addition, increased GAD67 mRNA levels in the internal segment of the pallidum support the hypothesis of an increased activity of GABAergic neurons in the output structures of the basal ganglia in parkinsonism.

The cholinergic structures in the forebrain of the rat were studied immunohistochemically with a monoclonal antibody to choline acetyltransferase (ChAT) which has proven to be particularly suited to reveal the trajectory of ChAT-positive fibers and their terminations. The habenulo-interpeduncular tract contained the most distinctly ChAT-positive fibers. Here, ChAT-positivity was demonstrated in the perikarya, fibers and terminal field. From the basal forebrain, ChAT-positive fiber bundles could be followed rostrally to the olfactory bulb, dorsomedially to the neocortex and hippocampus, and dorsocaudally to the hippocampus. Terminations of ChAT-positive fibers in these regions were either boutons terminaux (in the interpeduncular nucleus), finely granular (in the hippocampus) or a network of varicose fibers (in the olfactory bulb and neocortex). Cholinergic terminal fields were also demonstrated in the following regions: the nucleus of the lateral olfactory tract, the olfactory tubercle, the pyriform cortex, the lateral nucleus and part of the basal nucleus of the amygdala, the caudate-putamen, the nucleus accumbens, some thalamic nuclei, and the suprachiasmatic nucleus of the hypothalamus. In most of these regions, ChAT-positivity appeared as either fine granules or a meshwork of varicose fibers.

We have developed a novel fluorescent histochemical method to localize the enzyme neutral endopeptidase-24.11 (NEP, E.C. 3.4.24.11, enkephalinase) in the rat brain in order to directly compare the relative distributions of the enzyme and its putative peptide substrate, the enkephalins. The method is based on the sequential cleavage of the synthetic peptide substrate, glutaryl-alanyl-alanyl-phenylanyl-4-methoxy-2-naphthylamide, by NEP and exogenous aminopeptidase M to yield free 4-methoxy-2-naphthylamine (MNA). In the presence of nitrosalicylaldehyde, free MNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of NEP activity. The specificity of the method was demonstrated using the selective NEP inhibitors thiorphan, phosphoramidon, and JHF26. All NEP staining throughout the brain was abolished using a 50-nM concentration of these inhibitors. The enzyme was richly localized to many regions, including the cerebral cortex, caudate putamen, globus pallidus, hippocampus, substantia nigra, periaqueductal gray, several cranial nerve nuclei, nuclei of the reticular formation of the medulla. In most regions, reaction product was associated with cell bodies of varying size and morphology. In a number of regions, colchicine increased the amount of NEP staining, particularly in cell processes. The regional distribution pattern of the enzyme, however, did not change in response to colchicine and was similar to that of untreated animals. The histochemical localization of NEP was combined with fluorescent immunocytochemical visualization of the enkephalins in order to localize both in the same tissue section. In the globus pallidus, this combined fluorescent technique revealed numerous NEP-positive cell bodies surrounded by fiber pathways displaying intense enkephalin-like immunoreactivity. The source of the NEP in the globus pallidus was studied using the neurotoxic agent, N-methyl-D-aspartate (NMDA). A pronounced decrease in NEP cellular staining was observed within 7 d in response to NMDA, persisted for at least 16 weeks, and correlated with injury of pallidal neurons. There was no apparent change in enkephalin-like immunoreactivity in the globus pallidus in response to NMDA. These data provide evidence that NEP and enkephalin in the globus pallidus derive from different sources. This study supports the hypothesis that NEP localizes to enkephalin-rich regions of the rat brain, and that the enzyme may be involved in the inactivation of synaptically released enkephalins.