Comparative distribution of messenger RNAs encoding glutamic acid decarboxylases (Mr 65,000 and Mr 67,000) in the basal ganglia of the rat.

Glutamic acid decarboxylase, the enzyme required for GABA synthesis, exists as distinct isoforms, which have recently been found to be encoded by different genes. The relative expression of messenger RNAs encoding two isoforms of glutamic acid decarboxylase (Mr 67,000 and Mr 65,000) was measured at the single-cell level in neurons of the rat basal ganglia with in situ hybridization histochemistry. Both messenger RNAs were expressed in neurons of the striatum, pallidum, and substantia nigra pars reticulata, but marked differences in the relative level of labelling were observed with the two probes. In striatum, efferent neurons were more densely labelled for the messenger RNA encoding glutamic acid decarboxylase (Mr 65,000) than for the messenger RNA encoding glutamic acid decarboxylase (Mr 67,000), whereas the reverse was observed for GABA-ergic interneurons. Neurons of the entopeduncular nucleus were much more densely labelled for messenger RNA encoding glutamic acid decarboxylase (Mr 65,000) than for messenger RNA encoding glutamic acid decarboxylase (Mr 67,000). In addition, labelling for messenger RNA encoding glutamic acid decarboxylase (Mr 65,000) was higher in the entopeduncular nucleus (internal pallidum) than in the globus pallidus (external pallidum), a structure which expressed similar levels of both mRNAs. In contrast to neurons of the internal pallidum, efferent neurons of the substantia nigra pars reticulata expressed slightly more messenger RNA encoding glutamic acid decarboxylase (Mr 67,000) than that encoding the other isoform of the enzyme. The results suggest a differential expression of the messenger RNAs encoding the two isoforms of glutamic acid decarboxylase in subpopulations of basal ganglia neurons in rats.     

Subpopulations of mesencephalic dopaminergic neurons express different levels of tyrosine hydroxylase messenger RNA.

Subpopulations of mesencephalic dopamine containing neurons possess different electrophysiological, pharmacological, biochemical, and anatomical properties. In order to determine whether such differences are related to the regulation of tyrosine hydroxylase, the rate limiting enzyme in the synthesis of catecholamines, the regional distribution of tyrosine hydroxylase messenger RNA in these neurons was examined using in situ hybridization histochemistry. In the mouse, labelling for tyrosine hydroxylase messenger RNA associated with individual neurons was significantly less in the lateral substantia nigra pars compacta than in the medial substantia nigra pars compacta and the ventral tegmental area. A similar pattern of labelling was observed in the rat. Labelling for tyrosine hydroxylase messenger RNA was significantly less in the lateral substantia nigra pars compacta than in medial pars compacta (a densely cellular region), the area dorsal to the medial substantia nigra pars compacta (a less cell dense region), and the ventral tegmental area. Differences in levels of labelling for messenger RNA in mesencephalic dopamine neurons were not related to differences in cell size as measured in sections processed for tyrosine hydroxylase immunohistochemistry. The results suggest that tyrosine hydroxylase messenger RNA is differentially regulated in subpopulations of mesencephalic dopamine neurons, supporting the view that these neurons are physiologically distinct.     

Messenger RNAs encoding glutamate-decarboxylases are differentially affected by nigrostriatal lesions in subpopulations of striatal neurons.

Dopaminergic nigrostriatal neurons constitute one of the major inputs to the striatum, and play a role in the regulation of gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD), the GABA-synthesizing enzyme, in striatal neurons. The effect of nigrostriatal lesions on the level of expression of messenger RNAs encoding two distinct isoforms of glutamate decarboxylase was examined at the single cell level with in situ hybridization histochemistry. Rats received a unilateral injection of the neurotoxin 6-hydroxydopamine in the substantia nigra and were sacrificed 2 or 3 weeks later. Sections of the striatum were processed for in situ hybridization histochemistry with radiolabeled RNA probes selective for mRNAs encoding glutamate decarboxylase with molecular weights of 65,000 and 67,000, respectively. In addition, immunohistochemistry with a monospecific antibody for the latter glutamate decarboxylase isoform was performed. In agreement with previous reports, we observed increased labeling for the messenger RNA encoding glutamate decarboxylase (M(r) 67,000) in a population of medium-sized striatal efferent neurons normally expressing low levels of this messenger RNA. We now show that this effect occurred in two striatal compartments, the striosomes and the extrastriosomal matrix, and was accompanied by increased immunostaining for the corresponding protein with a monospecific antibody. In contrast, labeling for messenger RNA encoding GAD (M(r) 67,000) was decreased in a population of medium-sized neurons normally expressing high levels of this messenger RNA and corresponding to GABAergic interneurons. Labeling for messenger RNA encoding glutamate decarboxylase (M(r) 65,000) was not modified in the dopamine-depleted striatum. The results show that dopamine depletion differentially affects gene expression for different isoforms of glutamate decarboxylase in distinct subpopulations of striatal neurons in rat.     

Substance P synaptic interactions with GABAergic and dopaminergic neurons in rat substantia nigra: An ultrastructural doublelabeling immunocytochemical study

The distribution of substance P (SP), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD) immunoreactivity in the substantia nigra of the rat was studied by means of an ultrastructural double-labeling immunocytochemical method. Direct synaptic contact between SP-immunoreactive terminals and GAD-positive nigral neurons was more often observed in the pars lateralis than the pars reticularis and was rarely observed in the pars compacta. Substance P-positive terminals also formed synapses with cell bodies and dendrites of TH-positive, dopaminergic neurons in the pars compacta and pars reticulata. Multiple SP-immunoreactive terminals were often observed with symmetrical and, less frequently, asymmetrical synapses on individual TH-containing dendrites. Evidence of SP-containing terminals contacting both GABAergic and dopaminergic neurons in the substantia nigra suggests a direct excitatory action upon nigral projection neurons.     

Substance K (NKA) increases tyrosine hydroxylase mRNA in cultured substantia nigra

Cultured explants of the mouse substantia nigra were used to analyze mechanisms underlying the depolarization-induced increase in tyrosine hydroxylase activity. Steady-state levels of messenger RNA encoding tyrosine hydroxylase were detected using an antisense riboprobe in an RNase protection assay. Explants exposed to the depolarizing agent, veratridine, exhibited an approximate 2-fold increase in tyrosine hydroxylase messenger RNA. Moreover, the native presynaptic excitatory agonist substance K also elicited a significant increase in tyrosine hydroxylase message. We conclude that depolarizing influences induce tyrosine hydroxylase in the cultured substantia nigra in association with an elevation of enzyme messenger RNA.     

Increased expression of glutamic acid decarboxylase mRNA in rat substantia nigra after an ibotenic acid lesion in the caudate-putamen

In situ hybridization histochemistry and RNA blots were used to study expression of glutamic acid decarboxylase (GAD) mRNA in rat caudate-nucleus and substantia nigra. In situ hybridization combined with computerized image analysis revealed that in the intact substantia nigra reticulata the cross-section area of GAD mRNA positive neurons were 25% larger in the dorsolateral part as compared with the ventromedial part. A unilateral ibotenic acid injection in caudate-putamen lesioned neurons, some of which project to the ipsilateral substantia nigra. An increased level of GAD mRNA was observed in substantia nigra ipsilateral to the lesion. Computerized image analysis of sections from in situ hybridization revealed an increase in the number of silver grains over GAD mRNA positive neurons in the dorsolateral substantia nigra reticulata ipsilateral to the lesion. However, no change was observed in the ventromedial part suggesting that GAD mRNA expression in this part of the nigra is less sensitive to inhibition by caudate-putamen afferents. In agreement with in situ experiments, RNA blots showed a 2-fold increased level of GAD mRNA in substantia nigra ipsilateral to the lesion. The increased GAD mRNA expression in the deafferented substantia nigra suggests a disinhibition of nigral GABA neurons, resulting in an increased utilization of GABA in these substantia nigra neurons.     

Striatonigral projection neurons contain D1 dopamine receptor-activated c-fos

Dopamine receptor agonists which stimulate the D1 receptor have been shown to activate c-fos in the striatum ipsilateral to a 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway. In the present study, striatal neurons ipsilateral to a 6-OHDA lesion of the medial forebrain bundle were retrogradely labelled by injection of the fluorescent tracer Fluoro-Gold into the substantia nigra pars reticulata. Five days later, c-fos was induced in the 6-OHDA-denervated striatum by injection of the selective D1 agonist SKF 38393. C-fos-positive nuclei were frequently found in medium-sized striatal cell bodies labelled with Fluoro-Gold. These results indicate that D1 agonists activate c-fos in medium-sized neurons that project to the substantia nigra pars reticulata.     

c-fos and tyrosine hydroxylase expression after an excitotoxic lesion on the nigrostriatal system: a study on the effects of hypoxia used as a preconditioning stimulus

Hypoxia-ischemia during the perinatal period causes excitotoxic lesions in sensitive brain areas, such as the striatum. The impact of hypoxia-ischemia on nigral neurons is less well known. Hypoxia alone, a less traumatic event without overt histological sequelae, has neuroprotective properties when used as a preconditioning stimulus. In some pathologies, injured neurons of the nigrostriatal system in the adult may be the result of neurodegenerative processes that originated at early stages of life. The effects of hypoxia on the immunoreactivity to tyrosine hydroxylase of the dopaminergic neurons of the substantia nigra pars compacta and the effects of a period of hypoxia previous to an excitotoxic lesion were examined by means of histological and Western blot methods, at immediate and late periods of the episode. By counting the number of tyrosine hydroxylase-stained neurons and c-fos-positive nuclei a short period after injection of quinolinic acid into the striatum, we observed that hypoxia induced a more marked decrease in the number of tyrosine hydroxylase-stained neurons. On the contrary, c-fos-positive profiles decreased in the substantia nigra pars reticulata of the quinolinic acid-injected animals after the preconditioning hypoxia. Hypoxia alone did not affect the number of tyrosine hydroxylase-positive neurons in the pars compacta nor did hypoxia induce c-fos expression in the pars reticulata. More sensitive Western blot analysis of tissue blocks that included the whole substantia nigra demonstrated the same trend as the immunohistochemical results. We conclude that the responses of the substantia nigra neurons to hypoxia are regionalized and potential neuroprotective effects may depend on the vulnerability of each neuronal type.     

The origin of tyrosine hydroxylase-immunoreactive fibers in the regions of the nucleus basalis magnocellularis of the rat

The simultaneous use of acetylcholinesterase (AChE) histochemistry and tyrosine hydroxylase (T-OH) immunocytochemistry permitted demonstration of the existence of a dense catecholaminergic network surrounding cholinergic neurons within the nucleus basalis magnocellularis (NBM) of the rat. The origin of this catecholaminergic network was investigated by combining T-OH immunocytochemistry with horseradish peroxidase (HRP) retrograde labelling using a slow release gel, unilaterally implanted in the area of the NBM. Retrogradely transported HRP was detected in some of the aminergic cell groups of the substantia nigra (A9) and locus coeruleus (A6). In these areas, approximately 1% of the tyrosine hydroxylase immunoreactive neurons were retrogradely labelled with HRP. In the substantia nigra, dually labelled neurons were found predominantly in the pars lateralis.     

Sprouting of dopaminergic fibers from spared mesencephalic dopamine neurons in the unilateral partial lesioned rat

A unilateral partially lesioned rat model of Parkinson's disease was developed following selective lesioning of the dopamine neurons of the substantia nigra pars compacta by stereotactic injection of the neurotoxin 6-hydroxydopamine. In this animal model the dopamine neurons of the ventral tegmental area and medial substantia nigra are spared. The neuronal loss in such partial lesioned models mimics more closely that seen in human mid-stage parkinsonism. Cografts of adrenal medullary cells and sciatic nerve to the partially lesioned striatum induced a sprouting response in grafted animals that was confirmed by immunocytochemical staining with antibodies to tyrosine hydroxylase (TH) and by quantification of the high affinity dopamine uptake complex using [³H]GBR 12935 binding. Enhanced TH fiber immunostaining was evident even in the presence of poor cograft survival. The origin of the TH-like immunostained fibers in the striatum was determined using Lucifer yellow retrograde axonal transport. Following discrete tracer injections into the striatum adjacent to a cograft, neurons in the medial substantia nigra and ventral tegmental area (areas A9 and A10, respectively) were labelled with Lucifer yellow. These labelled neurons displayed a morphology characteristics of dopamine neurons and, in double-labelling experiements, also immunostained for TH. These results support the utility of unilateral partially lesioned rat models of Parkinson's disease for studies investigating a host sprouting or upregulation response and confirm that the immunostained striatal fibers originate from spared dopamine neurons in the ventromedial midbrain.     

Compartmental distribution of cytochrome oxidase in the striatum of the rat.

Endogenous cytochrome oxidase activity was investigated in the adult rat striatum at the light microscope level to see if it was distributed in accordance with the established striatal patch/matrix compartmentalisation. Striatal sections stained to visualise cytochrome oxidase activity were compared with serial sections stained to visualise tyrosine hydroxylase and calbindinD28k-like immunoreactivity, established markers of the matrix compartment. The distribution of endogenous cytochrome oxidase activity was found to coincide with the immunocytochemical staining pattern seen for tyrosine hydroxylase and calbindinD28k whereby areas of intense tyrosine hydroxylase and calbindinD28k-like immunoreactivity (termed the matrix) corresponded to areas of intense cytochrome oxidase activity. Conversely, areas of less intense tyrosine hydroxylase and calbindinD28k-like immunoreactivity (termed patches) corresponded to areas of low cytochrome oxidase activity. In addition, the distribution of two other oxidative enzymes involved in the regulation of mitochondrial respiration, succinic dehydrogenase and NADH-diaphorase, was examined in the striatum and substantia nigra by using histochemical techniques. Both NADH-diaphorase and succinic dehydrogenase histochemistry showed an uneven pattern of neuropil staining in the striatum. In the substantia nigra a few intensely stained cell bodies were seen in the dorsal-lateral tip of the pars reticulata with both histochemical techniques. By using an anti-cytochrome oxidase antibody an abundance of immunoreactive cell bodies and processes were seen in the substantia nigra, particularly in the dorso-medial rim and dorsal tip of the pars reticulata. The substantia nigra pars lateralis contained many intensely stained cytochrome oxidase-like immunoreactive cell bodies and processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The GABA and substance P input to dopaminergic neurones in the substantia nigra of the rat.

In order to examine the synaptic input to dopaminergic neurones in the substantia nigra from GABAergic terminals and terminals that contain substance P, double and triple immunocytochemical studies were carried out at the light and electron microscopic levels in the rat. In a first series of experiments sections of the substantia nigra were incubated to reveal axon terminals containing either substance P or glutamate decarboxylase and then incubated to reveal dopaminergic neurones using tyrosine hydroxylase immunocytochemistry. Examination of this material in the light microscope revealed that many substance P- and glutamate decarboxylase-immunoreactive boutons were associated with the dopaminergic cells. In the electron microscope it was found that the perikarya and dendrites of the dopaminergic neurons received symmetrical synaptic input from terminals that displayed immunoreactivity for substance P or glutamate decarboxylase. A small proportion of the substance P-positive boutons formed asymmetrical synapses. In a second series of experiments sections of the substantia nigra were processed by the pre-embedding immunocytochemical technique for tyrosine hydroxylase and then the post-embedding immunogold technique for gamma-aminobutyric acid (GABA). Examination in the electron microscope revealed that the tyrosine hydroxylase-positive neurons received symmetrical synaptic input from many GABA-positive terminals. Quantitative analyses demonstrated that a minimum of 50-70% of all boutons afferent to the dopaminergic neurones display glutamate decarboxylase or GABA immunoreactivity. Triple immunocytochemical studies i.e. pre-embedding immunocytochemistry for tyrosine hydroxylase and substance P, combined with post-embedding immunogold staining for GABA, revealed that some of the substance P-immunoreactive boutons that were in contact with the dopaminergic neurones also displayed GABA immunoreactivity. In a third series of experiments the combination of anterograde transport of lectin-conjugated horseradish peroxidase or biocytin with post-embedding GABA immunocytochemistry demonstrated that at least one of the sources of GABA-containing terminals in the substantia nigra is the striatum. The results of the present study: (1) demonstrate that dopaminergic neurones in the substantia nigra receive symmetrical synaptic input from GABAergic and substance P-containing terminals, (2) show that a proportion of these terminals contain both substance P and GABA and (3) suggest that the major synaptic input to dopaminergic neurones is from GABAergic terminals and that a part of this innervation is derived from the striatum.     

Two-color immunohistochemistry for dopamine and GABA neurons in rat substantia nigra and zona incerta

Dopaminergic and GABAergic neurons were visualized in the same section of rat substantia nigra (SN) and zona incerta (ZI) by a two-color double immunoperoxidase procedure or by double immunofluorescence. Rabbit antiserum to tyrosine hydroxylase (TH) and sheep antiserum to glutamic acid decarboxylase (GAD), markers for catecholaminergic and GABAergic neurons, respectively, were used as primary antisera. These techniques rely on species difference of primary antisera and non-crossreactivity of linking antisera. In normal and colchicine pretreated rats, SN pars compacta (SNC), SN pars lateralis (SNL), and ZI pars medialis (area A13) contained high densities of TH-positive neurons. Relatively few TH-positive cells were scattered in SN pars reticulata (SNR) and ZI pars lateralis (ZIL). In normal rats, GAD-positive boutons were more numerous throughout SNR and ZIL than in SNC, SNL, and area A13. In colchicine pretreated rats, the majority of neurons in SNR and ZIL and few neurons in SNC, SNL, and area A13 were GAD-positive and TH-negative. This study suggests a dichotomy of both SN and ZI into a predominantly dopaminergic and a predominantly GABAergic part.     

Direct demonstration of interactions between substance P immunoreactive terminals and tyrosine hydroxylase immunoreactive neurons in the substantia nigra of the rat: an ultrastructural study

The results of many anatomical, physiological, and pharmacological studies suggest that substance P-containing neurons of the striatum project to the substantia nigra, and that substance P influences the activity of dopaminergic nigrostriatal neurons. The purpose of the present ultrastructural study was to employ dual immunocytochemical labeling to determine the morphological basis for the observed actions of substance P on nigral dopaminergic neurons. Substance P-like and tyrosine hydroxylase-like immunoreactivities were localized simultaneously at the ultrastructural level in the substantia nigra of the rat. A double label method was utilized which relied on a combination of the peroxidase-antiperoxidase method (Sternberger, 1979) for substance P, and immunogold or silver enhanced immunogold labeling for tyrosine hydroxylase. The present results indicate that tyrosine hydroxylase immunoreactive (THLI) dendrites in the substantia nigra receive synaptic input from terminals exhibiting substance P-like immunoreactivity. These findings support the idea that substance P is a major neurotransmitter in the striatonigral loop, and suggest that striatal substance P neurons act directly upon nigral dopaminergic cells.     

Transient increase of brain derived neurotrophic factor mRNA expression in substantia nigra reticulata after partial lesion of the nigrostriatal dopaminergic pathway

By using non-isostopic in situ hybridization we have demonstrated a transient increase of BDNF mRNA in the lateral subregion of the substantia nigra pars reticulata 1 week after intrastriatal application of 6-OH-DA. These changes correlate with a partial reduction of dopamine (DA) content in the striatum but with a normal tyrosine hydroxylase immunoreactivity in substantia nigra pars compacta. Our data suggest that non-DA, BDNF expressing cells in substantia nigra pars reticulata may play a role in neuronal protection after partial lesions of the DA nigrostriatal pathway.     

Effects of morphine on axonal transport in nigrostriatal neurons.

Axonal transport in the nigrostriatal tract of protein labeled with [³H]-leucine (10 h and 5 days) or of [³H]dopamine (10 h) was not significantly different from controls in rats treated with morphine by acute injection [20 mg/kg, intraperitoneal (ip)] or chronically implanted with 75-mg pellets. This applied both to the amount of label accumulated in the striatum and to the gel electrophoretic protein patterns in both the striatum and the substantia nigra. Rats chronically treated with morphine gave more variable results in all tests than the controls but the results did not seem to correlate with the observable behavioral effects. The difference between our results and the significant increase in protein transport reported by others in morphine-dependent animals probably depends on the lower doses of morphine used in the present experiments. The animals, however, seemed dependent as judged by their response to a naloxone challenge. There was also no significant difference between morphine-treated and control rats in the activities of tyrosine hydroxylase, acetylcholinesterase, monoamine oxidase, dopa decarboxylase, or glutamic decarboxylase in the striatum or of glutamic decarboxylase or tyrosine hydroxylase in the substantia nigra. Striatal choline acetyltransferase was elevated in the rats chronically treated with morphine as previously reported by others.     

Decreased tyrosine hydroxylase content in the dopaminergic neurons of MPTP–intoxicated monkeys: Effect of levodopa and GM1 ganglioside therapy

Parkinson's disease is characterized by the degeneration of melanized dopaminergic neurons of the substantia nigra. The functional capacity of the surviving dopaminergic neurons is affected, as suggested by the subnormal levels of tyrosine hydroxylase messenger RNA and protein found in the remaining cells. The reduced expression of tyrosine hydroxylase may be due to either the evolving neurodegenerative process or its downregulation, possibly secondary to chronic levodopa treatment. The cellular content of tyrosine hydroxylase was determined in the mesencephalon from 16 Macaca fascicularis monkeys, using a semiquantitative immunocytochemical method. Thirteen monkeys were rendered parkinsonian by weekly intravenous injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 2 (subacute treatment) or 20 (chronic treatment) weeks. Three of the monkeys received levodopa and 3 others received GM1 ganglioside. The loss of dopaminergic neurons in the mesencephalon of the MPTP-intoxicated monkeys was severe in the substantia nigra, intermediate in cell groups A8 and A10, and almost undetectable in the central gray substance. After both subacute and chronic treatment, the cellular content of tyrosine hydroxylase was reduced by 40% in the surviving neurons of the lesioned substantia nigra, but by less in the other mesencephalic dopaminergic regions. Neuronal survival and tyrosine hydroxylase content in monkeys that had received levodopa were not significantly different. The cellular content of tyrosine hydroxylase was increased in the substantia nigra of the monkeys that received GM1 ganglioside injections. The results show that the decreased expression of tyrosine hydroxylase found in nigral dopaminergic neurons after partial degeneration of the mesostriatal dopaminergic system is not influenced by levodopa treatment and is partially reversed by GM1 ganglioside administration.