Dopaminergic neurons in explants of substantia nigra in culture

Explants of substantia nigra and corpus striatum obtained from newborn rats were maintained in tissue culture for up to six days. Explants of substantia nigra exhibited a net increase in the ability to take up H³-dopamine, a process associated with the dopaminergic neurons; in contrast, the explants of corpus striatum showed a rapid loss in this ability to accumulate H³-dopamine. After three days in culture, the specific activity of tyrosine hydroxylase and monoamine oxidase had decreased 50% in explants of substantia nigra. A medium including fetal calf serum and chick embryo extracts was necessary for the increase in H³-dopamine uptake, and nerve growth factor had an inhibitory effect. Histofluorescent examination of nigral explants cultured for three days indicated morphologically normal dopaminergic neurons.     

Neurochemical sequelae of kainate injections in corpus striatum and substantia nigra of the rat.

Unilateral injection of 2 μg kainic acid into the substantia nigra of the rat results in a 45% decrease in tyrosine hydroxylase activity in the injected substantia nigra and in the ipsilateral corpus striatum. In contrast, the GABAergic nerve terminals in the substantia nigra are unaffected by this treatment. Injection of kainic acid into the striatum results in a 60% decrement in the activity of glutamate decarboxylase and of endogenous GABA levels in the ipsilateral substantia nigra whereas tyrosine hydroxylase activity remains unchanged; in addition, dopamine-sensitive adenylate cyclase activity in the ipsilateral substantia nigra decreases by 74%. These findings further support the hypothesis that intracerebral injections of kainic acid cause degeneration of neurons with cell bodies near the injection site while sparing axons passing through or terminating in the region.     

Determination of monoamines and both forms of monoamine oxidase in the rat's substantia nigra during postnatal development

Changes in biogenic amine content in the substantia nigra and in both forms of monoamine oxidase in substantia nigra and striatum of the rat during postnatal development (15-180 days) have been studied. Dopamine and serotonin had the same levels at day 15, however, each monoamine showed a different developmental profile. Dopamine levels and their metabolites (except 3-methoxytyramine) decreased during postnatal development. Serotonin levels and their main metabolite, 5-hydroxyindolacetic acid, underwent an increase during all stages studied. There were no statistically significant changes in noradrenaline levels until day 180 when they increased with respect to day 15. The highest activity of the monoamine oxidase-A in substantia nigra coincided with the highest 5-hydroxyindolacetic acid:serotonin ratio. Monoamine oxidase-A in the striatum did not change contrary to that which happened in substantia nigra. The monoamine oxidase-B:monoamine oxidase-A ratio increased during development both in the substantia nigra and the striatum. The significance of these changes is discussed.     

Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine-induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.     

Stress-induced increase in 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the cerebral cortex and in n. accumbens: reversal by diazepam

The effect of electrical foot shock stress on dopamine and DOPAC levels was examined in the frontal cortex, nucleus accumbens, striatum, substantia nigra and medial basal hypothalamus of rats. DA content did not change after stress in any of the structures analyzed except in the substantia nigra in which DA level decreased by about 35% following 20, 60 or 180 min of stress. DOPAC level did not change in the striatum, medial basal hypothalamus and substantia nigra, but increased in the frontal cortex and in n. accumbens by about 75% and 40%, respectively. Pretreatment with diazepam, but not with pentobarbital, prevented stress-induced increased in DOPAC levels.     

Regional Distribution of Neuropeptide Y and Its Receptor in the Porcine Central Nervous System

The regional distribution of neuropeptide Y (NPY) immunoreactivity and receptor binding was studied in the porcine CNS. The highest amounts of immunoreactive NPY were found in the hypothalamus, septum pellucidum, gyrus cinguli, cortex frontalis, parietalis, and piriformis, corpus amygdaloideum, and bulbus olfactorius (200-1,000 pmol/g wet weight). In the cortex temporalis and occipitalis, striatum, hippocampus, tractus olfactorius, corpus mamillare, thalamus, and globus pallidus, the NPY content was 50-200 pmol/g wet weight, whereas the striatum, colliculi, substantia nigra, cerebellum, pons, medulla oblongata, and medulla spinalis contained less than 50 pmol/g wet weight. The receptor binding of NPY was highest in the hippocampus, corpus fornicis, corpus amygdaloideum, nucleus accumbens, and neurohypophysis, with a range of 1.0-5.87 pmol/mg of protein. Intermediate binding (0.5-1.0 pmol/mg of protein) was found in the septum pellucidum, columna fornicis, corpus mamillare, cortex piriformis, gyrus cinguli, striatum, substantia grisea centralis, substantia nigra, and cerebellum. In the corpus callosum, basal ganglia, corpus pineale, colliculi, corpus geniculatum mediale, nucleus ruber, pons, medulla oblongata, and medulla spinalis, receptor binding of NPY was detectable but less than 0.5 pmol/mg of protein. No binding was observed in the bulbus and tractus olfactorius and adenohypophysis. In conclusion, immunoreactive NPY and its receptors are widespread in the porcine CNS, with predominant location in the limbic system, olfactory system, hypothalamoneurohypophysial tract, corpus striatum, and cerebral cortex.     

Low Selenium Diet Affects Monoamine Turnover Differentially in Substantia Nigra and Striatum

Abstract: Turnover of dopamine, noradrenaline. serotonin, and their metabolites has been measured in striatum and substantia nigra of adult female rats that were fed control or selenium-deficient diets for 15 days. In addition, the glutathione peroxidase activity has been studied. The most striking result was the increase of dopamine turnover (63%) and 3- methoxytyramine turnover (55%) in substantia nigra between control and experimental animals. On the other hand, no changes were found in the turnover rate of dopamine and its metabolites in the striatum. Likewise, no changes were found in noradrenaline turnover in substantia nigra. In the striatum, there was a significant increase of serotonin turnover versus no change for 5-hydroxy-3-indoleacetic acid. However, in the substantia nigra, serotonin turnover did not show significant changes, whereas 5-hydroxy-3-indoleacetic acid turnover decreased. At the same time, glutathione peroxidase activity significantly decreased in both structures after selenium-deficient diets. These results suggest that a selenium-deficient diet for a short period of time decreases brain protection. principally in the substantia nigra, against oxidative damage.     

Amino Acids in the Substantia Nigra of Rats with Striatal Lesions Produced by Kainic Acid

In an attempt to estimate the pool size of glutamate and other amino acids in γ-aminobutyric acid (GABA)-containing neurons, we determined the content of 12 amino acids in the bilateral substantia nigra of rats, in which unilateral striatal lesions had been made with kainic acid two weeks earlier. The assay of the amino acids (including glutamate, aspartate, glutamine, asparagine, glycine, and GABA) and ethanolamine was based on HPLC and fluorimetric detection after precolumn derivatization with o-phthaldialdehyde. The levels of all measured amino acids (except those of tyrosine, threonine, and ethanolamine) were decreased in the affected striatum, but only the levels of aspartate, taurine, and GABA were lowered in the ipsilateral substantia nigra. These results indicate that the pool size of the various amino acids in the striatonigral GABAergic pathway is small compared to their nigral content, and that in addition to GABA a significant fraction of aspartate and taurine may be confined to nerve terminals in the substantia nigra.     

Differential effects of acute and chronic haloperidol treatment on striatal and nigral 3, 4-dihydroxyphenylacetic acid (DOPAC) levels

Chronic treatment of rats with haloperidol (4 weeks, 0.5 or 1 mg/kg) resulted in a significant attenuation of the large DOPAC rise seen in the corpus striatum after acute treatment. This tolerance effect was observed both shortly following termination of chronic treatment and on challenge with a low dose (0.1 mg/kg) of the drug 6–8 days later. In contrast, acute haloperidol treatment resulted in only a small and nonsignificant elevation of DOPAC levels in the substantia nigra, while chronic treatment caused a larger and significant increase in levels of the metabolite. Moreover, the latter effect was also observed in response to haloperidol challenge 6–8 days after discontinuation of drug treatment. The differential pattern of response in these two brain regions is discussed in relation to possible mechanisms mediating striatal tolerance and to recent observations regarding changes in nigral dopamine cell firing after chronic haloperidol treatment.     

Striatal dopamine levels after unilateral lesions of the substantia nigra: evidence for a contralateral decrease

Unilateral electrolytical and chemical (6-hydroxydopamine) lesions in the substantia nigra (SN) of rats were followed 7 days later by considerable bilateral decreases of neostriatal dopamine (DA) levels. Similarly, the DA content of the substantia nigra decreased not only ipsilaterally but contralaterally as well. Positive correlations were found between ipsi- and contralateral nigral DA levels, ipsi- and contralateral striatal DA and between the DA level of the SN and the striatum of the corresponding side both ipsi- and contralaterally to the lesion.     

Nigrostriatal Modifications After Vanadium Inhalation: An Immunocytochemical and Cytological Approach

Vanadium (V) has increased in the air as a component of suspended particles originated from fuel combustion. In this report, a model of inhaled V in mice was implemented to identify the effect that V has in the corpus striatum and substantia nigra, structures with high concentrations of dopamine and scarce antioxidants burden. Mice inhaled 0.02 M V2O5 1 h twice a week and were sacrificed at points from 1 to 8 weeks after inhalation, perfused, and processed for Golgi method and for tyroxine hidroxylase (TH) inmunocytochemistry. Cytological analysis consisted in counting the number of dendritic spines in 20 medium-size spiny neurons and the number of TH immunoreactive neurons in the substatia nigra pars compacta. Dendritic spine density decreased drastically after V exposure; the same was observed with the TH-positive neurons, which decreased in a time-dependent mode. No previous morphological studies about V and nervous system have been reported. The decrease in spine density and in TH-positive neurons might have functional repercussions that should be studied because the trend of this element in the atmosphere is to increase.     

Habenular modulation of dynorphinergic systems in rat ventral mesencephalon

Bilateral electrolytic lesion of the striatonigral pathways (which convey massive afferents to the substantia nigra) caused a marked lowering of alpha-neo-endorphin (alpha-Neo) and dynorphin A(1-8) [Dyn A(1-8)] levels in the substantia nigra without affecting the alpha-Neo content in the ventral tegmental area. Moreover, unilateral infusion of the axon sparing neurotoxin ibotenate into the striatum, but not into the substantia nigra, decrease these two opioid peptides in the substantia nigra on the side ipsilateral to the lesion, failing to modify the alpha-Neo levels in the ventral tegmental area. Bilateral electrolytic lesion of the habenula augmented alpha-Neo content in the substantia nigra and ventral tegmental area at 8-30 days postlesion without affecting the nigral Dyn A(1-8). These results add further support to the view that alpha-Neo- and Dyn A(1-8)-containing neurons projecting to the substantia nigra originate in the striatum and descend through striatonigral pathways. The present data provide evidence that the habenula may participate in the regulation of the activity of alpha-Neo-immunoreactive neurons in the substantia nigra and ventral tegmental area.     

The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats

Recent studies of Parkinson's disease indicate that dorsal motor nucleus of nerve vagus is one of the earliest brain areas affected by alpha-synuclein and Lewy bodies pathology. The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats is investigated. Synchrotron radiation based X-ray fluorescence was applied to the elemental micro-imaging and quantification in thin tissue sections. It was found that elements such as P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br and Rb are present in motor cortex, corpus striatum, nucleus accumbens, substantia nigra, ventral tectal area, and dorsal motor nucleus of vagus. The topographic analysis shows that macro-elements like P, S, Cl and K are highly concentrated within the fiber bundles of corpus striatum. In contrast the levels of trace elements like Fe and Zn are the lowest in these structures. It was found that statistically significant differences between the animals with electrical stimulation of vagus nerve and the control are observed in the left side of corpus striatum for P (p = 0.04), S (p = 0.02), Cl (p = 0.05), K (p = 0.02), Fe (p = 0.04) and Zn (p = 0.02). The mass fractions of these elements are increased in the group for which the electrical stimulation of vagus nerve was performed. Moreover, the contents of Ca (p = 0.02), Zn (p = 0.07) and Rb (p = 0.04) in substantia nigra of right hemisphere are found to be significantly lower in the group with stimulation of vagus nerve than in the control rats.     

Regional distribution of glucose in mouse brain

After rapid inactivation of the enzymes responsible for glucose metabolism by microwave irradiation, concentrations of glucose in 20 regions of the mouse brain were estimated with combined gas chromatography-mass spectrometry (GC-MS). The highest concentrations of glucose were found in the periventricular nuclei of the hypothalamus and nucleus preopticus (P<0.05). The septum and nucleus amygdaloideus showed significantly higher glucose concentration compared with the cerebral neocortex, olfactory bulb, corpus striatum, cingulum, fornix, colliculus inferior, cerebellar cortex, corpus geniculatum laterale, substantia nigra, and nucleus ruber (P<0.05). The glucose concentration in the substantia nigra and nucleus ruber was significantly lower than in the other regions (P<0.01).     

Polyamines, Ornithine Decarboxylase, and Diamine Oxidase in the Substantia Nigra and Striatum of the Male Rat After Hemitransection

Partial hemitransection at the mesodiencephalic junction in the rat increased striatal and nigral putrescine concentrations on the lesioned side for at least 168 h, with maximal increases between 24 and 48 h. Spermidine and spermine levels declined at 24 h in the striatum, rising above control values at 48 h and further at 168 h. In the substantia nigra, they remained unchanged for the first 48 h and then increased by 168 h. Cadaverine in the striatum also increased at 48 h. On the intact side putrescine increased but to a much lesser extent (at 48 h in the striatum and at 24 and 48 h in the substantia nigra). Ornithine decarboxylase and diamine oxidase activities showed maximal increases at 24 h in the striatum of the lesioned side, whereas in the substantia nigra ornithine decarboxylase attained a very high value as early as 4 h after the operation and diamine oxidase activity peaked at 48 h. The enzyme activities returned toward the basal values at 168 h. On the intact side, ornithine decarboxylase showed a small increase starting at 4 h and diamine oxidase was enhanced at 48 h. These results indicate that the stimulation of biosynthetic and degradative enzymes of polyamine metabolism accompanied by marked and prolonged increases in putrescine may be essential events in the early phases of neuronal response to mechanical injury in the CNS.     

Semichronic Inhibition of Glutathione Reductase Promotes Oxidative Damage to Proteins and Induces both Transcription and Translation of Tyrosine Hydroxylase in the Nigrostriatal System

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

Semichronic inhibition of glutathione reductase promotes oxidative damage to proteins and induces both transcription and translation of tyrosine hydroxylase in the nigrostriatal system

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

Long-term post-synaptic consequences of methamphetamine on preprotachykinin mRNA expression

Exposure to repeated high doses of methamphetamine produces long-term toxicity to central monoamine systems and alters striatonigral pathway function 3 weeks after exposure. To determine whether these changes in the striatonigral pathway persist for longer we examined neuropeptide mRNA expression in the striatum and cytochrome oxidase activity in the output nuclei of the basal ganglia after treatment with multiple high doses of methamphetamine. Rats exposed to multiple high doses of methamphetamine had significant depletion in dopamine and serotonin content, decreases in tyrosine hydroxylase immunoreactivity, and decreases in preprotachykinin mRNA expression, 6 and 12 weeks after methamphetamine treatment. Preprotachykinin mRNA expression was significantly reduced by approximately 20% in the middle striatum and approximately 32% in the caudal striatum, 6 weeks after treatment. Twelve weeks after treatment, preprotachykinin mRNA expression continued to be significantly reduced by approximately 20% in the middle striatum and approximately 14% in the caudal striatum. Cytochrome oxidase histochemical staining in the entopeduncular nucleus and substantia nigra pars reticulata was not significantly different from that in controls at either time point. These data suggest that neurotoxic regimens of methamphetamine induce changes in striatonigral neurons that persist for up to 3 months, although there is some recovery.     

LOCALIZATION OF GABAERGIC, CHOLINERGIC AND AMINERGIC STRUCTURES IN THE MESOLIMBIC SYSTEM

Abstract— The localization of cholinergic, GABAergic and aminergic structures in the 'mesolimbic' system has been discussed from studies on the topographical distribution of choline acetyltransferase, glutamate decarboxylase and aromatic amino acid decarboxylase in normal rat brain and in brains hemitransected at the level of globus pallidus. The structures analysed included nucleus accumbens, olfactory tubercle, septum, medial forebrain bundle, striatum, substantia nigra, ventral tegmental area and nucleus interpeduncularis.
Choline acetyltranferase was highly concentrated in the nucleus interpeduncularis, but it did also exhibit considerable activity in the nucleus accumbens, the olfactory tubercle and the striatum. The activities did not change after hemitransection. Aromatic amino acid decarboxylase was highly concentrated in the ventral tegmental area, but high activities were also found in the striatum, the nucleus accumbens, the olfactory tubercle and the pars compacta of the substantia nigra. The activity decreased in all areas rostral to the hemitransection. Glutamate decarboxylase was highly concentrated in the dopamine innervated regions, moreso in the limbic structures than in the striatum. Much higher activity was found in the substantia nigra than in the ventral tegmental area. After hemitransection the activity in the substantia nigra was decreased whereas in the ventral tegmental area it was unchanged. Our results thus suggest that dopaminergic cells in the ventral tegmental area do not receive GABAergic fibres from the terminal regions of the ascending dopaminergic fibres. In addition, we found a very high concentration of glutamate decarboxylase in a region traversed by the rostral medial forebrain bundle. Here the activity was mainly confined to the paniculate fraction, probably the synaptosomes. This fraction also displayed a very active high affinity uptake of y-aminobutyric acid.