Toxicity of dopamine to striatal neurons in vitro and potentiation of cell death by a mitochondrial inhibitor.

Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 microM) nor dopamine (2.5 microM) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 microM) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 microM, reduced the ATP/ADP ratio by approximately 30% after 6 h; this decrease was not augmented by coincubation with 25 microM dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.

Glutamic acid decarboxylase gene expression in thalamic reticular neurons transplanted as a cell suspension in the adult thalamus.

The goal of the present study was to determine whether alterations in neuronal morphology and connections in thalamic grafts were accompanied by changes in the expression of mRNA encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of GABA, the normal neurotransmitter of neurons of the thalamic reticular nucleus. Cell suspensions of rat fetal tissue containing both thalamic reticular nucleus and ventrobasal primordia were transplanted into the excitotoxically lesioned somatosensory thalamus of adult rats. Levels of messenger RNA (mRNA) encoding GAD (Mr 67,000; GAD67) were measured 7 days to 4 months following transplantation via quantitative in situ hybridization with 35S-radiolabeled antisense RNAs. Expression of GAD67 mRNA in the thalamic reticular nucleus was analyzed in parallel in rat pups between 0 and 30 days postnatally, and in adult animals. As already observed with immunohistochemistry, transplanted neurons of the thalamic reticular nucleus did not group in specific clusters but rather mingled with unlabeled (putatively ventrobasal) neurons. Levels of labelling for GAD67 mRNA per neuron increased over time and reached adult levels during the third week post-grafting, i.e. 2 weeks after the theoretical birthdate of the neurons (grafted at embryonic days 15-16). Similar values were observed and a plateau was reached at similar time points during normal ontogeny. The results suggest that, in contrast to morphology and size of the neuronal cell bodies, gene expression of GAD67 develops normally despite the ectopic location of neurons of the thalamic reticular nucleus in the somatosensory thalamus, the abnormal connectivity and the lack of segregation from non-GABAergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain.

Neurotransmitter-related messenger RNAs were detected by in situ hybridization in sections of rat and mouse brains by using 35S-radiolabelled RNA probes transcribed from cDNAs cloned in SP6 promoter-containing vectors. The distribution of messenger RNAs for glutamic acid decarboxylase, tachykinins (substance P and K), and tyrosine hydroxylase was examined in the striatum, pallidum, and substantia nigra. Dense clusters of silver grains were observed with the RNA probe complementary of the cellular messenger RNA for glutamic acid decarboxylase (antisense RNA) over most large neurons in the substantia nigra pars reticulata and medium-sized to large neurons in all pallidal subdivisions. A few very densely and numerous lightly labelled medium-sized neurons were present in the striatum. Among the areas examined, only the striatum contained neurons labelled with the antisense tachykinin RNA. Most of these neurons were of medium size, and a few were large. With the antisense tyrosine hydroxylase RNA, silver grains were found over neurons of the substantia nigra pars compacta and adjacent A10 and A8 dopaminergic cell groups. No signal was observed with RNAs identical to the cellular messenger RNA for glutamic acid decarboxylase or tachykinin (sense RNA). These results show a good correlation with immunohistochemical studies, suggesting that documented differences in the distribution and the level of glutamic acid decarboxylase, tyrosine hydroxylase, and substance P immunoreactivities in neurons of the basal ganglia are related to differences in the level of expression of the corresponding genes rather than to translation accessibility, stability, or transport of the gene products.

Involvement of the thalamus in the asymmetric effects of unilateral sensory stimuli on the two nigrostriatal dopaminergic pathways in the cat.

The effects of unilateral sensory stimuli on dopamine (DA) release from nerve terminals and dendrites of the two nigrostriatal dopaminergic pathways were estimated in halothane-anaesthetized cats without or with sagittal transections. In control animals, the electrical stimulation of the right forelimb enhanced DA release in the right caudate nucleus (CN) and decreased DA release in the right substantia nigra (SN). Opposite effects were observed in the contralateral structures. Sagittal transections of the corpus callosum and commissura anterior, or of the mesencephalic decussations or of the thalamic massa intermedia were made to investigate mechanisms involved in the reciprocal regulation of the two dopaminergic pathways. These sections were without effect on the spontaneous release of DA from nerve terminals and dendrites. The transection of the thalamic massa intermedia was the only one which interrupted the asymmetric changes in DA release induced by unilateral sensory stimuli; an increased dendritic release of DA was only seen in the left SN but it was significantly less pronounced than that observed in control cats. The other transections did not prevent the asymmetric changes in DA release evoked by the sensory stimulation. However, the mesencephalic sagittal transection significantly reduced the stimulatory effect on DA release induced in the left SN. These results suggest that the thalamus is involved in the transfer of information implicated in the reciprocal regulation of the two dopaminergic pathways. In the light of electrophysiological data, the role of nigrothalamic neurones in this phenomenon is discussed.

Stimulating effect of angiotensin II on the spontaneous release of newly synthetized [3H]dopamine in rat striatal slices.

Angiotensin II (A II) (10(-6) M, 10(-5) M, 5 . 10(-5) M) stimulated the spontaneous release of [3H]dopamine (DA) continuously synthetized from [3H]tyrosine in striatal slices of the rat. The A II-evoked release of [3H]-DA was prevented when slices were superfused with a calcium-free medium containing EGTA 5 . 10(-4) M. It was also suppressed in the presence of the potent antagonist Sar1-Ile8-angiotensin.