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Glial cell line-derived neurotrophic factor (GDNF) promotes the survival or differentiation of several types of neurons. This study examines GDNF-induced signal transduction and biological effects in cultured striatal neurons. Results show that GDNF addition to striatal cultures transiently increased the protein levels of phosphorylated p42/p44, but did not change the levels of phosphorylated Akt. GDNF effects on phosphorylated p42/p44 levels were blocked by the mitogen-activated protein kinase (MAPK) pathway specific inhibitors (PD98059 and U0126). Activation of the p42/p44 MAPK pathway by GDNF led to an increase in the degree of dendritic arborization and axon length of both GABA- and calbindin-positive neurons but had no effect on their survival and maturation. These GDNF-mediated effects were suppressed in the presence of the inhibitor of the MAPK pathway (PD98059). Furthermore, the addition of the phosphatidylinositol 3-kinase pathway specific inhibitor (LY294002) blocked GDNF-mediated striatal cell differentiation suggesting that the basal activity of this pathway is needed for the effects of GDNF. Our results indicate that treatment of cultured striatal cells with GDNF specifically activates the p42/p44 MAPK pathway, leading to an increase in the arborization of GABA- and calbindin-positive neurons.

Excitotoxicity has been involved in the pathogenesis of several neurodegenerative disorders. Using intrastriatal quinolinic acid (QUIN) injection as an animal model of Huntington's disease, we attempt to identify the neurotransmitter phenotype of striatal projection neurons protected by neurturin (NRTN). Control or NRTN-secreting cell lines were grafted in the striatum before QUIN injection and striatal projection neurons were examined by retrograde Fluorogold labeling and in situ hybridization. Intrastriatal grafting of NRTN-secreting cell line selectively prevented the loss of striatopallidal neurons and also the decrease in the mRNA levels for their markers (glutamic acid decarboxylase 67 and preproenkephalin) induced by QUIN, without affecting striatonigral neurons. Thus, our findings show that NRTN is a selective neuroprotective factor for striatopallidal neurons, suggesting that it might be a candidate for the treatment of movement disorders in which this neuronal population is affected.

The neostriatum is one of the areas with relatively high levels of glial cell line-derived neurotrophic factor (GDNF) messenger RNA expression in the developing and adult brain. GDNF expression in the neostriatum has been suggested to be involved in promoting the survival of nigral dopaminergic neurons, acting as a target-derived neurotrophic factor. However, GDNF messenger RNA expression in the striatum starts several days before dopaminergic and other afferent neurons reach the striatum, suggesting additional trophic effects of this factor on striatal neurons. In the present report, we have examined whether GDNF is able to prevent the degeneration of striatal calbindin- and parvalbumin-immunoreactive neurons in a lesion model of Huntington's disease. Fischer 344 rat 3T3 fibroblast cell line expressing high levels of GDNF (F3A-GDNF) was used to assess the protective effect of this factor, on striatal neurons, against excitotoxicity. Quinolinate (34 nmol) was injected at two different coordinates, and calbindin, parvalbumin and tyrosine hydroxylase immunoreactivity were examined seven days after lesion. Dopaminergic afferents were spared after quinolinate injection, but the number of calbindin- and parvalbumin-immunoreactive neurons was decreased. Interestingly, implantation of F3A-GDNF cells increased the density of tyrosine hydroxylase staining in the intact and also in the quinolinate-lesioned striatum. Furthermore, GDNF partially protected calbindin- but not parvalbumin-immunoreactive neurons from quinolinate excitotoxicity. Instead, mock-transfected fibroblasts did not affect any of these parameters. Our results show that GDNF specifically protects a subpopulation of striatal calbindin-immunoreactive neurons against quinolinate lesion, suggesting that GDNF administration may have a potential therapeutic application in the prevention and treatment of striatonigral degenerative disorders.

The interaction between excitatory amino acids (EAAs) and nerve growth factor (NGF) levels were studied on neostriatal cholinergic neurons during postnatal development. Striatal choline acetyltransferase (ChAT) activity and NGF levels were determined 7 days following EAA injection in 7-, 15-, 21-, 30-, and 50-day-old rats. ChAT activity was decreased 7 days after kainate (KA), quinolinate (QUIN), or quisqualate (QUIS) lesion. The reduction was most pronounced in 30-day-old rats. KA injection produced the greatest decrease in ChAT activity. Conversely, KA did not change NGF levels. QUIN and QUIS increased NGF protein and these effects were maximal with lesions in 21-day-old rats. In order to further characterize the effect of EAAs on NGF levels and ChAT activity, the time-course of the lesion was studied. We used 30-day-old rats as the maximal sensitivity of cholinergic neurons to EAAs was observed at this age. ChAT activity decreased 2 days following QUIN or QUIS injection and 1 day after KA. The EAA agonists also changed NGF levels. QUIN induced an increase in NGF levels 1 day after lesion. This effect was maintained to the last time point examined. In contrast, KA and QUIS induced transient increases in NGF levels that were only detected 2 and 4 days after injection, respectively. To study whether NGF is able to regulate EAA excitotoxicity on striatal cholinergic neurons, we studied ChAT activity 7 days after simultaneous injection of NGF plus QUIN, KA, or QUIS. Intrastriatal injection of exogenous NGF was able to block the decrease in ChAT activity observed following EAA injection alone.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present work we have characterized a possible mechanism leading to the early survival of neostriatal cholinergic neurons after quinolinic acid injection. Different doses of quinolinic acid were injected in rat neostriatum and two different parameters were analysed 7 days after the lesion: choline acetyltransferase (ChAT) activity and nerve growth factor (NGF) levels. We have observed that ChAT activity decreased (until 68 nmol quinolinic acid) and NGF levels increased (until 34 nmol quinolinic acid) in a dose-dependent manner. In order to characterize the time-course of the lesion on NGF levels and ChAT activity, and the possible protective effect of NGF and basic fibroblast growth factor (bFGF) on cholinergic neurons, we have used the quinolinic acid dose (68 nmol) at which the first decrease of ChAT activity was observed. ChAT activity and NGF levels showed different patterns of response to quinolinic acid injection, since the maximal effect was reached at 1 day for ChAT activity and at 2 days for NGF levels. NGF or bFGF simultaneously injected with quinolinic acid (68 nmol) completely prevented the decrease in ChAT activity in a dose-dependent manner but NGF was more effective than bFGF. Furthermore, differences observed in ChAT activity after NGF but not bFGF treatment were correlated with changes in the number of ChAT immunoreactive cells. Finally, we have also observed that, although bFGF alone was not able to modify NGF levels, bFGF simultaneously injected with quinolinic acid produced an increase of NGF levels higher than that observed after quinolinic acid injection alone.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present work we have studied the postnatal development of functional dopamine, opioid and tachykinin receptors, which regulate cholinergic activity in the neostriatum. The release of endogenous acetylcholine from rat striatal slices was measured using a chemiluminescent method. We have observed that the inhibition mediated by dopamine through D2 receptors was not detectable until postnatal day 10, whereas the inhibition mediated by opioid receptors was detectable at postnatal day 15 for delta-receptors ([D-Pen2,D-Pen5]-enkephalin) and at postnatal day 21 for mu-receptors ([D-Ala2,Gly(ol)5]-enkephalin). Excitatory effect mediated by tachykinins through NK1 ([Sar9,Met(O2)11]- Substance P), NK2 ([Nle10]-Neurokinin A4-10), or NK3 (senktide) receptors was already detectable at postnatal day 5. In order to examine the influence of dopamine in the development of tachykinin and opioid systems in the neostriatum, we induced dopamine deficiency by intraventricular injection of 6-hydroxydopamine at postnatal day 3. We observed an increase in senktide-evoked acetylcholine release at postnatal day 30. The effect produced by [Sar9,Met(O2)11]-Substance P and [Nle10]-Neurokinin A4-10 was not modified. Furthermore, at postnatal day 35, we could observed that the two opioid receptor agonists have no effect. Our results show that dopamine, tachykinins and opioids are already able to mediate the modulation of acetylcholine release in early stages of development with a different pattern of postnatal development. Furthermore, the integrity of a dopaminergic system plays an important role in the functional development of the neostriatal cholinergic neurons which are differentially modulated by opioids or tachykinins.

The regulation of tachykinin-evoked acetylcholine release by the dopaminergic system in the neostriatum was examined. We studied the effect of selective and potent tachykinin agonists for each subtype of receptor ([Sar9,Met(O2)11]-Substance P for NK1; [Nle10]-Neurokinin A4-10 for NK2; and senktide for NK3) on endogenous acetylcholine release from rat striatal slices where the dopaminergic system was modified either by 6-hydroxydopamine lesion or by dopamine receptor antagonists. Unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway induced a decrease in senktide-evoked acetylcholine release and an increase in the effect of [Nle10]-Neurokinin A4-10. The same results were obtained after chronic haloperidol treatment, whereas SCH-23390 or clozapine treatment had no effect on tachykinin-evoked acetylcholine release, suggesting an involvement of D2 receptors. 6-hydroxydopamine lesion induced a diminution in the density of NK3 receptor, which could be related to the reduction in senktide-evoked acetylcholine release.

We have studied changes on endogenous acetylcholine (ACh) release evoked by different agents from rat neostriatal slices after quinolinic acid (QA) injections. QA lesions induced a biphasic decrease on ACh release evoked by 1 microM glutamate and 50 mM KCl. ACh release evoked by selective tachykinin agonists was only significantly decreased by 250 nmol QA. These results suggest the presence of different functional cholinergic cell populations, with tachykinin-responsive cholinergic neurons selectively spared.