Long-term exposure to GDNF induces dephosphorylation of Ret, AKT, and ERK1/2, and is ineffective at protecting midbrain dopaminergic neurons in cellular models of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) promotes differentiation, proliferation, and survival in different cell types, including dopaminergic neurons. Thus, GDNF has been proposed as a promising neuroprotective therapy in Parkinson's disease. Although findings from cellular and animal models of Parkinson's disease were encouraging, results emerging from clinical trials were not as good as expected, probably due to the inappropriate administration protocols. Despite the growing information on GDNF action mechanisms, many aspects of its pharmacological effects are still unclear and data from different studies are still contradictory. Considering that GDNF action mechanisms are mediated by its receptor tyrosine kinase Ret, which activates PI3K/AKT and MAPK/ERK signaling pathways, we aimed to investigate Ret activation and its effect over both signaling pathways in midbrain cell cultures treated with GDNF at different doses (0.3, 1, and 10 ng/ml) and times (15 min, 24 h, 24 h (7 days), and 7 continuous days). The results showed that short-term or acute (15 min, 24 h, and 24 h (7 days)) GDNF treatment in rat midbrain neurons increases Tyrosine hydroxylase (TH) expression and the phosphorylation levels of Ret (Tyr 1062), AKT (Ser 473), ERK1/2 (Thr202/Tyr204), S6 (Ser 235/236), and GSK3-ß (Ser 9). However, the phosphorylation level of these kinases, TH expression, and dopamine uptake, decreased below basal levels after long-term or prolonged treatment with 1 and 10 ng/ml GDNF (7 continuous days). Our data suggest that long-term GDNF treatment inactivates the receptor by an unknown mechanism, affecting its neuroprotective capacity against degeneration caused by 6-OHDA or rotenone, while short-term exposure to GDNF promoted dopaminergic cell survival. These findings highlight the need to find new and more effective long-acting therapeutic approaches for disorders in which GDNF plays a beneficial role, including Parkinson's disease. In this regard, it is necessary to propose new GDNF treatment guidelines to regulate and control its long-term expression levels and optimize the clinical use of this trophic factor in patients with Parkinson's disease.

Tetrahydrobiopterin stimulates L-DOPA release from striatal tissue.

In the present study we have analyzed the effect of tetrahydrobiopterin (BH4) essential cofactor for tyrosine hydroxylase and nitric oxide synthase, on the 3,4-dihydroxyphenylalanine (L-DOPA) release from in vitro incubated striatal tissue. dl-6-methyl-5,6,7,8 tetrahydropterine (6-MPH4)-stimulated L-DOPA release in a concentration-dependent manner in the range from 25 to 100 microM. At these concentrations 6-MPH4 did not have any effect on dopamine release. Presence of Nomega-Nitro-L-arginine methyl ester (L-NAME, 200 microM), a nitric oxide synthase inhibitor, but not of alpha-methyl-rho-tyrosine (alpha-MPT, 100 microM), a tyrosine hydroxylase inhibitor, blocked L-DOPA release induced by 6-MPH4 (200 microM). Also, the addition to the incubation medium of melatonin (MEL, 300 microM), which is a scavenger of NO and other free radicals, blocked the L-DOPA release induced by 6-MPH4 (200 microM) but this effect did not occur with the addition of the peroxynitrite scavenger uric acid (UA, 300 microM). Sodium nitroprusside (SNP, 100 muM), a NO generator and l-DOPA releaser as previously reported, potentiated the L-DOPA releasing effect of 6-MPH4 (200 microM) which was also blocked by melatonin. In summary 6-MPH4 stimulates L-DOPA release from striatal fragments incubated in vitro by a mechanism which involves NO or other free radicals derived from NO but not peroxynitrite.

Consequences of unilateral nigrostriatal denervation on the thalamostriatal pathway in rats.

The position of the caudal intralaminar nuclei within basal ganglia circuitry has largely been neglected in most studies dealing with basal ganglia function. During the past few years, there has been a growing body of evidence suggesting that the thalamic parafascicular nucleus in rodents (PF) exerts a multifaceted modulation of basal ganglia nuclei, at different levels. Our aim was to study the activity of the thalamostriatal pathway in rats with unilateral dopaminergic depletion. The experimental approach comprised first unilateral delivery of 6-OHDA in the medial forebrain bundle. Thirty days post-lesioning, animals showing a clear asymmetry were then subjected to bilateral injection of Fluoro-Gold (FG) within the striatum. Subsequently, expression of the mRNA encoding the vesicular glutamate transporter 2 (vGLUT2) was detected within thalamostriatal-projecting neurons (FG-labeled) by in situ hybridization and the results were confirmed by laser-guided capture microdissection microscopy followed by real-time PCR. The data showed that there was a marked neuronal loss restricted to PF neurons projecting to the dopamine-depleted striatum. Moreover, PF neurons innervating the dopamine-depleted striatum were intensely hyperactive. These neurons showed a marked increase on the expression of vGLUT2 mRNA as well as for the mRNA encoding the subunit I of cytochrome oxidase as compared with those neurons projecting to the striatum with normal dopamine content. Thus, the selective neurodegeneration of PF neurons innervating the striatum together with the increased activity of the thalamostriatal pathway coexist after nigrostriatal denervation.

Effect of intracerebroventricular injection of lipopolysaccharide on the tuberoinfundibular dopaminergic system of the rat.

Numerous studies indicate that monoaminergic systems are sensitive to both peripheral and central inflammatory stimuli, and in particular dopaminergic neurons in the nigrostriatal system degenerate after local injection of lipopolysaccharide (LPS). However, data about the response of other dopaminergic groups to local inflammation are very sparse. In this study, we have examined the effect of i.c.v. injection of LPS on the tuberoinfundibular dopaminergic (TIDA) system by using biochemical and morphological parameters. Our results show that 6 h after i.c.v. injection of LPS, in parallel to a transient and intense immunoreaction to interleukin-1beta in arcuate microglial cells, there is a decrease in tyrosine hydroxylase (TH) activity in the median eminence and in the number of TH- and TH mRNA-positive cells in the arcuate nucleus, and at 12 h, an increase of prolactin levels in serum. Posterior changes were found in the TH mRNA labeling pattern, mostly in the ventrolateral region of the arcuate nucleus, but they were not accompanied by any changes in TH activity and immunoreactivity and TH-cell count. This suggests that the TIDA system is functionally susceptible to local inflammation, but the effects are transient and do not induce neurodegeneration.

Response of GABAergic cells in the deep mesencephalic nucleus to dopaminergic cell degeneration: an electrophysiological and in situ hybridization study.

The deep mesencephalic nucleus (DMN) is a large midbrain reticular region located between the substantia nigra compacta and the superior colliculus. It contains GABAergic cells that share striatal afferents, thalamic and collicular efferents, as well as neurochemical and electrophysiological similarities, with those of the substantia nigra reticulata. In the present paper we used electrophysiological (firing rate and firing pattern) and morphological (densitometric analysis of in situ hybridization histochemical labeling for glutamic acid decarboxylase (GAD)65 and GAD67 mRNA) techniques, to study the response of DMN GABAergic cells to the degeneration of nigral dopaminergic cells. Our results showed that unilateral dopaminergic cell loss (after injection of 6-hydroxydopamine in the medial forebrain bundle) induces a bilateral and symmetrical increase in both firing rate and GAD67 mRNA levels and a decrease in GAD65 mRNA levels. These findings support the involvement of DMN GABAergic cells in the basal ganglia modifications that follow dopaminergic cell loss, also suggesting its participation in the pathophysiology of Parkinson's disease. The symmetry of effects, together with its recently reported bilateral projections to the thalamus and superior colliculus, suggest that unlike substantia nigra reticulata, DMN is involved in the interhemispheric regulation of basal ganglia, probably keeping their functional symmetry even after asymmetric lesions.

The deep mesencephalic nucleus as an output center of basal ganglia: morphological and electrophysiological similarities with the substantia nigra.

The deep mesencephalic nucleus (DMN) is a large midbrain reticular region between the superior colliculus, the substantia nigra compacta, the periaqueductal gray, and the medial geniculate body. Although some data suggest that it is involved in nociception and visceral control, its functions remain unclear. In the present study, by using morphological (combination of anterograde and retrograde tracers with immunocytochemistry and in situ hibrydization) and electrophysiological (firing activity and transynaptic response to striatal stimulation) methods, we show that a subpopulation of DMN cells shares many morphological and electrophysiological characteristics with those of the substantia nigra reticulata (SNR). These similarities include the following: 1) firing rate, firing pattern, and conduction velocity; 2) expression of GAD65, GAD67, and PV; 3) excitatory and inhibitory inputs from the striatum; and 4) projections to the ventral thalamus, superior colliculus, and pedunculopontine tegmental nucleus. Some differences were also found. In comparison with SN, DMN cells and striatal afferents are more sparsely distributed and they show conspicuous contralateral projections to the thalamus and superior colliculus. This suggests that, similarly to the SNR, the DMN acts as an output center of basal ganglia and probably facilitates the inter-hemispheric regulation of these centers.

Dopamine cell degeneration induced by intraventricular administration of 6-hydroxydopamine in the rat: similarities with cell loss in parkinson's disease.

In an attempt to find a convenient rat model to study cell vulnerability in Parkinson's disease, we have investigated the cell-loss profile in different midbrain dopaminergic nuclei and subnuclei of rats injected with 6-hydroxydopamine (6-OHDA) in the third ventricle. Following administration of different doses (5-1000 microgram) of 6-OHDA, motor behavior was evaluated and tyrosine hydroxylase-immunostained neurons were counted in the A8 group and different subdivisions of A9 and A10 groups. Animals developed hypokinesia, repetitive chewing movements, and catalepsia. Signs of cell degeneration were evident from the first day after injection, reaching the definitive pattern at the end of the first week. There was a similar degeneration in both brain sides, the A9 group showing the highest degree of cell-loss, followed by A8 and A10 groups. In the A9 group, the degeneration mostly affected those subgroups located in its ventral, lateral, and posterior regions. In the A10 group the degeneration mainly affected the parabrachial pigmented nucleus, the paranigral nucleus and the ventral tegmental area. This topographic pattern of degeneration is very similar to that previously described in Parkinson's disease, suggesting that this model may be a useful tool in the study of the cell vulnerability mechanisms in this neurodegenerative disorder. In addition, our results also showed that small dopaminergic neurons are more resistant to degeneration than the large ones. In some DA subgroups, the cells that contained calbindin but not calretinin were less vulnerable to the neurotoxic effect of 6-OHDA.

Motor behavioural changes after intracerebroventricular injection of 6-hydroxydopamine in the rat: an animal model of Parkinson's disease.

At the beginning of the 1970s, different studies reported behavioural disturbances after the intracerebroventricular (icv) administration of 6-hydroxydopamine (6-OHDA) in the rat. Despite the fact that this neurotoxic agent degenerates brain dopaminergic (DA-) cells, its potential utility to produce a rat model of Parkinson's disease (PD) was never systematically studied because the aphagia and adipsia were often observed. In the present study, a procedure that induces a marked DA-cell degeneration that bypasses these and other undesirable complications of icv injection of 6-OHDA is reported. Lesioned animals (50-500 microg of 6-OHDA) showed a persistent motor syndrome composed of hypokinesia, purposeless chewing and catalepsy. The intensity of motor signs was dose-dependent, and recovered partially after administration of DA-receptor agonists, exposure to sensorial stimuli and stress, three procedures that reduce motor dysfunctions in Parkinson's disease (PD). Lesioned animals showed bilateral and symmetrical midbrain DA-cell degeneration with the highest cell-loss in A9 group (substantia nigra), followed by A8 (retrorubral field) and A10 (ventral tegmental area) groups. The similarity between the behavioural syndrome and the topographical profile of cell-loss after icv injection of 6-OHDA in rats and the clinical and neuropathological features of PD indicates that this may be a convenient animal model of PD particularly useful for checking in rats the possible efficacy of new anti-parkinsonian drugs on specific parameters of motor dysfunctions.

Colocalization of tyrosine hydroxylase and GAD65 mRNA in mesostriatal neurons.

Although dopamine has been considered as the only neurotransmitter in the nigrostriatal pathway, studies carried out in the last two decades have suggested the existence of a nondopaminergic nigrostriatal projection, and more recently, gamma-aminobutyric acid (GABA) has been identified as its neurotransmitter. In this study, we used the combination of immunocytochemistry for tyrosine hydroxylase (TH; a marker of dopaminergic neurons), in situ hybridization (ISH) for two different isoforms of glutamic acid decarboxylase (GAD65 and GAD67, the rate-limiting enzyme in GABA synthesis) and retrograde tracing techniques to investigate the possible existence of nigrostriatal neurons containing both neurotransmitters (dopamine and GABA) in the rat. Our results revealed that approximately 10% of mesostriatal dopaminergic neurons, most of them lying in the medial region of the substantia nigra pars compacta (SNC) and neighbouring A10 region, contain GAD65 mRNA. These findings reveal a third nigrostriatal pathway formed by dopaminergic/GABAergic neurons. Contrasting with the idea that in the basal ganglia, dopamine and GABA are released from different cell populations, the results suggest a more complex dopamine/GABA interaction than previously assumed, probably including cotransmission.