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3,4-Dihydroxyphenyl ethanol, known as hydroxytyrosol (HTy), is a phenylpropanoid found in diverse vegetable species. Several studies have demonstrated that HTy is a potent antioxidant. Thus, our study is aimed to evaluate the antioxidant effect of HTy and its derivatives, hydroxytyrosol acetate (HTyA) and nitrohydroxytyrosol (HTyN), in a model of oxidative stress induced by 1-methyl-4-phenylpyridinium (MPP+) in rats. Rats were administered intravenously (i.v.) in the tail with 1 mL saline solution or polyphenol compound (1.5 mg/kg) 5 min before intrastriatal infusion of 10 µg MPP+/8 µL. We found that rats injured with MPP+, pretreatment with HTy, HTyA or HTyN significantly decreased ipsilateral turns. This result was consistent with a significant preservation of striatal dopamine levels and decreased lipid fluorescence products (LFP), a marker of oxidative stress. Brain GSH/GSSG ratio, from rats pretreated with HTy or HTyN showed a significant preservation of that marker, decreased as a consequence of MPP+-induced oxidative damage. These results show an antioxidant effect of HTy, HTyA and HTyN in the MPP+ model of Parkinson’s disease in the rat.

New neuroprotective treatments of natural origin are being investigated. Both, plant extracts and isolated compounds have shown bioactive effects. Hempseed is known for its composition of fatty acids, proteins, fibre, vitamins, as well as a large number of phytochemical compounds. After a defatting process of the seeds, hydroxycinnamic acids and its amine derivatives are the majoritarian compounds in an ethyl acetate fraction (EAF). In the present study, we investigated

According to Braak’s hypothesis, it is plausible that Parkinsońs disease (PD) starts in the enteric nervous system (ENS) to spread the brain via the vagus nerve. Thus, we were wondering whether human inflammatory bowel diseases (IBD) can progress with appearance of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. Analysis of human gastrointestinal tract sections from IBD patients demonstrated the presence of pathogenic phosphorylated α-syn in both myenteric (Auerbach’s) and submucosal (Meissner’s) plexuses. Remarkably, PD subjects exhibit α-syn pathology in identical gastrointestinal locations. Analysis of human midbrain sections from IBD subjects revealed a clear displacement of neuromelanin in some nigral neurons from the ventral mesencephalon, which were inherently associated with presence of α-syn aggregates reminiscent of pale bodies. We also used different dextran sodium sulfate (DSS)-based rat models of gut inflammation (subchronic and chronic) to study the appearance of phosphorylated α-syn inclusions in both Auerbach’s and Meissner’s plexuses (gut), and in dopaminergic neuritic processes (brain) along with degeneration of nigral dopaminergic neurons, which are considered classical hallmarks of PD. Vagotomized DSS-treated animals exhibited pathological α-syn in the gut but failed to show dopaminergic cells degeneration and α-syn aggregation in the ventral mesencephalon. Taken together, these results strongly suggest that Braak’s hypothesis is plausible.

Parkinson’s disease is a highly prevalent neurological disorder for which there is currently no cure. Therefore, the knowledge of risk factors as well as the development of new putative molecular targets is mandatory. In this sense, peripheral inflammation, especially the originated in the colon, is emerging as a predisposing factor for suffering this disease. We have largely studied the pleiotropic roles of galectin-3 in driving microglia-associated immune responses. However, studies aimed at elucidating the role of galectin-3 in peripheral inflammation in terms of microglia polarization are lacking. To achieve this, we have evaluated the effect of galectin-3 deletion in two different models of acute peripheral inflammation: intraperitoneal injection of lipopolysaccharide or gut inflammation induced by oral administration of dextran sodium sulfate. We found that under peripheral inflammation the number of microglial cells and the expression levels of pro-inflammatory mediators take place specifically in the dopaminergic system, thus supporting causative links between Parkinson’s disease and peripheral inflammation. Absence of galectin-3 highly reduced neuroinflammation in both models, suggesting an important central regulatory role of galectin-3 in driving microglial activation provoked by the peripheral inflammation. Thus, modulation of galectin-3 function emerges as a promising strategy to minimize undesired microglia polarization states., This work was supported by grants from the Spanish Ministerio de Ciencia, Innovación y Universidades (RTI 2018-098830-B-I00), from the Consejería de Economía y Conocimiento of Junta de Andalucía (P18-RT-1372 and US-1264806). MJP, MDVC and PGM were supported by a grant from the Junta de Andalucía (CTS 5884) and AEC by an associated post-doctoral grant.

3,4-Dihydroxyphenyl ethanol, known as hydroxytyrosol (HTy), is a phenylpropanoid found in diverse vegetable species. Several studies have demonstrated that HTy is a potent antioxidant. Thus, our study is aimed to evaluate the antioxidant effect of HTy and its derivatives, hydroxytyrosol acetate (HTyA) and nitrohydroxytyrosol (HTyN), in a model of oxidative stress induced by 1-methyl-4-phenylpyridinium (MPP

Apoptotic caspases are thought to play critical roles in elimination of excessive and non-functional synapses and removal of extra cells during early developmental stages. Hence, an impairment of this process may thus constitute a basis for numerous neurological and psychiatric diseases. This view is especially relevant for dopamine due to its pleiotropic roles in motor control, motivation and reward processing. Here, we have analysed the effect of caspase-3 depletion on the development of catecholaminergic neurons and performed a wide array of neurochemical, ultrastructural and behavioural assays. To achieve this, we performed selective deletion of the Casp3 gene in tyrosine hydroxylase (TH)-expressing cells using Cre-loxP-mediated recombination. Histological evaluation of most relevant catecholaminergic nuclei revealed the ventral mesencephalon as the most affected region. Stereological analysis demonstrated an increase in the number of TH-positive neurons in both the substantia nigra and ventral tegmental area along with enlarged volume of the ventral midbrain. Analysis of main innervating tissues revealed a rather contrasting profile. In striatum, basal extracellular levels and potassium-evoked DA release were significantly reduced in mice lacking Casp3, a clear indication of dopaminergic hypofunction in dopaminergic innervating tissues. This view was sustained by analysis of TH-labelled dopaminergic terminals by confocal and electron microscopy. Remarkably, at a behavioural level, Casp3-deficient mice exhibited impaired social interaction, restrictive interests and repetitive stereotypies, which are considered the core symptoms of autism spectrum disorder (ASD). Our study revitalizes the potential involvement of dopaminergic transmission in ASD and provides an excellent model to get further insights in ASD pathogenesis., This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (SAF2015-64171-R and RTI2018-098645-B-I00). J.A.A. and E.P.V. was supported by a MINECO (BFU2015-64536) grant. I.S.P., E.B. and L.B. were co-financed by the “Fondo Europeo de Desarrollo Regional” (FEDER)-UE “A way to build Europe” from the “Ministerio de Ciencia, Innovación y Universidades” (RTI2018-099778-B-I00) and the Ministerio de Salud-Instituto de Salud Carlos III (PI18/01691), as well as funding from the “Consejería de Salud de la Junta de Andalucía” (PI-0134-2018); the “Programa Operativo de Andalucía FEDER, Iniciativa Territorial Integrada ITI 2014-2020 Consejería Salud, Junta de Andalucía” (PI-0080-2017); the “Consejería de Economía, Innovación, Ciencia y Empleo de la Junta de Andalucía” (CTS-510); and the “Centro de Investigación Biomédica en Red de Salud Mental- CIBERSAM” (CB/07/09/0033).

Aims The natural phenolic oil compound hydroxytyrosol (HTy) is widely studied because of its antioxidant and neuroprotective properties. Nitroderivatives of HTy have been studied in order to evaluate their putative effects on catechol- O -methyltransferase (COMT) activity. Main methods To study its effect on dopamine metabolism, nitrohydroxytyrosol and its lipophilic derivatives (nitrohydroxytyrosyl acetate and ethyl nitrohydroxytyrosyl ether), were administered into the rat corpus striatum through a microdialysis probe. Other catechols (HTy and the known COMT inhibitor Ro 41-0960) were also studied for comparison. Key findings The olive oil phenolic compounds (nitroderivatives and HTy) increased extracellular levels of 3,4-dihydroxyphenylacetic acid during the perfusion with similar maximum values to that of Ro 41-0960 when comparing to basal dialysate levels (approximately 140%). None of the compound series produced a decrease in the homovanillic acid extracellular levels below 75%. Among all novel compounds studied, both lipophilic nitrocatechols (nitrohydroxytyrosyl acetate and ethyl nitrohydroxytyrosyl ether) showed a long-acting effect over time once the perfusion through the microdialysis probe ended. Significance In accordance with the actual design of novel COMT inhibitors with a long profile, our results suggest a certain influence of the side chain substituent on the COMT activity that could provide new lipophilic COMT inhibitors.

The impact of systemic inflammation in nigral dopaminergic cell loss remains unclear. Here, we have investigated the role of peripheral inflammation induced by systemic lipopolysaccharide (LPS) administration in the MPTP-based model of Parkinson’s disease. Brain inflammation, microglia and astroglia activation, disruption of the blood–brain barrier (BBB) and integrity of the nigrostriatal dopaminergic system were evaluated in response to i.p. injection of LPS, MPTP or the combination of both. Our results showed that combinative treatment exacerbates microglia activation and enhances (i) the appearance of galectin-3-positive microglia, recently identified as microglial disease-associated phenotypic marker, (ii) the up-regulation of pro-inflammatory cytokines, (iii) the occurrence of A1 neurotoxic astrocytes, (iv) the breakdown of the BBB, and (v) the loss of dopaminergic neurons in the substantia nigra. Microglia activation was triggered earlier than other degenerative events, suggesting that over-activation of microglia (including different polarization states) may induce dopaminergic neuron loss by itself, initiating the endless cycle of inflammation/degeneration. Our study revitalizes the importance of peripheral inflammation as a potential risk factor for Parkinson’s disease and raises the possibility of using new anti-inflammatory therapies to improve the course of neurodegenerative diseases, including those directly aimed at modulating the deleterious activity of disease-associated microglia., This work was supported by a grant from the Spanish Ministerio de Economia y Competitividad SAF2015-64171-R (MINECO/FEDER, EU).

Hydroxytyrosol is a well-known phenolic compound with antioxidant properties that is found in virgin olive oil. Studies have shown that virgin olive oil has neuroprotective effects in rats; thus the purpose of the present study was to investigate the neuroprotective effect of hydroxytyrosol in rats. Additionally, this study aimed to investigate the neuroprotective potential of a homologous series of compounds with better lipophilic profiles in order to increase the assortment of compounds with a putative effect against Parkinson's disease (PD). In this context, the inhibition of catechol-O-methyl transferase (COMT) activity by hydroxytyrosol, nitrohydroxytyrosol, nitrohydroxytyrosol acetate and ethyl nitrohydroxytyrosol ether was investigated by measuring intracellular dopamine and its metabolite levels in the corpus striatum by high performance liquid chromatography (HPLC) with electrochemical detection. The animals received an acute (single dose; 20 mg kg-1; i.p.) or chronic (one daily dose for 5 days; 20 mg kg-1; i.p.) treatment of hydroxytyrosol and its nitroderivatives. For comparison, a commercial COMT inhibitor, Ro 41-0960, was also included. Our data show that acute and chronic systemic administration of these compounds produced a clear and statistically significant increase in the intracellular levels of dopamine and its metabolite, 3,4-dihydroxyphenylacetic acid. The increase in dopamine levels was very similar to the increase seen with Ro 41-0960 treatment. The effect of chronic treatment was stronger than that of acute treatment. With respect to the intracellular level of homovanillic acid, Ro 41-0960 produced a statistically significant decrease which it was not observed when hydroxytyrosol and its nitroderivatives were systemically administered. However, the chronic homovanillic acid treatment effect was stronger than the acute treatment. The results suggest that these compounds could inhibit COMT activity.

The involvement of dopaminergic (DAergic) receptor drugs in the neuroprotection against the neurotoxic action of 1-methyl-4-phenylpyridinium (MPP+) in the DAergic terminals in striatum was studied using an intracerebral microdialysis technique. Twenty-four hours after surgery (day 1), apomorphine and haloperidol, alone or with 1 mmol/l of MPP+ perfusion through the microdialysis probe, were systemically administered. Forty-eight hours after surgery (day 2), 1 mmol/l of MPP+ was perfused for 15 min in all groups of animals and the output of dopamine was measured. The amount of dopamine was directly proportional to the remaining striatal DAergic terminals. The results show that: (1) subcutaneous administration of apomorphine before MPP+ perfusion prevented MPP+-induced neurotoxicity, and (2) intraperitoneal administration of haloperidol before MPP+ perfusion did not prevent MPP+-induced neurotoxicity.

Due to their potential role in preventing further deterioration of Parkinson's disease, anti-inflammatory strategies have attracted great interest. In this context, some studies point out the possible protective effect of anti-inflammatory compounds against the in vivo degeneration of dopaminergic neurons produced by lipopolysaccharide (LPS)-induced inflammatory processes and others. We have investigated the effect of the treatment of Zocor Forte® (simvastatin) in LPS and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurodegenerative models to identify neuroprotective drugs for Parkinson's disease. We have perfused different concentrations of LPS or 1 mM 1-methyl-4-phenylpyridinium ion (MPP + ) in the rat's striatum, 24 h after implanting a brain microdialysis probe, both with and without Zocor Forte® (simvastatin) treatment. Results show that LPS perfusion produced a decrease in the basal release of dopamine. Forty-eight hours after implanting the probe, we have perfused 1 mM MPP + to check the integrity of the dopaminergic terminals present around the cannula. Our model to study toxicity in the striatal dopaminergic terminals suggests that Zocor Forte® (simvastatin) could prevent the neurotoxic damage produced by LPS, but not that produced by MPP + .

Anti-inflammatory strategies have attracted much interest for their potential to prevent further deterioration of Parkinson's disease. Recent experimental and clinical evidence indicate that statins - extensively used in medical practice as effective lipid-lowering agents - have also anti-inflammatory effects. In this study, we investigated the influence of simvastatin on the degenerative process of the dopaminergic neurons of the rat following intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation that we have previously used as an animal model of Parkinson's disease. We evaluated TH positive neurons, astroglial, and microglial populations and found that simvastatin prevented the inflammatory processes, as the induction of interleukin-1beta, tumor necrosis factor-alpha, and iNOS and the consequent dopaminergic degeneration induced by LPS. Moreover, simvastatin produced the activation of the neurotrophic factor BDNF, along with the prevention of the oxidative damage to proteins. Moreover, it also prevents the main changes produced by LPS on different mitogen-activated protein kinases, featured as increases of P-c-Jun N-terminal protein kinase, P-extracellular signal-regulated kinase, p-38, and P-glycogen synthase kinase and the decrease of the promotion of cell survival signals such as cAMP response element-binding protein and Akt. Our results suggest that statins could delay the progression of dopaminergic degeneration in disorders involving inflammatory processes.

The present study is concerned with the hypothetical toxicity of β-N-methylamino- l -alanine (BMAA), a compound that has been hypothesized to produce amyotrophic lateral sclerosis/Parkinson-dementia complex. We have used the microdialysis technique to perfused different concentrations of BMAA in the rat's striatum 24 h after the implantation of a microdialysis probe (day 1). BMAA perfusion produced a dose-response increase in the extracellular output of dopamine. Forty-eight hours after implantation of the probe (day 2), we have perfused MPP+ 1 mM to check the integrity of the dopaminergic terminals present around the cannula. Only the highest concentration of BMAA studied, 50 mM, produced a clear decrease in the extracellular output of dopamine after MPP+ perfusion. However, this decrease was very similar, even smaller, to that obtained in a previous study carried out by us with MPP+ 1 mM, a dose much lower than that used for BMAA. Our model to study toxicity in the striatal dopaminergic terminal did not show that acute perfusion of BMAA at high doses produces a clear damage to the dopaminergic terminals.

We have evaluated the ability of the injured nigrostriatal dopaminergic system to produce highly reactive hydroxyl radicals (•OH) by the electrochemical detection of salicylate hydroxylation. Unilateral transection of the medial forebrain bundle transiently increased the formation of •OH in substantia nigra (SN) but not in striatum during the first 48 h postlesion, when most relevant changes in terms of oxidatively modified proteins take place. Short-term adaptive axotomy-induced changes in substantia nigra included downregulation of nigral tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA expression and more intense TH immunoreactivity. Maintained inhibition of monoamine oxidase activity with deprenyl totally prevented the axotomy-induced formation of •OH, thus demonstrating the dopaminergic nature of these radicals. In contrast, deprenyl treatment, which is associated with a diminution in free radical production, failed to delay the onset of dopaminergic degeneration. This observation highlights the importance of being extremely cautious when analyzing parameters of oxidative stress and extrapolating them as a primary cause of cell death in the context of neurodegeneration. Long-term adaptive changes included a dramatic downregulation of DAT mRNA expression along with a moderate decrease in TH mRNA levels in SN. We anticipate a key regulatory role of the DAT to maximally optimize dopaminergic transmission in the synaptic cleft under conditions of degeneration.

Microdialysis was used to evaluate the effect of desferrioxamine (DES) against 1-methyl-4-phenylpyridinium (MPP + ) toxicity. The presence of DES (40 fmol-40 nmol/15 min for a total of 90 min) in the Ringer solution, coperfused with MPP + (40 nmol/15 min) on day 1, produced on day 2 a higher extracellular dopamine output after perfusion of MPP + than in control MPP + perfusion experiments, in which no DES was administered on day 1. Both Ringer perfusion alone (control Ringer) and coperfusion of 40 nmol DES with 40 nmol MPP + on day 1 produced on day 2 similar increases in extracellular dopamine output after a second MPP perfusion. In the control Ringer experiment, note that the MPP + on day 2 is the first MPP + perfusion. Perfusion of 800 fmol FeCl 3 / 15 min along with 40 nmol MPP + and 400 fmol DES on day 1 completely abolished on day 2 the neuroprotective effect found with 40 nmol MPP + and 400 fmol DES; 800 fmol FeCl 3 did not increase the neurotoxic effect of 40 nmol MPP + perfusion. The ability of DES to protect against MPP toxicity may indicate a therapeutic strategy in the treatment of diseases when iron is implicated.

We have previously described a neuroprotective action of (2S,2′R,3′R)-2-(2′3′-dicarboxycyclopropyl)glycine (DCG-IV), an agonist for group-II metabotropic receptors, on dopaminergic nerve terminals against the degeneration induced by 1-methyl-4-phenylpyridinium (MPP+). This effect was accompanied by an up-regulation of brain-derived neurotrophic factor (BDNF) mRNA expression in the rat striatum. We have now analyzed the phenotypic nature of the BDNF mRNA-expressing cells in response to intrastriatal injection of DCG-IV. Dual in situ hybridization and immunohistochemistry revealed that microglial cells but not astrocytes were responsible for this induction. Subsequent analysis demonstrated that this effect was accompanied by striking loss of striatal glutamic acid decarboxylase (GAD) mRNA and massive appearance of internucleosomal DNA fragmentation, a hallmark of apoptosis. A dose–response study demonstrated that doses of DCG-IV as low as 5 nmol was very toxic in terms GAD mRNA and apoptosis. 0.5 nmol of DCG-IV did not induce toxicity at all in terms of GAD mRNA and apoptosis. Activation of group-II metabotropic receptors in striatum with N -Acetyl-Asp-Glu (NAAG; a mGlu3 agonist) and (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (a mGlu2 and mGlu3 agonist) did not induce neither loss of GAD mRNA nor appearance of apoptosis (doses up to 20 nmol). In additional experiments, NAAG, in contrast to DCG-IV, failed to protect the striatal dopaminergic system against the degeneration induced by MPP+ as studied by microdialysis. Finally, we studied the mechanism by which DCG-IV is highly toxic. For that, selective antagonists of either metabotropic – (R,S)-α-methyl-4-carboxyphenylglycine and LY 341495 – or ionotropic ( N -methyl- D -aspartate, NMDA) – DL -2-amino-5-phosphonovaleric acid (AP-5) glutamate receptors – were co-administered with DCG-IV. Only AP-5 highly protected the striatum against the degeneration induced by DCG-IV. Since DCG-IV also activates the NMDA receptor at concentrations higher than 3 μM, it is conceivable that a intrastriatal concentration equal or higher than 3 μM after a single striatal injection of 5–20 nmol of DCG-IV. Our findings suggest that much caution must be exerted when testing the numerous neuroprotective effects ascribed to group-II metabotropic receptor activation, in particular when using DCG-IV. We conclude that the neuroprotectant capability of a given compound on a specific system does not exclude the possibility of inducing toxicity on a different one.

Phenolic compounds were measured by in vivo brain microdialysis in rat striatum. Basal extracellular levels of hydroxytyrosol, homovanillic alcohol and nitro-hydroxytyrosol were not detectable by HPLC with electrochemical detection. However, systemic administration of hydroxytyrosol (20 and 40 mg/kg, i.p.) showed a clear increase in the extracellular level of this compound. This increase was accompanied by an increase in the extracellular level of homovanillic alcohol, a metabolite of hydroxytyrosol formed by catechol-O-methyltransferase activity. Perfusion of hydroxytyrosol (20 μM) through the microdialysis cannula also produced an increase in the extracellular level of homovanillic alcohol. Systemic administration of nitro-hydroxytyrosol (20 and 40 mg/kg, i.p.) produced a small increase in the extracellular level of this compound. Our data show that hydroxytyrosol is a more brain penetrant phenolic compound than nitro-hydroxytyrosol. Accordingly, there is high cerebral metabolism of hydroxytyrosol to produce homovanillic alcohol by catechol-O-methyltransferase activity, that is saturated at the higher administered dose of hydroxytyrosol.

We have studied the in vivo effect of the selective agonist for group II metabotropic glutamate receptors (2S, 2'R, 3'R)-2-(2'3'-dicarboxycyclopropyl)glycine (DCG-IV) against MPP+-induced toxicity on rat striatal dopaminergic nerve terminals by using both microdialysis and immunohistochemical techniques. Perfusion of 1 mM DCG-IV during 1 h protected dopaminergic nerve terminals against the degeneration induced by a 15-minute perfusion of 1 mM MPP+. In addition, the microglial cell population was markedly activated 24 h after DCG-IV perfusion. The astroglial cell population was only markedly activated around the microdialysis probe. This protective effect seems to be dependent on protein synthesis since 1 mM cycloheximide, an inhibitor of protein synthesis, abolished the neuroprotective effect of 1 mM DCG-IV against MPP+ toxicity. Perfusion of DCG-IV induced an upregulation of striatal brain-derived neurotrophic factor (BDNF) mRNA expressing cells which were confined precisely around the microdialysis probe. Taken together, our results suggest that the induction and release of brain-derived neurotrophic factor (BDNF) by activated glial cells induced by DCG-IV perfusion may account for its protective action against MPP+-induced dopaminergic terminal degeneration.

We have used the microdialysis technique to perfuse different concentrations of LPS in the rat's striatum 24 h after the implantation of a microdialysis probe. Dopamine metabolites in the dialysate obtained from the rat brain were measured by HPLC using electrochemical detection. Results show that intrastriatal perfusion of different concentrations of LPS produced a dose-dependent decrease in the extracellular DOPAC output, with no effect on the extracellular HVA output. Since DOPAC levels reflect the intraneuronal metabolism of dopamine while and HVA levels reflect the extraneuronal one, we suggest that in vivo intrastriatal LPS perfusion especially affects the level of newly synthesized dopamine or intraneuronal dopamine catabolism.

We have evaluated the effect of a vitamin E-deficient diet on the rat nigrostriatal dopaminergic system. After 15 days of deficient diet, the amount and activity of striatal and nigral tyrosine hydroxylase increased, which contrasted with a decreased messenger RNA expression for tyrosine hydroxylase and the dopamine transporter in the ventral mesencephalon. When we prolonged the deficiency of vitamin E for 30 days, dopamine levels did not differ in both areas. In contrast, messenger RNA levels for tyrosine hydroxylase and the dopamine transporter were markedly reduced in 30-day deficient rats. In addition, the number of oxidatively modified proteins significantly increased in the striatal and nigral areas studied. Overall, we propose that these changes suggest an important role of vitamin E in maintaining the normal equilibrium of the dopaminergic nigrostriatal system.

The neurotoxic effect of dopamine (DA) and iron(III) on DAergic terminals in striatum has been studied by intracerebral microdialysis technique. Twenty-four hours after surgery (day 1), DA and/or iron(III) with and without DA reuptake inhibitor, nomifensine, were perfused for 1 h. Forty-eight hours after surgery (day 2), MPP(+) 1 mM was perfused for 15 min and the output of DA was measured, its amount being directly proportional to the remaining striatal DAergic terminals, supported by tyrosine hydroxylase immunohistochemistry technique. Perfusion of exogenous DA, as well as iron(III) 10 and 100 microM, did not produce any neurotoxic effect. However, perfusion of iron(III) (333 and 1000 microM) produced a concentration-dependent toxic effect. Co-perfusion of iron(III) at non-toxic concentration (100 microM) with DA (15 microM) produced a toxic effect. Elevation of the endogenous extracellular levels of DA by inhibiting its uptake with nomifensine increased the neurotoxic effect of iron(III) in a dose-dependent manner. The use of tetrodotoxin after elevation of DA with nomifensine partially prevented the neurotoxic effect of its co-perfusion with iron(III) (100 microM). These results suggest that DAergic system could be synergistically damaged by DA and iron(III). Thus, alterations in the clearance of DA from extracellular space along with an increase of iron may have significant consequences for DAergic system toxicity.

In vivo microdialysis was used to investigate the mechanism behind the increase in extracellular dopamine (DA) induced by increase in extracellular serotonin (5-HT) level and 5-HT1 and 5-HT2 receptor activation. The following serotoninergic drugs were perfused in the absence or presence of nomifensine (5 microM) or tetrodotoxin (TTX; 2 microM): clomipramine (10, 500 and 1,000 microM), a selective 5-HT reuptake inhibitor; 8-OH-DPAT (50 and 500 microM), a 5-HT1A receptor agonist; and alpha-methyl-5-HT (1, 5 and 50 microM), a 5-HT2 receptor agonist. All the serotoninergic drugs studied increased DA extracellular output in a dose-dependent manner. The presence of nomifensine attenuated the effect of perfusion of clomipramine (500 microM) and completely abolished the effect of perfusion of 8-OH-DPAT (500 microM) and alpha-methyl-5-HT (5 microM) on DA extracellular output. Clomipramine (100-1,000 microM) perfusion produced a dose dependent increase in DOPAC extracellular output, which was stronger when clomipramine (500 microM) was co-perfused with nomifensine. 8-OH-DPAT and alpha-methyl-5-HT perfusion decreased DOPAC overflow. Addition of TTX to the perfusion fluid one hour before serotoninergic drugs perfusion, did not completely abolish the effect on dopamine extracellular output produced by the serotoninergic drugs. These data seem to indicate that increase in extracellular 5-HT level and 5-HT1 and 5-HT2 receptor activation increase in vivo DA extracellular output in the striatum mainly by a nonexocytotic mechanism involving DA uptake sites and, secondarily, by activation of 5-HT receptors.

The present study concerns the possible relationship between glutamate excitotoxicity and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenylpyridinium (MPTP/MPP+) neurotoxicity on striatal dopaminergic terminals. MPP+ neurotoxicity has been studied by means of two MPP+ perfusions separated by 24 h. After the second MPP+ 1 mM perfusion, dopamine extracellular output, measured by microdialysis, was considered to be an index of the dopaminergic neurone damage produced by the first MPP+ 1 mM perfusion. High concentration (10 mM) of glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) stimulated basal release of dopamine and protected against the neurotoxic effect of MPP+. PDC 10 mM perfusion produced an increase in the extracellular output of glutamate and aspartate, and a decrease in that of ascorbate. The protective effect against MPP+ toxicity observed with PDC 10 mM was completely abolished when this glutamate uptake inhibitor was co-perfused with ascorbate 0.5 mM. These results suggest that glutamate-induced neurotoxicity is not involved in MPP+ toxicity. The protective effect found with the glutamate uptake inhibitor could be due to a decrease in extracellular ascorbate levels.

The involvement of dopaminergic (DAergic) receptor drugs in the neuroprotection against the neurotoxic action of 1-methyl-4-phenylpyridinium (MPP(+)) in the DAergic terminals in striatum was studied using an intracerebral microdialysis technique. Twenty-four hours after surgery (day 1), apomorphine and haloperidol, alone or with 1 mmol/l of MPP(+) perfusion through the microdialysis probe, were systemically administered. Forty-eight hours after surgery (day 2), 1 mmol/l of MPP(+) was perfused for 15 min in all groups of animals and the output of dopamine was measured. The amount of dopamine was directly proportional to the remaining striatal DAergic terminals. The results show that: (1) subcutaneous administration of apomorphine before MPP(+) perfusion prevented MPP(+)-induced neurotoxicity, and (2) intraperitoneal administration of haloperidol before MPP(+) perfusion did not prevent MPP(+)-induced neurotoxicity.

Research indicates that inflammation and microglial activation are involved in the initiation and progression of Parkinson's disease (PD). Neuroinflammation contributes to the infiltration of peripheral immune cells and blood–brain barrier (BBB) leakage, linking peripheral and central inflammatory events in the pathogenesis of PD. Dopamine (DA) likely plays a role in this process. In the present study, the dopaminergic toxin 6-hydroxydopamine (6-OHDA) was used to damage dopaminergic neurons. Injection of 6-OHDA within the nigrostriatal pathway produced loss of astrocytes, disruption of the BBB, microglia activation and a reduction in osteopontin (OPN) immunoreactivity. Depletion of DA content by alpha-methylparatyrosine (α-MPT, a tyrosine hydroxylase inhibitor) reduced the infiltration of peripheral macrophages as well as the 6-OHDA-induced increase in microglial cells. DA could therefore be relevant in sustaining inflammation and lymphocyte recruitment induced by 6-OHDA, supporting DA implication in the degeneration of dopaminergic neurons induced by inflammatory processes.

1-methyl-4-phenylpyridinium (MPP+) is the bioactivated product of 1-methyl-4-phenyl- 1, 2, 3, 6-tetrahydropyri dine (MPTP). The neurotoxic action of MPP+ injected intracerebroventricularly (ICV) in the rat has been stud ied, using dopaminergic systems in the substantia nigra, striatum, olfactory bulb, median eminence and hypoph ysis. The following results were obtained: (1) Rats with ICV administration of 1 μl MPP+ solution (62.5 μg of MPP+ rat) showed 50% mortality; (2) The ICV administration of MPP+ produced a decrease in dopamine (DA) concentra tion in different areas of rat CNS studied: striatum (83%), hypophysis (95%) and median eminence (70%). However, olfactory bulb and substantia nigra were not affected; (3) MPP+ by ICV administration produced neurotoxic effect on the dopaminergic system. We also studied the possible protective action of acetyl-L-carnitine (ALC) against the neurotoxic action of MPP+. Rats were intraperitoneally injected daily for 8 days with 100 mg kg-1 of ALC and 3 days from the beginning of the MPP+ treatment; (4) We found that the ALC treatment significantly protected against mortality produced by the ICV injection of MPP+. Rats treated with ALC showed no mortality; (5) We did not find a protective effect on the dopaminergic system study ing either catecholamine concentration or measuring tyro sine hydroxylase, neurofilament or glial fibrillary acid protein; (6) The results suggest that the ALC protective action could be related to energy metabolism.

1. In vivo microdialysis was used to study the effect of phenylbiguanide (PBG), a 5-hydroxytryptamine3 receptor agonist, on the extracellular output of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the corpus striatum. 2. PBG produced a dose-related (10-500 microM) increase in the release of dopamine (280-2000%). DOPAC and HVA output decreased with the perfusion of PBG. This decrease was similar with 50-500 microM PBG. 5-HIAA output was not affected by any PBG concentration used. 3. When nomifensine (5 microM) was included in the Ringer solution, the effect of PBG on the release of dopamine was ameliorated or inhibited. However, the effect of PBG (50-500 microM) on the extracellular output of DOPAC and HVA was similar in the absence and in the presence of nomifensine (5 microM). 4. Perfusion of MDL 72222, a 5-hydroxytryptamine3 receptor antagonist, at doses of 50 and 100 microM produced similar decreases (50% of controls) and increases (120% of controls) in the extracellular output of dopamine and DOPAC, respectively. HVA and 5-HIAA output levels were not affected by either concentration of MDL 72222. MDL 72222 (10 microM) produced a slight and transient increase in the release of dopamine and a decrease in the extracellular output of DOPAC. HVA and 5-HIAA extracellular output was not affected by MDL 72222 (10 microM) perfusion. 5. Co-perfusion of MDL 72222 (10 and 100 microM) or tetrodotoxin (1 microM) with PBG (50 microM) did not modify the effect produced by PBG (50 microM) alone on the release of dopamine. 6 These results suggest that the effect of PBG on the release of dopamine is mainly carrier-mediated.

Evidence supports the role of inflammation in the development of neurodegenerative diseases. In this work, we are interested in inflammation as a risk factor by itself and not only as a factor contributing to neurodegeneration. We tested the influence of a mild to moderate peripheral inflammation (injection of carrageenan into the paws of rats) on the degeneration of dopaminergic neurons in an animal model based on the intranigral injection of lipopolysaccharide (LPS), a potent inflammatory agent. Overall, the treatment with carrageenan increased the effect of the intranigral injection of LPS on the loss of dopaminergic neurons in the SN along with all the other parameters studied, including: serum levels of the inflammatory markers TNF-α, IL-1β, IL-6 and C-reactive protein; activation of microglia, expression of proinflammatory cytokines, the adhesion molecule ICAM and the enzyme iNOS, loss of astrocytes and damage to the blood brain barrier (BBB). The possible implication of BBB rupture in the increased loss of dopaminergic neurons has been studied using another Parkinson's disease animal model based on the intraperitoneal injection of rotenone. In this experiment, loss of dopaminergic neurons was also strengthened by carrageenan, without affecting the BBB. In conclusion, our data show that a mild to moderate peripheral inflammation can exacerbate the degeneration of dopaminergic neurons caused by a harmful stimulus. © 2012 Elsevier Inc., This work was supported by grants SAF2007-62425, SAF2008-04366, SAF2009-13778 and a Proyecto de Excelencia de la Junta de Andalucía. MS and MJDC thank Junta de Andalucía for a Beca de Proyecto de Excelencia.

The question was investigated whether dopamine release-controlling receptors are evenly distributed over somatodendritic sites and nerve terminals of nigrostriatal dopaminergic neurons of the rat. Prototypical drugs of 5 different (sub)types of receptors (D-2, cholinergic, GABA(B), and non-NMDA) were infused via a microdialysis probe into the striatum, and effects on dopamine released from nerve terminals were determined by microdialysis. In separate experiments the same drugs were infused into the substantia nigra and effects on dendritic release of dopamine were recorded. In addition, the effect of calcium depletion and tetrodotoxin infusion (1 mu mol/l) was studied in both areas. Infusion of(-)-N0437 (1 mu mol/l), (-)-sulpiride (1 mu mol/l), NMDA (300 mu mol/l), AMPA (100 mu mol/l), kainic acid (30 mu mol), tetrodotoxin and depletion of calcium induced comparable changes in the release of dopamine when applied into the striatum as well as into the nigra. Carbachol (100 mu mol/l) and baclofen (5 mu mol/l) inhibited dendritic dopamine release when administered into the nigra; however, the latter drugs were not effective when infused into the striatum. It is concluded that the release-controlling receptors are not evenly distributed over somata and nerve terminals of dopaminergic neurons.

We have developed an animal model of degeneration of the nigrostriatal dopaminergic neurons, the neuronal system involved in Parkinson’s disease (PD). The implication of neuroinflammat ion on this disease was originally established in 1988, when the presence of activated microglia in the substantia nigra (SN) of parkinsonians was reported by McGeer et al. Neuroinflammation could be involved in the progression of the disease or even has more direct implications. We injected 2 μ gofthepotent proinflammatory compound lipopolysaccharide (LPS) in di ff erent areas of the CNS, finding that SN displayed the highest inflammatory response and that dopaminergic (body) neurons showed a special and specific sensitivity to this process with the induction of selective dopaminergic degeneration. Neurodegeneration is induced by inflammation since it is prevented by anti- inflammatory compounds. The special sensitivity of dopaminergic neurons seems to be related to the endogenous dopaminergic content, since it is overcome by dopamine depletion. Compounds that activate microglia or induce inflammation have similar e ff ects to LPS. This model suggest that inflammation is an import ant component of the degeneration of the nigrostriatal dopaminergic system, probably also in PD. Anti-inflammatory treatments could be useful to prevent or slow down the rate of dopaminergic degeneration in this disease

The gamma-aminobutyric acid-dopamine (GABA-DA) relationship was studied by intracerebral microdialysis in the prefrontal cortex. Nomifensine (5 microM) was included in the Ringer solution during all the dialysis experiments. Muscimol, a GABAA receptor agonist (50 and 500 microM) did not affect the extracellular output of DA and 3,4-dihydroxyphenylacetic acid (DOPAC). Baclofen, a GABAB receptor agonist (50 microM) significantly decreased the extracellular output of DA and DOPAC. On the other hand, picrotoxin and phaclofen, GABAA and GABAB receptor antagonists respectively, at a concentration of 50 microM, both significantly increased the release of DA. While the DOPAC level was affected only by picrotoxin perfusion. The present study indicates that GABA could control the release of DA in the prefrontal cortex.

In the present study, we have infused 6 prototypical drugs known to affect nigrostriatal dopaminergic neurons (kainate, baclofen, muscimol (10 mumol/l), picrotoxin (50 mumol/l), tetrodotoxin (TTX) (5 mumol/l) and 1-methyl-4-phenylpyridinium ion (MPP+) (10 mmol/l) via a microdialysis probe unilaterally into the left substantia nigra. During the infusion of these compounds, the extracellular content of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) was recorded simultaneously via microdialysis cannulas in both left and right striatum. Intranigral infusion of TTX, MPP+ and baclofen decreased dopamine release in the ipsilateral striatum, whereas muscimol, picrotoxin and kainate increased dopamine release. No changes were seen in the extracellular content of dopamine in the contralateral striatum. During all experiments, extracellular DOPAC increased in the ipsilateral striatum. No changes were seen in the extracellular content of DOPAC in the contralateral striatum. The present data provide no evidence that the bilateral nigrostriatal dopaminergic pathways are functionally linked in the rat brain.

1. The effect of baclofen, a GABAB receptor agonist, on the release of dopamine from the striatum (ST), substantia nigra (SN) and prefrontal cortex (PFC) of the rat was examined by intracerebral microdialysis. 2. Perfusion of baclofen 50 microM did not affect the striatal release of dopamine. However, dopamine release was markedly reduced in the SN and PFC. 3. 3,4-Dihydroxyphenylacetic acid and homovanillic acid output increased in the ST and decreased in the SN and PFC when baclofen was perfused through the microdialysis probe. 5-Hydroxyindoleacetic acid levels were not affected in any experimental condition by baclofen perfusion. 4. The results suggest that GABAB receptors modulate the release of dopamine in the SN and PFC, but do not affect the striatal release of dopamine, which indicates that the role of GABA receptor activation is different in the dopaminergic terminals of the ST and PFC.

The in vivo microdialysis technique was used to measure the extracellular output of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in adult and aged rats; as well as in response to the perfusion of dopamine agonists and antagonists, high-potassium Ringer solution and 1-methyl-4-phenyl-pyridinium ion (MPP+). The DA basal release before the drug treatment was higher in adult rats compared with the aged animals. The perfusion of dopaminergic drugs (nomifensine, sulpiride and quinpirole) through the probe at 1 microM concentration produced the same effect on DA, DOPAC and HVA outflow in adult and aged rats. 5-HIAA levels were not affected. The application of a high-potassium (60 mM) Ringer solution did not produce any significant difference on DA, DOPAC, HVA and 5-HIAA extracellular content when adult and aged rats were compared. The perfusion of MPP+ (1 mM) for 15 min produced a greater increase in DA release in aged than in adult rats. Greater susceptibility in the adult rats could explain these results.

In previous studies we found that intrastriatal DCG-IV administration, an agonist for group II metabotropic glutamate receptor: (i) protected striatal dopaminergic terminals against MPP(+)-induced neurotoxicity (Matarredona et al., 2001); (ii) selectively destroyed striatal GABAergic neurons (Venero et al., 2002) and (iii) induced early robust up-regulation of brain-derived neurotrophic factor (BDNF) in nigral dopaminergic neurons afferents in a target-dependent manner (Rite et al., 2005). Considering that BDNF protein is anterogradely transported to dopaminergic nerve endings, an autocrine role of BDNF could account for the neuroprotective effect of DCG-IV against the MPP(+)-induced toxicity of dopaminergic terminals. To test this possibility, we first performed a previous insult with quinolinic acid (QA) to specifically damage the striatal GABAergic neuronal cell bodies in order to remove the nigral BDNF target. Fourteen days later, we explored the potential in vivo neuroprotective action of DCG-IV against MPP(+)-induced toxicity on striatal dopaminergic nerve terminals by in vivo microdialysis. Integrity of GABAergic system was evaluated by glutamic acid decarboxylase (GAD) in situ hybridization. We demonstrate that previous striatal target ablation with QA prevented the neuroprotective effect of DCG-IV perfusion against the MPP(+)-induced neurotoxicity on dopaminergic terminals. Our results strongly suggest an important autocrine neuroprotective role of BDNF on striatal dopaminergic nerve terminals. In addition, we found an unexpected regulatory response of surviving striatal GABAergic neurons in terms of high levels of GAD mRNA expression.

The present study was designed to assess by microdialysis whether N-methyl-D-aspartate (NMDA) non-competitive receptor antagonist, MK-801, is able to protect dopaminergic neurons against 1-methyl-4-phenylpyridinium ion (MPP+) neurotoxicity. An intraperitoneal injection of MK-801, 10 mumol/kg, half an hour before striatal MPP+, 10 mM, perfusion did not protect against its neurotoxicity. Afterwards, rats received an intraperitoneal injection of MK-801 every 4 h, during 24 h. Under these conditions, one day after MPP+ perfusion, DA basal extracellular levels were close to the detection limit of our HPLC equipment in both control and MK-801 treated rats. 3,4-Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) basal output were lower in the former than in the latter rats. A second MPP+ perfusion statistically increased extracellular levels of DA and decreased DOPAC and HVA output in both groups of rats. However, the increase in extracellular DA overflow was higher in MK-801 treated rats than in the control group, indicating a higher number of surviving dopaminergic terminals. These results suggest that MK-801 is not able to protect against the primary direct neurotoxic action of MPP+, but a second MPP+ neurotoxic action mediated by excitatory amino acids could be partially prevented by MK-801.

The possible localization of excitatory amino acid (EAA) receptors on dopaminergic neurons was studied by microdialysis in conscious male rats. Varying concentrations of 3 specific EAA agonists, N-methyl-D-aspartate (NMDA), kainate and amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), were infused into the striatum or into the substantia nigra, and the extracellular dopamine (DA) was recorded by the same probe. All 3 compounds induced a dose-dependent increase in both striatal and nigral extracellular DA. Kainate and AMPA were more potent than NMDA. Nigral DA release was stimulated by lower concentrations of kainate and AMPA than striatal DA release. The effects of two concentrations of NMDA and kainate on the release of DA were analyzed in terms of tetrodotoxin (TTX) dependency and sensitivity to ibotenic acid-induced striatal lesion. It appeared that NMDA and kainate stimulated DA release by 3 different mechanisms. The first mechanism is seen at low concentrations of kainate, it fulfills the criteria for a functional receptor-interaction: it is TTX-sensitive and independent of the ibotenic acid lesion. The second mechanism was observed when relatively low concentrations of NMDA stimulate the release of DA; in this effect postsynaptic structures are involved. The third mechanism lacks specificity as it is seen after high concentrations of kainate as well as of NMDA. The latter mechanism is TTX-independent and is probably of a toxic nature. Finally NMDA and kainate were infused into the nigra, whereas DA was recorded with a second probe implanted into the striatum. Kainate and NMDA induced an increase of striatal DA, but kainate was about 100 times more potent in this model than NMDA. The present data therefore support localization of kainate and (probably) AMPA-receptors on nigrostriatal dopaminergic neurons. The receptors on the somatodendritic sites were observed to be more sensitive than those on the nerve terminals.

The extracellular concentration of dopamine (DA) and 3,4-hydroxyphenylacetic acid (DOPAC) were estimated in the substantia nigra and striatum by microdialysis. The dialysate content of DA increased at least 10-fold when nomifensine (5 μmol/l) was infused into the nigra via the dialysis membrane. To improve the analytical chemical detection of DA, nomifensine was infused continually during all the dialysis experiments that were carried out in the substantia nigra. Dendritic as well as terminal release of DA were inhibited for several hours when the nerve impulse flow in dopaminergic neurons was blocked by systemic administration of gamma-butyrolacton (750 mg/kg, i.p.). Perfusion of tetrodotoxin through the nigra (1 μmol/l) produced a complete disappearance of nigral DA release and a somewhat variable decrease in the release of striatal dopamine. When tetrodotoxin was infused into the nigra, the DOPAC output from the nigra was unchanged, but striatal DOPAC increased to about 300% of controls. These results show that the dendritic release of DA fulfills classical release criteria: it possesses an effective uptake-mechanism, it is dependent on the opening of fast-sodium channels and it is related to drug-induced changes in impulse-flow activity.

Drugs were infused into the substantia nigra of the rat brain via a microdialysis probe, and the extracellular concentration of dopaminc (DA) and 3,4-dihydroxyphenylacelic acid was recorded from a second dialysis probe implanted in the ipsilateral striatum. This approach allowed the evaluation of the sensitivity of nigral autoreceptors to various d 1 and D 2 agonists and antagonists in conscious animals. Intranigral administration of the specific D 2 agonists. quinpirole (LY 171555: 1, 10 and 50 μmol/l) and (−)-N-0437 (10 μmol/l), decreased the output of striatal dopamine, whereas the specific D 2 antagonist, (—)-sulpiridc (10 and 50 μmol/l), stimulated slightly the release of dopamine from the ipsilateral corpus striatum. Intranigral administration of the D 1 agonist, SKF 38393 (100 μmol/l). and the D 1 antagonist, SCH 23390 (KM) μmol/l), was without effect on the release of striatal dopamine. The results indicate that release-controlling autoreceptors on nerve terminals and autoreceptors controlling impulse flow in the nigra contribute equally to the decrease of striatal DA release caused by a high systemic dose of D 2 agonists. The stimulating effect of D 2 antagonists on the release of DA is mainly caused by release-controlling autoreceptors localized on nerve terminals. The autoreceptors in the nigra controlling impulse flow contributed somewhat less (about 20%) to this effect. Advantages and disadvantages of the use of a microdialysis probe to deliver drugs to restricted brain areas are discussed.

The extracellular concentration of dopamine (DA) and 3,4-dihydroxyphenylacetic acid in the substantia nigra (SN) and striatum was estimated by microdialysis. The dialysate content of DA from the SN was recorded during infusion of a DA uptake blocker (nomifensine; 5 mumol/L) dissolved in the perfusion fluid. Perfusion of tetrodotoxin (1 mumol/L) produced a virtually complete disappearance of nigral and striatal DA release. Dendritic as well as terminal release of DA was inhibited for several hours when the nerve impulse flow in dopaminergic neurons was blocked by systemic administration of gamma-butyrolactone (750 mg/kg, i.p.). The systemic administration (0.3 mg/kg, i.p.) as well as infusion (1 mumol/L) of the D2 agonist (-)-N-0437 [2-(n-propyl-N-2-thienylethylamino)-5-hydroxytetralin] produced a significant decrease in the release of DA in both the striatum and the SN. DA levels were recorded in the striatum both with and without addition of nomifensine to the perfusion fluid. The decrease in the striatum after (-)-N-0437 was suppressed in the presence of nomifensine. Infusion (1 mumol/L) as well as systemic administration (40 mg/kg) of sulpiride caused a similar increase in the release of striatal DA; this increase was, in both experiments, potentiated by nomifensine coinfusion. Sulpiride administration induced a small increase in the release of nigral DA. Infusion of (-)-N-0437 or (-)-sulpiride into the nigra caused a moderate decrease and increase, respectively, of striatal DA level.(ABSTRACT TRUNCATED AT 250 WORDS)

We have measured the level of catecholamines and serotonin and their principal metabolites, and the activities of tyrosine hydroxylase, tryptophan hydroxylase and monoamine oxidase-A and -B in the rat superior colliculus during postnatal development after bilateral removal of the eyes. The visual deprivation has different effects on the catecholamines and serotonin metabolism. The major changes in both amines were at day 15. Tyrosine hydroxylase and tryptophan hydroxylase activities increased during postnatal development but this increase was higher in enucleated compared with controls. An increase of the MAO-B to MAO-A ratio during postnatal development was found. The significance of these changes has been discussed.

3-Nitropropionic acid (3-NP), an inhibitor of the mitochondrial enzyme succinate dehydrogenase, induces neuronal degeneration in the striatum. It is known that dopamine (DA) enhances this toxic effect. In this work, we study how the increase of DA influences the toxic effect of 3-NP on DAergic terminals, GABAergic neurons, astroglia and microglia in the striatum. We increased the content of DA through the inhibition of its uptake by nomifensine or the inhibition of its catabolism by deprenyl. We found that although nomifensine and deprenyl enhanced the DA overflow produced by 3-NP perfusion, they protected against the damage induced by 3-NP in the DAergic terminals and the GABAergic neurons in the striatum. Moreover, there was a decrease of apoptotic cells, astrogliosis and activation of microglia as index of damage. We also found that depletion of DA by reserpine and alpha-methyl-p-tyrosine produced a significant reduction of the inhibition of the respiratory rate and of the production of superoxide radical induced by 3-NP in synaptosomes from the striatum. All these results suggest that endogenous dopamine within the dopaminergic terminals of the striatum enhances the mitochondrial production of radical oxygen species along with the respiratory inhibition produced by 3-NP and thus increases the toxicity produced by 3-NP in the striatum.

The development of a technique that allows the direct quantitative study of the damage produced by a toxin on a specific neurotransmitter system is very important. For that, we have used the microdialysis technique to validate a method to study the specific drug's toxicity on dopaminergic (DAergic) striatal terminals. We perfused different MPP(+) and 6-hydroxydopamine (6-OHDA) concentrations, with different toxicity for DAergic terminals, 24 h after the implantation of the microdialysis probe (day 1). One day later (day 2), MPP(+) was perfused through the microdialysis probe and DA extracellular output measured. We hypothesize that the amount of extracellular dopamine (DA) obtained on day 2 is directly proportional to the neurotoxic damage produced on day 1. To corroborate this hypothesis tyrosine hydroxylase (TH) immunohistochemistry was also carried out on day 2. There was a clear correlation index between the amount of DA measured after MPP(+) perfusion and the lack of TH immunoreactivity measured as the radius of the area showing decrease in TH immunoreactivity around the cannula. These results show the possibility to measure DAergic remaining terminals after a toxic drug exposure by in vivo MPP(+) perfusion. The possibility to extend this neurotoxic study to another neurotransmitter systems is suggested.

Somatodendritic release of dopamine (DA) was studied by microdialysis. The basal release of endogenous extracellular DA in the rat's substantia nigra was 1.0 ± 0.14 fmol/min (mean ± S.E.M.; n = 6). Compared with the basal extracellular level of DA found in the striatum (12.9 ± 0.94; n = 12), the former value was about 13 times smaller than the latter value. The addition to the Ringer solution of the fast sodium channel inhibitor, tetrodotoxin (TTX), at a concentration of 1 μM, produced the total disappearance of DA in the substantia nigra. When TTX was removed from the Ringer solution, the DA extracellular level came back slowly to the control value. This result suggests that the somatodendritic basal endogenous DA release measured by microdialysis is dependent on the sodium channel conductance.

We have evaluated the effects of a direct infusion of histamine, as mediator of inflammatory response, in substantia nigra, striatum, medial septum, and medial lemniscus. Injection of 100 and 250 nmol of histamine in substantia nigra produced a selective damage in dopaminergic neurons evidenced by the loss of tyrosine hydroxylase mRNA-expressing cells, tyrosine hydroxylase-immunolabeled-positive cell bodies, and dopamine and 3,4-dihydroxyphenylacetic acid levels. In parallel we found an acute inflammatory response manifested by a loss of glial fibrillary acidic protein-immunolabeled astrocytes and, at precisely the same area, an activation of microglia. In the striatum, only high doses (500 nmol) produced an evident terminal degeneration. The selective neurotoxicity of histamine for dopaminergic cells was demonstrated by the unaltered transcription of glutamic acid decarboxylase mRNA in substantia nigra. Moreover, intraseptal injection of 100 nmol of histamine failed to alter the pattern of choline acetyltransferase mRNA-expressing cells, and intraparenchymal injection of histamine in medial lemniscus failed to alter the pattern of serotonin-immunolabeled cells. We conclude that the substantia nigra is highly sensitive to histamine-derived neurotoxicity, where inflammatory processes mediated by histamine could be important in the pathological changes that lead to dopaminergic neuronal damage after histamine infusion.

Desferrioxamine (DES) protective effect against 1-methyl-4-phenylpyridinium (MPP+) toxicity was evaluated by microdialysis in the substantia nigra. DES (1 microM to 10 mM) co-perfused with MPP+ (2.5 mM) on day 1, produced on day 2 a higher dopamine extracellular output after perfusion of MPP+ than in control-MPP+ perfusion experiments, in which no DES was administered on day 1. Both Ringer's perfusion alone (control-Ringer) and co-perfusion of DES (10 mM) with MPP+ (2.5 mM) on day 1 produced on day 2 similar increases in dopamine extracellular output after a second MPP+ perfusion. In the control-Ringer experiment, note that the MPP+ on day 2 is the first MPP+ perfusion. Perfusion of FeCl3 (200 microM) along with MPP+ (2.5 mM) and DES (100 microM) on day 1 completely abolished on day 2 the neuroprotective effect found with MPP+ (2.5 mM) and DES (100 microM). The ability of DES to protect against MPP+ toxicity may indicate a therapeutic strategy in the treatment of diseases when iron is implicated.