The protective effects of Citrus aurantium flower extract against 6-hydroxydopamine-mediated cell damage in human neuroblastoma SH-SY5Y cells / Los efectos protectores del extracto de flor de Citrus aurantium contra el daño celular mediado por 6-hidroxidopamina en células humanas de neuroblastoma SH-SY5Y

Parkinson's disease (PD) is described as a neurological condition, resulting from continuous degeneration of dopaminergic neurons. Currently, most treatments for neurodegenerative diseases are palliative. In traditional Iranian medicine, Citrus aurantium flower extract is used to treat some neural diseases, such as sleep disorders and anxiety. The tendency towards the use of medicinal herbs for the treatment of diseases (eg, seizure) is growing. Accordingly, we evaluated the antioxidant effects of C. aurantium flowers and analyzed their protective effects against 6-hydroxydopamine (6-OHDA)-mediated oxidative stress. In this study, 150 mM of 6-OHDA was used to induce cellular damage. Also, MTT assay was performed to analyze cellular viability. Fluorescence spectrophotometry was performed to measure the intracellular reactive oxygen species (ROS) and calcium levels. Based on the findings, 6-OHDA could reduce cell viability. We also analyzed the effects of C. aurantium against neurotoxicity. The intracellular levels of ROS and calcium greatly improved in cells exposed to 6-OHDA. SH-SY5Y cell incubation with C. aurantium (400 and 600 mg/mL) induced protective effects and decreased the biochemical markers of cell apoptosis. According to the findings, C. aurantium showed protective effects against neurotoxicity, caused by 6-OHDA; these protective properties were accompanied by antiapoptotic features. According to the findings, it seems that hydromethanolic C. aurantium extract can be used to prevent seizures.

La enfermedad de Parkinson (EP) se describe como una afección neurológica que resulta de la degeneración continua de las neuronas dopaminérgicas. Actualmente, la mayoría de los tratamientos para las enfermedades neurodegenerativas son paliativos. En la medicina tradicional iraní, el extracto de flor de Citrus aurantium se usa para tratar algunas enfermedades neurológicas, como los trastornos del sueño y la ansiedad. La tendencia hacia el uso de las medicinas para el tratamiento de enfermedades (por ejemplo, convulsiones) está creciendo. Por consiguiente, el objetivo de este trabajo consistió en evaluar los efectos antioxidantes de las flores de C. aurantium y analizar sus efectos protectores contra el estrés oxidativo mediado por la 6- hidroxidopamina (6-OHDA). En este estudio, se usó 150 mM de 6-OHDA para inducir daño celular. Además, se realizó un ensayo de MTT para analizar la viabilidad celular. La espectrofotometría de fluorescencia se realizó para medir las especies reactivas de oxígeno (ROS) intracelulares y los niveles de calcio. En base a los hallazgos, 6-OHDA podría reducir la viabilidad celular. También analizamos los efectos de C. aurantium contra la neurotoxicidad. Los niveles intracelulares de ROS y calcio se expandieron a las células expuestas a 6-OHDA. La incubación de células SH-SY5Y con C. aurantium (400 y 600 mg / ml) indujo efectos protectores y disminuyó los marcadores bioquímicos de la apoptosis celular. De acuerdo con los hallazgos, C. aurantium mostró efectos protectores contra la neurotoxicidad, causada por 6-OHDA; estas propiedades protectoras fueron acompañadas por características antiapoptóticas. Según los hallazgos, parece que el extracto hidrometanólico de C. aurantium se puede usar para prevenir las convulsiones.

Striatal cholinergic cell ablation attenuates L-DOPA induced dyskinesia in Parkinsonian mice.

3,4-Dihydroxyphenyl-L-alanine (L-DOPA)-induced dyskinesia (LID) is a debilitating side effect of long-term dopamine replacement therapy in Parkinson's Disease. At present, there are few therapeutic options for treatment of LID and mechanisms contributing to the development and maintenance of these drug-induced motor complications are not well understood. We have previously shown that pharmacological reduction of cholinergic tone attenuates the expression of LID in parkinsonian mice with established dyskinesia after chronic L-DOPA treatment. The present study was undertaken to provide anatomically specific evidence for the role of striatal cholinergic interneurons by ablating them before initiation of L-DOPA treatment and determining whether it decreases LID. We used a novel approach to ablate striatal cholinergic interneurons (ChIs) via Cre-dependent viral expression of the diphtheria toxin A subunit (DT-A) in hemiparkinsonian transgenic mice expressing Cre recombinase under control of the choline acetyltransferase promoter. We show that Cre recombinase-mediated DT-A ablation selectively eliminated ChIs when injected into striatum. The depletion of ChIs markedly attenuated LID without compromising the therapeutic efficacy of L-DOPA. These results provide evidence that ChIs play a key and selective role in LID and that strategies to reduce striatal cholinergic tone may represent a promising approach to decreasing L-DOPA-induced motor complications in Parkinson's disease.

Puerarin protects dopaminergic neurons against 6-hydroxydopamine neurotoxicity via inhibiting apoptosis and upregulating glial cell line-derived neurotrophic factor in a rat model of Parkinson's disease.

Neurodegeneration is one of the primary etiologies in the onset of Parkinson's disease. In the quest for a new antiparkinsonism treatment the potential benefits of puerarin from the roots of Pueraria lobata have been recognized. Thus, we examined whether puerarin is capable to protect dopaminergic neurons of the substantia nigra against 6-hydroxydopamine induced neuronal cell death. Our data showed that the intraperitoneal administration of 0.12 mg/kg/day puerarin over 10 days reduced the 6-hydroxydopamine-induced decrease of tyrosine hydroxylase-positive cell counts. Analysis of apoptosis via DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay proved that puerarin could prevent 6-hydroxydopamine-induced apoptosis. As an additional apoptotic cell death marker, the BAX and BCL-2 expression levels were investigated using immunohistochemistry. Whereas 6-hydroxydopamine increased the level of Bax (p < 0.05), the coadministrated puerarin significantly antagonized this effect in a dose-dependent manner. Bcl-2 expression was not affected. Analysis of the dopamine, dihydroxyphenylacetic acid, and L-dihydroxy-phenyl-alanine contents of 6-hydroxydopamine-treated animals by HPLC revealed that puerarin was capable to restore the contents of dopamine and its metabolites. Moreover, the expression level of glial cell line-derived neurotrophic factor in the striatum was higher in puerarin than in rats treated with 6-hydroxydopamine alone. These results suggest that puerarin develops its neuroprotective effect against 6-hydroxydopamine-induced neurotoxicity in the substantia nigra through the inhibition of apoptotic signaling pathways and upregulation of glial cell line-derived neurotrophic factor expression in the striatum.

Role of the central ascending noradrenergic system in the regulation of luteinizing hormone responsiveness to testosterone negative feedback in the adult male rat.

It is well established that testosterone (T) feeds back on the brain and the anterior pituitary to inhibit gonadotropin secretion. However, the precise mechanism by which T exerts its central inhibitory action is poorly understood. We hypothesized that central noradrenergic activity decreases hypothalamic sensitivity to T negative feedback. To test this hypothesis, we compared the dose-response relationships between T and luteinizing hormone (LH) and between T and follicle-stimulating hormone (FSH) in adult male rats chronically depleted of hypothalamic norepinephrine (NE) to the dose-response curves exhibited by control animals. Depletion of hypothalamic NE was achieved by two independent methods: 1) by bilateral transection of the ascending noradrenergic system at the level of the mesencephalon, and 2) by intracerebroventricular infusion of the neurotoxin, 6-hydroxydopamine. After allowing 2-3 weeks for recovery from this initial surgery, all animals were castrated, and 3 weeks later were outfitted with subcutaneous T-containing or empty (sham) implants for a period of 48 hours. We observed that despite a profound chronic reduction in hypothalamic noradrenergic activity, the dose-response relationship between plasma T and the gonadotropins remained unaltered. These data demonstrate that normal amounts of hypothalamic noradrenergic activity are not essential for T to exert its negative feedback effect on gonadotropin secretion. Furthermore, they suggest that chronic removal of hypothalamic NE does not alter gonadotropin sensitivity to T negative feedback.

The potential use of vitamin E and selenium in parkinsonism.

Parkinson's disease (PD) is a chronic neurodegenerative illness which affects a significant number of the older population. Its treatment, which consists mostly of the dopamine precursor, L-Dopa, is associated with various complications. Research which has been conducted in order to develop drugs which might be without these toxic side-effects has not so far been met with a great degree of success. Recently the development of a parkinson-like syndrome in addicts who were using the compound methyl-phenyl-pyridine has refocused attention on the possible participation of free radicals in the etiology of PD. Herein it is postulated that the use of the free radical scavengers, vitamin E and selenium, might be effective in the early treatment of PD and might help to circumvent some of the complications associated with agonists therapy.

Patterns of destruction of mouse neuroblastoma cells by extracellular hydrogen peroxide formed by 6-hydroxydopamine and ascorbate.

The patterns of the cytolytic effects of 6-hydroxydopamine (6-OHDA), with/without ascorbate, on C-1300 and three other cloned mouse neuroblastoma cell lines (N1E-115, NS-20, N-18) were studied in vitro. The sensitivity to 6-OHDA differed and the three cloned cell lines were more sensitive than the wild type C-1300 cell line. Ascorbate synergistically potentiated the cytolytic effect of 6-OHDA to all four cell lines. The 6-OHDA cytotoxicity was eliminated by the addition of exogenous catalase but not by addition of other oxygen free radical scavengers, thereby suggesting that the hydrogen peroxide formed might influence the cells, extracellularly. In addition, the critical time for tumor cell lysis was the first 60 min of the reaction. The cytotoxicity induced by the unmasked cyclophosphamide, 4-hydroperoxycyclophosphamide, was synergistically enhanced in the presence of a nontoxic concentration of 6-OHDA and ascorbate. These data suggest that reactive oxygen intermediates may prove to be a good tool for destroying neuroblastoma cells.

Neuroleptic-like disruption of the conditioned avoidance response requires destruction of both the mesolimbic and nigrostriatal dopamine systems.

An examination of the ability to learn an active avoidance response was made in rats subjected to 6-hydroxydopamine (6-OHDA) lesions of the individual terminal areas of the midbrain dopamine (DA) system or a lesion to all these terminal regions in one group. Lesions were made by infusing 8 micrograms (base) of 6-OHDA in 2 microliter of vehicle into the following forebrain regions (each region representing a separate group of rats); frontal cortex, nucleus accumbens, corpus striatum and a double lesion of nucleus accumbens and corpus striatum. A separate group of rats received a smaller 6-OHDA lesion of the ventral substantia nigra. Only those rats with the combined double lesion of both the nucleus accumbens and corpus striatum (90% total depletion of dopamine) showed a severe deficit in acquisition of active avoidance. However, the rats with the separate 6-OHDA lesions to the mesolimbic or nigrostriatal DA systems did show the appropriate blockade of the amphetamine-induced locomotion or stereotyped behavior, respectively. In contrast, the rats with the double lesion showed no response to a low or high dose of amphetamine, remained cataleptic for the duration of the experiment but rapidly recovered from transient aphagia and adipsia (less than 10 days post lesion). Results suggest that a severe deficit in acquisition of an active avoidance response, similar to that observed with high doses of neuroleptics, requires destruction of all of the dopamine innervation of nucleus accumbens and corpus striatum. Results also suggest that both the mesolimbic and nigrostriatal dopamine systems act in concert to produce response enabling to important environmental events, and that the severe response enabling deficits observed in Parkinson's disease involves not only degeneration of the nigrostriatal dopamine system, but of the mesolimbic dopamine system as well.

Regional brain catecholamine levels and the development of hypertension in the spontaneously hypertensive rat: the effect of 6-hydroxydopamine.

To investigate the role of central catecholaminergic pathways in the development of hypertension in the spontaneously hypertensive rat (SHR) the effects of intracerebroventricular (i.c.v.) injections of 6-hydroxydopamine (6-OHDA) were compared with those of local injections near the main ascending noradrenergic pathways. The parameters studied were systolic blood pressure, heart rate and regional catecholamine concentrations in micropunched brain areas. I.c.v. treatment with 6-OHDA (three 200 micrograms injections) of young SHR attenuated the development of hypertension and caused widespread depletion of noradrenaline and to a lesser extent of dopamine and adrenaline. 6-OHDA-induced lesions of the dorsal and ventral noradrenergic bundles did not affect the rise in blood pressure but induced a depletion of forebrain noradrenaline comparable to that after the i.c.v. treatment. Dopamine and adrenaline levels were, however, not substantially affected. These results suggest that forebrain noradrenergic innervation may not be of major importance for the development of hypertension in the SHR.

Turning behavior, barrel rolling, and sensory neglect induced by picrotoxin in the thalamus.

Microinjections of picrotoxin in the ventromedial thalamic nucleus (VMT) in the rat elicited a slow contralateral turning plus a stereotyped upward sniffing. The animals showed a tendency to stand on the hind legs, increased grooming, and a tilt of the head toward the injected side. Similar injections in the parafascicular nuclear complex (Pf) initiated a faster ipsilateral turning and all the behaviors described plus another motor component: an ipsilateral barrel rolling. A 6-OHDA lesion did not impair the barrel rolling induced by picrotoxin injection in the Pf nucleus. Animals injected with picrotoxin in the Pf and VMT showed a marked increase in the current needed to evoke an orienting response after stimulation of the contralateral flank. Unlike rats treated with 6-OHDA, this response to ipsilateral stimulation was also reduced.

Multifocal brain sites for apomorphine-induced circling and other stereotyped motor behaviour in the 6-hydroxydopamine-lesioned rat.

Rats pretreated with 6-hydroxydopamine (6-OHDA, 8 micrograms in 4 microliters) in one medial forebrain bundle exhibited an assortment of stereotyped activities and pronounced circling towards the unlesioned side when apomorphine was administered either subcutaneously (0.5 mg/kg), or by discrete stereotaxic injection (5 micrograms in 0.2 microliter) into the caudate nucleus, nucleus accumbens, amygdala, lateral habenula, ventromedial thalamus, substantia nigra zona reticulata, periaqueductal grey or superior colliculus (but not a variety of other areas) ipsilateral to the lesion. These rotational responses were absent in unlesioned animals and, where tested, were attenuated by intraperitoneal haloperidol (0.5 mg/kg). It is suggested that multiple brain sites become sensitive to apomorphine following dopamine depletion by 6-OHDA.

Biochemical specificity of septal hyperreactivity: a behavioral discrimination.

Intra-septal injections of specific monoamine and cell body neurotoxins were used to determine the anatomical and biochemical elements critical to both short and longer term changes in reactivity normally seen after septal lesions. A multivariate discrimination performed on the basis of tactile, foot-shock and heat responsiveness revealed that septal lesions have a unique effect not accounted for by specific monoamine induced degeneration. Injections of 6-hydroxydopa and monosodium glutamate had slight effects on heat and shock responsiveness not at all comparable in degree or kind to septal lesion effects. Decreased hippocampal, but not hypothalamic, levels of norepinephrine may be associated with the longer term increased responsiveness to shock and heat in animals with septal lesions.

Evaluation of methods for determining 6-hydroxydopamine cytotoxicity.

The toxic effects of 6-hydroxydopamine on the human neuroblastoma cell line SK-N-SH-SY5Y(SY5Y) and the Chinese hamster ovary (CHO) cell line were measured with five viability assays. Four of the assays (attachment efficiency, plating efficiency, amino acid incorporation into acid-precipitable proteins, and Trypan Blue dye exclusion) showed higher drug susceptibility in SY5Y cells than CHO cells. Only growth inhibition (proliferation index) gave results indicating greater sensitivity in CHO cells. Over a time span of 48 hr, injured cell populations lost vital functions in the following order: attachment ability, amino acid incorporation, proliferative capacity, and dye exclusion. Recovery of each of the functions occurred in sublethally injured populations. Monitoring the extinction and recovery of vital functions permitted the accurate determination of a drug concentration (30 micrograms/ml) selectively toxic for SY5Y cells. A strong correlation was noted between relative values for amino acid incorporation 3 hr after drug treatment, attachment efficiency at 24 hr, and dye exclusion at 24 and 48 hr. We concluded that Trypan Blue dye exclusion and amino acid incorporation were suitable methods for comparing the effects of cytotoxins on different cell lines, provided they were performed at the appropriate time after treatment with the toxin. The combined techniques yield both population and individual cell data, are simple to do, and are applicable to nondividing cell populations.

Age as a factor in 6-hydroxydopamine-induced plasticity in the hypothalamus.

The question of age as a possible factor influencing the regenerative response of catecholaminergic varicosities in the hypothalamus was investigated in the supraoptic commissure and the paraventricular, periventricular, and dorsomedial hypothalamic nuclei of rats that had received intraventricular injections of the neurotoxin 6-hydroxydopamine when they were (1) neonates, (2) young adults, or (3) senescent adults. After post-neurotoxin survival for 4, 21, 56, or 180 days, the animals were perfused, and the hypothalamic tissue sections were cut and processed using a glyoxylic acid method for localizing catecholamines. Four days following neurotoxin administration, counts of fluorescent varicosities showed a significant loss of catecholamine varicosities in each of the four areas. Subsequently, at least partial restoration of numbers of catecholamine varicosities occurred in all hypothalamic areas in all three age groups. It is concluded that, following selective lesions induced by the neurotoxin 6-OH-DA, catecholamine varicosities were restored both in immature and mature groups. According to the evidence obtained experimentally, the rate of restoration was greater in the neonate group, whereas the percentage restoration attained varies according to the hypothalamic area studied and the age of the animal.

Conditioning compensates the neglect due to unilateral 6-OHDA lesions of substantia nigra in rats.

The degree of the contralateral sensory neglect in rats with unilateral 6-OHDA lesions of substantia nigra was assessed by a conditioning procedure, employing lateralized CS. In the first experiment visual neglect (revealed by failure of the visually elicited placing reaction contralateral to the lesion) was shown to be accompanied by slower acquisition of a brightness discrimination task. The impairment was due to ipsilateral turning tendency rather then to visual deficit, however, since monocular relearning yielded equal savings with the ipsilateral and contralateral eyes. The second experiment showed that rats anesthetized with urethane reacted to noxious skin stimulation contralateral to the lesion with shorter-lasting EEG arousal than to ipsilateral stimulation of the same intensity. The electrophysiological asymmetry could be compensated by classical conditioning, i.e. by pairing habituated tactile stimuli with noxious tail shock. The conditioned arousal reaction could be elicited with the same efficiency from the neglected and intact body surface. It is concluded that neglect is due neither to a sensory nor to a motor failure, but that 6-OHDA lesions of substantia nigra in one hemisphere reduce the arousing efficiency of unconditioned stimuli and interfere with sensorimotor integrating mechanisms on the side contralateral to the lesion. Compensation of the neglect by conditioning indicates that the role of the nigrostriatal system can be partly substituted by other circuits.

The use of neurotoxins to characterize the rates and subcellular distributions of axonally transported dopamine-beta-hydroxylase, tyrosine hydroxylase and norepinephrine in the rat brain.

The effect of 6-hydroxydopamine (6-OHDA), colchicine and cytochalasin B on the transport and subcellular distribution of proteins, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and norepinephrine (NE) were studied in the noradrenergic neurons of the rat locus coeruleus (LC). Four waves of transported 3H-labeled proteins and glycoproteins, defined by previous studies, as well as hypothalamic levels of TH, DBH and NE, were examined after injection of each neurotoxin into the ascending dorsal noradrenergic bundle. Blockade of subcellular components of TH, DBH and NE was compared to their endogenous hypothalamic distributions. 6-Hydroxydopamine variably blocked transport of all 4 waves of 3H protein and bilateral injections decreased hypothalamic levels of TH, DBH and NE by 58.2, 56.9 and 52.2% of controls, respectively. Cytochalasin B blocked transport of protein waves I (72--192 mn/day) and III (13--20 mm/day) and decreased hypothalamic levels of TH to 60.1% of control after bilateral injections. Colchicine blocked transport of waves I, II (24--48 mm/day) and V (1.4--2.9 mm/day) and blocked [3H]NE transport, while decreasing hypothalamic levels of DBH and NE to 56.6 and 69.3% of control after bilateral injections. Colchicine and 6-OHDA, but not cytochalasin B, caused a backup of DBH immunofluorescence proximal to the injection site. DBH and NE appeared to be transported primarily in particulate form, while TH transport was predominantly soluble in distribution. None of the toxins differentially affected the transport of one particular subcellular component of TH, DBH or NE. Based on the differential blocking effects of these toxins, DBH and NE appeared to be associated with wave II, and TH with wave III, travelling at 24--48 mm/day and 13--20 mm/day respectively.

The neuroanatomy of eating and drinking behavior.

For a number of years, the hypothalamus has been assigned the central and virtually exclusive role in regulating eating and drinking behavior. Now this concept is being challenged by new experimental evidence showing that ingestive as well as other behaviors are profoundly influenced by several neural tracts, particularly the neuroamine pathways, that may pass through the hypothalamus.

The comparison of fluoxetine and nisoxetine with tricyclic antidepressants in blocking the neurotoxicity of p-chloroamphetamine and 6-hydroxydopamine in the rat brain.

Fluoxetine prevents the loss of 5-hydroxytryptamine (5HT) uptake in synaptosomes of cerebral cortex after intraperitoneally administered p-chloroamphetamine (p-CA) with an ED50 of 3.8 mg/kg i.p. in rats. However, at 50 mg/kg, it does not prevent the loss of norepinephrine (NE) uptake in synaptosomes of hypothalamus after intraventricularly administered 6-hydroxydopamine (6-OHDA). Nisoxetine, on the other hand, centrally protects NE uptake from the neurotoxic effect of 6-OHDA with an ED50 value of 5 mg/kg i.p. At 50 mg/kg, it gives only 35% protection of 5HT uptake from the neurotoxic effect of p-CA. In comparison with the ED50 values of tricyclic antidepressants, both fluoxetine and nisoxetine are more potent and selective blockers of neurotoxicity resulting from the central actions of p-CA and 6-OHDA, respectively, in vivo.