OBJETIVO: Desenvolver e validar a técnica de CLAE-DE para o doseamento de dopamina (DA), ácido diidrofenilacético (DOPAC), ácido homovanílico (HVA) e 3-metoxitiramina (3-MT); validar a técnica de microdiálise (MD) cerebral em animais conscientes e avaliar o efeito do tratamento agudo com um extrato lipofílico das partes aéreas de Hypericum caprifoliatum (HCP) sobre os níveis cerebrais de DA e seus metabólitos. MATERIAIS E MÉTODOS: A validação analítica foi realizada conforme o preconizado pela ANVISA, com análise dos parâmetros linearidade, precisão, exatidão, especificidade e limite inferior de quantificação. A técnica de microdiálise foi realizada de forma clássica: guias de sonda foram implantadas nas regiões cerebrais de interesse por cirurgia estereotáxica; os dialisados foram obtidos, 48 horas após a cirurgia, através da perfusão da sonda com líquido cérebroespinhal artificial (1 μL/min) e coleta durante três horas, em intervalos de 20 min; as três primeiras coletas foram utilizadas para determinação da linha de base e só então foram administrados os tratamentos. Para a validação da MD a sonda foi implantada no estriado (A: -0,3; L: + 3,2; P: - 4,5) e o tratamento foi sulfato de anfetamina (2 mg/kg, s.c.). Para a avaliação do efeito de HCP, a sonda foi implantada no núcleo acumbens (A: +2,2; L: -1,5; P: -5,8) e o tratamento foi uma dose de 270 mg/kg do extrato (v.o.). RESULTADOS E DISCUSSÃO: Os parâmetros avaliados para validar o método analítico ficaram dentro dos limites especificados pela ANVISA. O sulfato de anfetamina produziu um aumento nos níveis extracelulares de DA em 160% e uma redução de DOPAC e de HVA em 80% e 50%, respectivamente. O extrato HCP não alterou o conteúdo intersticial de DA, DOPAC e HVA no núcleo acumbens. Os valores basais encontrados estão de acordo com dados relatados na literatura. Não foi possível quantificar 3-MT. CONCLUSÃO: A metodologia analítica e as condições experimentais de microdiálise utilizadas permitiram a mensuração de mudanças induzidas por anfetamina nos níveis extracelulares de DA e seus principais metabólitos demonstrando que os fundamentos da técnica estão estabelecidos em nossas condições. O regime de tratamento empregado com HCP não foi suficiente para alterar os níveis de DA e seus principais metabólitos no núcleo acumbens de ratos. / OBJECTIVE: The objectives of this work were to validate the HLPC-ED technique for measuring dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3- methoxytyramine (3-MT); to put into operation the brain microdialysis technique (MD) in freely moving rats; to evaluate, by using MD, the effect of a single treatment with lipophilic extract from aerial parts of Hypericum caprifoliatum (HCP) on DA, DOPAC, HVA and 3-MT brain levels. MATERIALS AND METHODS: In order to validate the analytical technique we followed the parameters established by ANVISA for bioanalytical samples. The parameters were: precision, accuracy, specificity and also the lower limit of quantitation. The microdialysis technique was carried out following classical procedures: male Wistar rats were submitted to stereotaxic surgery for the guide cannula implantation in the brain regions of interest; 48 hours later, the microdiaysis probe was inserted and perfused with cerebrospinal fluid (1 μL/min). After baseline samples collecting (1 h), the treatments were administrated and samples were collected every 20 min, during 2h. To accomplish MD technique validation the animals were treated with amphetamine sulfate (2 mg/kg, s.c.) and the extracellular levels of DA, DOPAC e HVA were measured in striatum (A: -0,3; L: + 3,2; P: - 4,5). The effect of HCP acute treatment (270 mg/kg, p.o.) was evaluated in nucleus accumbens (A: +2,2; L: -1,5; P: -5,8). RESULTS AND DISCUSSION: The analytical parameters values found were within the acceptable ranges for bio-analytical limits specified by ANVISA. As expected, amphetamine sulfate administration significantly increased DA (160%) extracellular levels and decreased DOPAC (80%) and HVA (50%) levels. The quantification of 3-MT was not possible. Acute HCP administration did not affected DA, DOPAC and HVA levels in the nucleus accumbens. CONCLUSION: The HPLC-ED methodology and microdialysis procedures employed allowed the detection of amphetamineinduced changes in extracellular levels of DA and its metabolites demonstrating that the technical starting point was acceptable. The acute administration of 270 mg/kg, p.o. of HCP to rats was not sufficient to modify the extracellular levels of DA, DOPAC and HVA in the nucleus accumbens.

OBJETIVO: Desenvolver e validar a técnica de CLAE-DE para o doseamento de dopamina (DA), ácido diidrofenilacético (DOPAC), ácido homovanílico (HVA) e 3-metoxitiramina (3-MT); validar a técnica de microdiálise (MD) cerebral em animais conscientes e avaliar o efeito do tratamento agudo com um extrato lipofílico das partes aéreas de Hypericum caprifoliatum (HCP) sobre os níveis cerebrais de DA e seus metabólitos. MATERIAIS E MÉTODOS: A validação analítica foi realizada conforme o preconizado pela ANVISA, com análise dos parâmetros linearidade, precisão, exatidão, especificidade e limite inferior de quantificação. A técnica de microdiálise foi realizada de forma clássica: guias de sonda foram implantadas nas regiões cerebrais de interesse por cirurgia estereotáxica; os dialisados foram obtidos, 48 horas após a cirurgia, através da perfusão da sonda com líquido cérebroespinhal artificial (1 μL/min) e coleta durante três horas, em intervalos de 20 min; as três primeiras coletas foram utilizadas para determinação da linha de base e só então foram administrados os tratamentos. Para a validação da MD a sonda foi implantada no estriado (A: -0,3; L: + 3,2; P: - 4,5) e o tratamento foi sulfato de anfetamina (2 mg/kg, s.c.). Para a avaliação do efeito de HCP, a sonda foi implantada no núcleo acumbens (A: +2,2; L: -1,5; P: -5,8) e o tratamento foi uma dose de 270 mg/kg do extrato (v.o.). RESULTADOS E DISCUSSÃO: Os parâmetros avaliados para validar o método analítico ficaram dentro dos limites especificados pela ANVISA. O sulfato de anfetamina produziu um aumento nos níveis extracelulares de DA em 160% e uma redução de DOPAC e de HVA em 80% e 50%, respectivamente. O extrato HCP não alterou o conteúdo intersticial de DA, DOPAC e HVA no núcleo acumbens. Os valores basais encontrados estão de acordo com dados relatados na literatura. Não foi possível quantificar 3-MT. CONCLUSÃO: A metodologia analítica e as condições experimentais de microdiálise utilizadas permitiram a mensuração de mudanças induzidas por anfetamina nos níveis extracelulares de DA e seus principais metabólitos demonstrando que os fundamentos da técnica estão estabelecidos em nossas condições. O regime de tratamento empregado com HCP não foi suficiente para alterar os níveis de DA e seus principais metabólitos no núcleo acumbens de ratos. / OBJECTIVE: The objectives of this work were to validate the HLPC-ED technique for measuring dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3- methoxytyramine (3-MT); to put into operation the brain microdialysis technique (MD) in freely moving rats; to evaluate, by using MD, the effect of a single treatment with lipophilic extract from aerial parts of Hypericum caprifoliatum (HCP) on DA, DOPAC, HVA and 3-MT brain levels. MATERIALS AND METHODS: In order to validate the analytical technique we followed the parameters established by ANVISA for bioanalytical samples. The parameters were: precision, accuracy, specificity and also the lower limit of quantitation. The microdialysis technique was carried out following classical procedures: male Wistar rats were submitted to stereotaxic surgery for the guide cannula implantation in the brain regions of interest; 48 hours later, the microdiaysis probe was inserted and perfused with cerebrospinal fluid (1 μL/min). After baseline samples collecting (1 h), the treatments were administrated and samples were collected every 20 min, during 2h. To accomplish MD technique validation the animals were treated with amphetamine sulfate (2 mg/kg, s.c.) and the extracellular levels of DA, DOPAC e HVA were measured in striatum (A: -0,3; L: + 3,2; P: - 4,5). The effect of HCP acute treatment (270 mg/kg, p.o.) was evaluated in nucleus accumbens (A: +2,2; L: -1,5; P: -5,8). RESULTS AND DISCUSSION: The analytical parameters values found were within the acceptable ranges for bio-analytical limits specified by ANVISA. As expected, amphetamine sulfate administration significantly increased DA (160%) extracellular levels and decreased DOPAC (80%) and HVA (50%) levels. The quantification of 3-MT was not possible. Acute HCP administration did not affected DA, DOPAC and HVA levels in the nucleus accumbens. CONCLUSION: The HPLC-ED methodology and microdialysis procedures employed allowed the detection of amphetamineinduced changes in extracellular levels of DA and its metabolites demonstrating that the technical starting point was acceptable. The acute administration of 270 mg/kg, p.o. of HCP to rats was not sufficient to modify the extracellular levels of DA, DOPAC and HVA in the nucleus accumbens.

OBJETIVO: Desenvolver e validar a técnica de CLAE-DE para o doseamento de dopamina (DA), ácido diidrofenilacético (DOPAC), ácido homovanílico (HVA) e 3-metoxitiramina (3-MT); validar a técnica de microdiálise (MD) cerebral em animais conscientes e avaliar o efeito do tratamento agudo com um extrato lipofílico das partes aéreas de Hypericum caprifoliatum (HCP) sobre os níveis cerebrais de DA e seus metabólitos. MATERIAIS E MÉTODOS: A validação analítica foi realizada conforme o preconizado pela ANVISA, com análise dos parâmetros linearidade, precisão, exatidão, especificidade e limite inferior de quantificação. A técnica de microdiálise foi realizada de forma clássica: guias de sonda foram implantadas nas regiões cerebrais de interesse por cirurgia estereotáxica; os dialisados foram obtidos, 48 horas após a cirurgia, através da perfusão da sonda com líquido cérebroespinhal artificial (1 μL/min) e coleta durante três horas, em intervalos de 20 min; as três primeiras coletas foram utilizadas para determinação da linha de base e só então foram administrados os tratamentos. Para a validação da MD a sonda foi implantada no estriado (A: -0,3; L: + 3,2; P: - 4,5) e o tratamento foi sulfato de anfetamina (2 mg/kg, s.c.). Para a avaliação do efeito de HCP, a sonda foi implantada no núcleo acumbens (A: +2,2; L: -1,5; P: -5,8) e o tratamento foi uma dose de 270 mg/kg do extrato (v.o.). RESULTADOS E DISCUSSÃO: Os parâmetros avaliados para validar o método analítico ficaram dentro dos limites especificados pela ANVISA. O sulfato de anfetamina produziu um aumento nos níveis extracelulares de DA em 160% e uma redução de DOPAC e de HVA em 80% e 50%, respectivamente. O extrato HCP não alterou o conteúdo intersticial de DA, DOPAC e HVA no núcleo acumbens. Os valores basais encontrados estão de acordo com dados relatados na literatura. Não foi possível quantificar 3-MT. CONCLUSÃO: A metodologia analítica e as condições experimentais de microdiálise utilizadas permitiram a mensuração de mudanças induzidas por anfetamina nos níveis extracelulares de DA e seus principais metabólitos demonstrando que os fundamentos da técnica estão estabelecidos em nossas condições. O regime de tratamento empregado com HCP não foi suficiente para alterar os níveis de DA e seus principais metabólitos no núcleo acumbens de ratos. / OBJECTIVE: The objectives of this work were to validate the HLPC-ED technique for measuring dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3- methoxytyramine (3-MT); to put into operation the brain microdialysis technique (MD) in freely moving rats; to evaluate, by using MD, the effect of a single treatment with lipophilic extract from aerial parts of Hypericum caprifoliatum (HCP) on DA, DOPAC, HVA and 3-MT brain levels. MATERIALS AND METHODS: In order to validate the analytical technique we followed the parameters established by ANVISA for bioanalytical samples. The parameters were: precision, accuracy, specificity and also the lower limit of quantitation. The microdialysis technique was carried out following classical procedures: male Wistar rats were submitted to stereotaxic surgery for the guide cannula implantation in the brain regions of interest; 48 hours later, the microdiaysis probe was inserted and perfused with cerebrospinal fluid (1 μL/min). After baseline samples collecting (1 h), the treatments were administrated and samples were collected every 20 min, during 2h. To accomplish MD technique validation the animals were treated with amphetamine sulfate (2 mg/kg, s.c.) and the extracellular levels of DA, DOPAC e HVA were measured in striatum (A: -0,3; L: + 3,2; P: - 4,5). The effect of HCP acute treatment (270 mg/kg, p.o.) was evaluated in nucleus accumbens (A: +2,2; L: -1,5; P: -5,8). RESULTS AND DISCUSSION: The analytical parameters values found were within the acceptable ranges for bio-analytical limits specified by ANVISA. As expected, amphetamine sulfate administration significantly increased DA (160%) extracellular levels and decreased DOPAC (80%) and HVA (50%) levels. The quantification of 3-MT was not possible. Acute HCP administration did not affected DA, DOPAC and HVA levels in the nucleus accumbens. CONCLUSION: The HPLC-ED methodology and microdialysis procedures employed allowed the detection of amphetamineinduced changes in extracellular levels of DA and its metabolites demonstrating that the technical starting point was acceptable. The acute administration of 270 mg/kg, p.o. of HCP to rats was not sufficient to modify the extracellular levels of DA, DOPAC and HVA in the nucleus accumbens.

To explore interactive deleterious effects of the teratogens ethanol and cadmium, pregnant rats were given cadmium (CdCl2, 50 ppm) and/or ethanol (10%), or tap water (controls) in the drinking water for the entire 21 days of pregnancy. At 3 months after birth, in vivo microdialysis was used to determine that there was a 4000% evoked release of DA by AMPH (AMPH, 4.0 mg/kg i.p.) in the striatum of rats exposed prenatally to both ethanol and cadmium, vs. a 2000% evoked release by AMPH in rats exposed prenatally to only ethanol or cadmium or tap water. Haloperidol (HAL)-evoked DA release was suppressed in groups exposed prenatally to ethanol, while HAL-evoked DOPAC and HVA release was greatest after co-exposure to prenatal cadmium and ethanol. These in vivo microdialysis results indicate that ontogenetic co-exposure to cadmium, and ethanol produces a long-lived suppressive effect on HAL-evoked DA release and a long-lived enhancing effect on AMPH-evoked DA release in rat striatum. These findings clearly demonstrate that there is marked alteration in dopaminergic regulation after ontogenetic cadmium and ethanol co-exposure, which in this regard resembles the reaction of the striatonigral pathway on AMPH-evoked DA release in rats with behavioral sensitization.

The intracellular second messenger nitric oxide (NO) is implicated in a variety of physiological functions, including release and uptake of dopamine (DA). In the described study, in vivo microdialysis and differential pulse voltammetric techniques were used to determine the involvement of NO in release of DA and its metabolites (dihydroxyphenylalanine, DOPAC; homovanillic acid, HVA) in neostriatum of freely moving rats. While the NO donor molsidomine (30.0 mg/kg; MOLS) and neuronal NO synthase- (nNOS-) inhbitor 7-nitroindazole (10.0 mg/kg; 7-NI) had no effect on the basal in vivo microdialysate level of DA, 7-NI specifically enhanced D,L-amphetamine- (1.0 mg/kg i.p.; AMPH) evoked release of DA. Basal or AMPH effects on DOPAC and HVA levels were not influenced by MOLS or 7-NI. Findings indicate that nitrergic systems have an important role in mediating effects of AMPH on dopaminergic systems.

Suicidal behavior has to be considered as a multifactorial phenomenon, which can be analyzed in a classifying-phenomenological manner. We have examined the relation of parasuicide typology to whole blood concentrations of serotonin, HVA, and tryptophan in 58 patients classified into 4 groups of parasuicide typology compared to 22 nonsuicidal depressed patients and 20 healthy subjects. Suicidal patients classified as impetuous, desperate and ambivalent types had significantly reduced whole blood 5-HT levels in comparison with the appealing type as well as nonsuicidal subjects. No differences were detected in the HVA content, but whole blood tryptophan concentrations were significantly reduced in impetuous suicidal patients and depressed patients compared to healthy subjects. This study provides evidence for reduced whole blood serotonin content based on different types of parasuicide. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Suicidal behavior has to be considered as a multifactorial phenomenon, which can be analyzed in a classifying-phenomenological manner. We have examined the relation of parasuicide typology to whole blood concentrations of serotonin, HVA, and tryptophan in 58 patients classified into 4 groups of parasuicide typology compared to 22 nonsuicidal depressed patients and 20 healthy subjects. Suicidal patients classified as impetuous, desperate and ambivalent types had significantly reduced whole blood 5-HT levels in comparison with the appealing type as well as nonsuicidal subjects. No differences were detected in the HVA content, but whole blood tryptophan concentrations were significantly reduced in impetuous suicidal patients and depressed patients compared to healthy subjects. This study provides evidence for reduced whole blood serotonin content based on different types of parasuicide. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

La 3,4-metilendioximetanfetamina (MDMA), una sustancia popularmente conocida como éxtasis, es una droga ilícita consumida habitualmente por adolescentes y adultos jóvenes. Además, el policonsumo es una práctica habitual entre los usuarios de la MDMA, siendo la cocaína una de las drogas más frecuentemente asociadas a esta sustancia. El objetivo del presente trabajo fue evaluar los efectos a corto y largo plazo que se producen tras la administración de la MDMA (5, 10, o 20 mg/kg) sola o en combinación con cocaína (25 mg/kg) en ratones adolescentes. En el estudio sobre el efecto agudo, observamos que ambas drogas administradas individual o simultáneamente incrementan la actividad motora. La dosis alta de MDMA disminuye de los contactos sociales en la prueba de la interacción social afectando igualmente el test de retención de la evitación pasiva. Sin embargo, sólo la co-administración de MDMA en combinación con cocaína produjo un efecto ansiolítico caracterizado por un aumento del tiempo de permanencia en los brazos abiertos del laberinto elevado en cruz. Igualmente, el análisis neuroquímico reveló que los ratones que recibieron MDMA en combinación con cocaína mostraron un incremento en el turnover de DA en el estriado, pero una disminución del de serotonina en la corteza. Los estudios de los efectos a largo plazo, realizados tres semanas después de haber finalizado un tratamiento con MDMA sola o en combinación con cocaína (2 administraciones por día durante 3 días consecutivos), mostraron que los ratones expuestos a la MDMA, sola o más cocaína, incrementaron el tiempo dedicado a las conductas sociales, aunque los que también habían recibido cocaína presentaban además conductas de amenaza. Observamos un efecto neurotóxico en los ratones tratados únicamente con 20 mg/kg de MDMA que mostraron un decremento de la concentración de DA en el estriado, no observándose este deterioro en los tratados además con cocaína. Por otra parte, mediante el condicionamiento de la preferencia de lugar (CPL) confirmamos que la MDMA posee efectos reforzantes y que esta sustancia es capaz de reinstaurar la preferencia de lugar una vez que esta se ha extinguido. La expresión y la reinstauración del CPL inducido por la MDMA dependen de la pauta de condicionamiento que condiciona los efectos neurotóxicos producidos por esta droga. Para finalizar, el tratamiento con MDMA sola o en combinación con cocaína durante la adolescencia favorece los efectos reforzantes de la MDMA durante el periodo adulto. Además, la exposición previa a la cocaína aumenta el tiempo necesario para extinguir la preferencia de lugar inducida por la MDMA. / 3,4-methylene-dioxy-methamphetamine (MDMA), commonly known as ecstasy, is an illicit recreational drug consumed by teenagers and young adults. The poly-drug pattern is the most common among those observed in MDMA users, with cocaine being a frequently associated drug. The aim of the present study was to evaluate the short- and long-term effects of exposure to MDMA (5, 10, or 20 mg/kg), alone or plus cocaine (25 mg/kg), on adolescent mice. In the acute phase, both drugs produced hyperactivity whether administered alone or concurrently. The highest MDMA dose decreased social contacts and affected the passive avoidance task. However, an anxiolytic effect, studied by means of the elevated plus maze and expressed as an increase in the time spent on the open arms, was observed only in animals treated with both cocaine and MDMA. Neurochemical analyses revealed an increase in striatal DA turnover and a decrease in serotonin cortical turnover in mice treated with MDMA plus cocaine. The studies conducted to establish long-term effects, which were performed 3 weeks after a 3-day treatment of two daily injections of MDMA alone or plus cocaine (6 administrations) during the adolescent period, showed that mice treated with MDMA alone and plus cocaine spent more time engaged in social contact, although those also treated with cocaine exhibited threat behaviors. Furthermore, we observed a neurotoxic effect in mice exposed to 20 mg/kg of MDMA, evident in a decrease in DA levels in the striatum, but this effect was not detected in mice additionally treated with cocaine. On the other hand, using the conditioned place preference (CPP), we have confirmed the rewarding effects of MDMA. In addition, we also show that MDMA can produce reinstatement of place preference after the extinction of this response. The expression and reinstatement of MDMA-induced CPP depend on the conditioning protocol, which conditions the neurotoxic effects produced by this drug. Finally, exposure during adolescence to MDMA alone or plus cocaine facilitates the rewarding effects of MDMA in adulthood. Moreover, previous experience of cocaine increases the time required to extinguish the MDMA-induced CPP.