RESUMEN
INTRODUCTION: Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype-phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost. AIM: The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs. METHODS: A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology. RESULTS: In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant. CONCLUSION: Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.
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Trastornos de la Coagulación Sanguínea Heredados/genética , Pruebas Genéticas/métodos , Adolescente , Adulto , Trastornos de la Coagulación Sanguínea Heredados/patología , Niño , Preescolar , ADN/química , ADN/aislamiento & purificación , ADN/metabolismo , Femenino , Mutación del Sistema de Lectura , Eliminación de Gen , Estudios de Asociación Genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mutación Missense , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
Recent studies have demonstrated a loss of cannabinoid CB1 receptors in the basal ganglia in Huntington's disease (HD), but there are no data on endocannabinoid levels in this disease. In the present study, we have addressed this question by using rats with bilateral intrastriatal injections of 3-nitropropionic acid (3-NP), a toxin that, through the selective damage of striatal GABAergic efferent neurons, produces a useful model of HD. Twelve days after the lesion, 3-NP-lesioned rats exhibited motor disturbances, characterized by an ambulatory hyperactivity accompanied by a loss of guided activities. Analysis of GABA contents in the basal ganglia showed a trend towards a reduction compatible with motor hyperactivity. In addition, CB1 receptor binding and, to a greater extent, CB1 receptor activation of GTP-binding proteins, were also reduced in the basal ganglia. These changes were paralleled by a decrease of the contents of the two endocannabinoids, anandamide and 2-arachidonoylglycerol, in the striatum, and by an increase, particularly of anandamide, in the ventral mesencephalon where the substantia nigra is located. Both CB1 receptors and endocannabinoid levels were not altered in the cerebral cortex, an area not affected by the lesion. In summary, behavioral and biochemical changes observed in rats intrastriatally lesioned with 3-NP were similar to those occurring in the brain of HD patients. As expected, a loss of CB1 receptor function was evident in the basal ganglia of these rats and this was accompanied by different changes in endocannabinoid levels.
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Ganglios Basales/fisiopatología , Cannabinoides/metabolismo , Modelos Animales de Enfermedad , Enfermedad de Huntington/metabolismo , Transmisión Sináptica/fisiología , Animales , Moduladores de Receptores de Cannabinoides , Convulsivantes/administración & dosificación , Endocannabinoides , Enfermedad de Huntington/inducido químicamente , Enfermedad de Huntington/fisiopatología , Inyecciones Intraventriculares , Masculino , Actividad Motora/efectos de los fármacos , Nitrocompuestos , Propionatos/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptores de Cannabinoides , Receptores de Droga/efectos de los fármacos , Receptores de Droga/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Recent evidence obtained in rat models of Parkinson's disease showed that the density of cannabinoid CB1 receptors and their endogenous ligands increase in basal ganglia. However, no data exists from post-mortem brain of humans affected by Parkinson's disease or from primate models of the disorder. In the present study, we examined CB1 receptor binding and the magnitude of the stimulation by WIN55,212-2, a specific CB1 receptor agonist, of [35S]GTPgammaS binding to membrane fractions from the basal ganglia of patients affected by Parkinson's disease. In Parkinson's disease, WIN55,212-2-stimulated [35S]GTPgammaS binding in the caudate nucleus, putamen, lateral globus pallidus and substantia nigra was increased, thus indicating a more effective activation of GTP-binding protein-coupled signalling mechanisms via CB1 receptors. This was accompanied by an increase in CB1 receptor binding in the caudate nucleus and the putamen, although no changes were observed in the lateral globus pallidus and the substantia nigra. Because Parkinson's disease patients had been chronically treated with l-DOPA, brains were studied from normal common marmosets and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated animals with and without chronic L-DOPA treatment. MPTP-lesioned marmosets had increased CB1 receptor binding in the caudate nucleus and the putamen compared to control marmosets, as well as increased stimulation of [35S]GTPgammaS binding by WIN55,212-2. However, following l-DOPA treatment these parameters returned towards control values. The results indicate that a nigro-striatal lesion is associated with an increase in CB1 receptors in the basal ganglia in humans and nonhuman primates and that this increase could be reversed by chronic l-DOPA therapy. The data suggest that CB1 receptor blockade might be useful as an adjuvant for the treatment of parkinsonian motor symptoms.
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Ganglios Basales/metabolismo , Dopaminérgicos/farmacología , Proteínas de Unión al GTP/metabolismo , Levodopa/farmacología , Neuronas/metabolismo , Trastornos Parkinsonianos/metabolismo , Receptores de Droga/metabolismo , Regulación hacia Arriba/fisiología , Anciano , Anciano de 80 o más Años , Animales , Ganglios Basales/efectos de los fármacos , Ganglios Basales/fisiopatología , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Callithrix , Femenino , Proteínas de Unión al GTP/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Neuronas/efectos de los fármacos , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/fisiopatología , Receptores de Cannabinoides , Receptores de Droga/agonistas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The incubation of cultured fetal mesencephalic neurons with Delta(9)-tetrahydrocannabinol (Delta(9)-THC) increased the activity of tyrosine hydroxylase (TH) and this increase was reversed by SR141716A, a specific antagonist for cannabinoid CB(1) receptors. In the present work, we extended these earlier observations by addressing two objectives. First, we characterized at a molecular level the presence of CB(1) receptors in cultured fetal mesencephalic neurons using two strategies: (i) analyzing the presence of CB(1) receptor gene transcripts by Northern blot, and (ii) measuring [3H]WIN-55,212-2 binding in membrane fractions obtained from these cells, as well as evaluating the potential increase in [35S]-guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding caused by the activation of these receptors with WIN-55,212-2, a synthetic agonist. Northern blot analyses demonstrated the presence of small, but measurable levels of CB(1) receptor mRNA in cultured fetal mesencephalic neurons. The presence of these transcripts was accompanied by the presence of receptor binding protein, as revealed by a small, but specific, [3H]WIN-55, 212-2 binding in membrane fractions obtained from these cells. These CB(1) receptors are coupled to GTP-binding proteins, as the incubation of membrane fractions obtained from these cells with WIN-55,212-2 slightly, but significantly increased [35S]GTPgammaS binding. This fact indicated the existence, not only of receptor binding, but also of a functional receptor transduction pathway. As a second objective, we examined the potential colocalization of CB(1) receptors and TH in these cells by double-labelling immunocytochemistry. We also determined by Western blotting whether the previously observed Delta(9)-THC-induced increase in TH activity was accompanied by increased TH protein levels. Cultured fetal mesencephalic neurons exhibit diverse cell phenotypes, with CB(1) receptors localized only on TH-containing neurons. In addition, we found that the incubation of fetal mesencephalic neurons with medium containing Delta(9)-THC increased TH protein levels, in concordance with the previously reported increase in TH activity. Collectively, our results support the notion that CB(1) receptors are present in cultured fetal mesencephalic TH-containing neurons, despite their absence in the corresponding neurons in the adult brain. Thus, it is likely that the effects of cannabinoids on TH activity are direct. All this data strengthen the view that cannabinoid receptors are atypically located during brain development and that they might play an important role during this process, in particular on the phenotypical expression of TH-containing neurons.
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Mesencéfalo/química , Mesencéfalo/enzimología , Neuronas/química , Neuronas/enzimología , Receptores de Droga/efectos de los fármacos , Receptores de Droga/genética , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/metabolismo , Animales , Células Cultivadas , Dronabinol/farmacología , Embrión de Mamíferos , Guanosina Trifosfato/metabolismo , Mesencéfalo/citología , Neuronas/citología , Psicotrópicos/farmacología , ARN Mensajero/análisis , Ensayo de Unión Radioligante , Ratas , Receptores de Cannabinoides , Receptores de Droga/metabolismo , Tirosina 3-Monooxigenasa/efectos de los fármacosRESUMEN
Recent studies have shown that the pharmacological tolerance observed after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope dilution gas chromatography/mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), and 2-arachidonoylglycerol (2-AG) in several brain regions of adult male rats treated daily with delta9-tetrahydrocannabinol (delta9-THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,212-2-stimulated guanylyl-5'-O-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to test the development of the well known down-regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased in most of the brain areas of delta9-THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of delta9-THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents observed in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2-AG were unchanged in brain regions from delta9-THC-tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.
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Ácidos Araquidónicos/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Dronabinol/análogos & derivados , Sistema Límbico/efectos de los fármacos , Abuso de Marihuana/metabolismo , Animales , Benzoxazinas , Moduladores de Receptores de Cannabinoides , Cannabinoides/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Dronabinol/metabolismo , Dronabinol/farmacología , Endocannabinoides , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Sistema Límbico/química , Sistema Límbico/metabolismo , Masculino , Morfolinas/farmacología , Naftalenos/farmacología , Fosfatidiletanolaminas/metabolismo , Alcamidas Poliinsaturadas , Ratas , Ratas Wistar , Receptores de Cannabinoides , Receptores de Droga/metabolismo , Radioisótopos de Azufre , TritioRESUMEN
The amounts, in nine different rat brain regions, of the two endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglycerol (2-AG), and of the putative AEA precursor N-arachidonoyl-phosphatidylethanolamine (NArPE), were determined by isotope-dilution gas chromatography-mass spectrometry and compared to the number of cannabinoid binding sites in each region. The distribution of NArPE, reported here for the first time, exhibited a good correlation with that of AEA, the former metabolite being 3-13 times more abundant than the endocannabinoid in all regions. The highest amounts of both metabolites (up to 358.5 and 87 pmol/g wet weight tissue, respectively) were found in the brainstem and striatum, and the lowest in the diencephalon, cortex, and cerebellum. These data support the hypothesis that, in the brain, AEA is a metabolic product of NArPE and may reach levels compatible with its proposed neuromodulatory function. The brain distribution of 2-AG, also described in this study for the first time, was found to correlate with that of AEA with levels ranging from 2.0 to 14.0 nmol/g (in the diencephalon and brainstem, respectively). The distribution of the endocannabinoids did not match exactly with that of cannabinoid binding sites, suggesting either that these compounds are not necessarily produced near their molecular targets, or that they play functional roles additional to the activation of cannabinoid receptors. Regional differences in the ligand/receptor ratios may also lead to predict corresponding differences in the efficiency of receptor activation, as shown by previous studies.
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Ácidos Araquidónicos/metabolismo , Encéfalo/metabolismo , Cannabinoides/metabolismo , Glicéridos/metabolismo , Fosfatidiletanolaminas/metabolismo , Envejecimiento , Animales , Ácidos Araquidónicos/análisis , Sitios de Unión , Química Encefálica , Moduladores de Receptores de Cannabinoides , Cannabinoides/análisis , Endocannabinoides , Cromatografía de Gases y Espectrometría de Masas , Glicéridos/análisis , Ligandos , Masculino , Fosfatidiletanolaminas/análisis , Alcamidas Poliinsaturadas , Ratas , Ratas Wistar , Receptores de Cannabinoides , Receptores de Droga/metabolismo , Factores de TiempoRESUMEN
In the present work, we analyzed cannabinoid receptor mRNA expression, binding and activation of signal transduction mechanisms in the fetal rat brain or in cultures of fetal neuronal or glial cells. Cannabinoid receptor binding and mRNA expression were already measurable at GD14, but they were only located in discrete regions at GD16. Among these, the hippocampus, the cerebellum and the caudate-putamen area, three regions that contain a marked signal for both binding and mRNA in the adult brain. Significant levels of binding and, in particular, of mRNA transcripts were also detected at GD16 in the cerebral cortex, midbrain and brainstem. These structures contain relatively low levels of binding and mRNA in the adult brain, suggesting that cannabinoid receptor gene is transiently expressed in atypical areas during the fetal period. The signal for cannabinoid receptor mRNA in the hippocampus, caudate-putamen and cerebral cortex progressively increased from GD16 up to GD21. At GD18 and GD21, mRNA transcripts could be measured in discrete nuclei, such as septum nuclei, ventromedial hypothalamic nucleus and others. The cerebral cortex exhibited the highest mRNA levels at GD21, although this was not accompanied by a parallel increase in binding. An important aspect is that binding measured at these ages represent binding to functional receptors because their activation by WIN-55,212-2 increased [35S]GTPgammaS binding in the same areas. This increase was reversed by a specific antagonist, SR141716. The areas where the stimulation was more marked were the midbrain and brainstem. Using cell cultures, we have observed that cannabinoid receptor mRNA is present in cortical and hippocampal neuronal cells, but not in the glial cells. However, WIN-55,212-2 was capable of stimulating [35S]GTPgammaS binding in membrane fractions obtained from cortical glial cells and this stimulation was reversed by SR141716. This was not seen with hippocampal glial cell cultures, but occurred in hippocampal and cortical neurons. In addition, the activation of these receptors with Delta9-tetrahydrocannabinol significantly reduced forskolin-stimulated cAMP production in cortical neuronal or glial cell cultures and this effect was reversed by SR141716. In summary, we have detected cannabinoid receptor binding, mRNA expression and activation of signal transduction mechanisms in the fetal rat brain (GD14-GD21), which support the view that the system constituted by these receptors and their putative endogenous ligands might play a role in specific molecular events of the brain development. Of relevance is that binding and mRNA expression appear atypically distributed in the fetal brain as compared with the adult brain, even, that their presence in white-matter-enriched areas might presumably indicate their location in non-neuronal cells. These studies with cell cultures suggest that CB1 receptor subtype is located in neuronal cells obtained from fetal brain, although preliminary evidence is provided of the existence of another receptor subtype operative in glial cells obtained from the cerebral cortex.
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Encéfalo/embriología , Regulación del Desarrollo de la Expresión Génica/genética , Receptores de Droga/fisiología , Transducción de Señal/fisiología , Animales , Autorradiografía , Benzoxazinas , Encéfalo/crecimiento & desarrollo , Colforsina/farmacología , AMP Cíclico/metabolismo , Ciclohexanoles/farmacología , Dronabinol , Edad Gestacional , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hibridación in Situ , Morfolinas/farmacología , Naftalenos/farmacología , Neuroglía/metabolismo , Unión Proteica/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de CannabinoidesRESUMEN
We have recently reported that perinatal cannabinoid exposure altered the normal development of dopaminergic neurons in the medial basal hypothalamus at early postnatal and peripubertal ages. Most of these effects tended to disappear in adulthood, although we suspect the existence of a persistent, but possibly silent, alteration in the adult activity of these neurons. To further explore this possibility, we evaluated the responsiveness of these neurons to pharmacological challenges with a variety of dopaminergic drugs administered to adult male and female rats that had been exposed to delta(9)-tetrahydrocannabinol (delta(9)-THC) or vehicle during the perinatal period. In the first experiment, we evaluated the sensitivity of hypothalamic dopaminergic neurons to amphetamine (AMPH), which causes enhancement of dopaminergic activity by a variety of mechanisms. The most interesting observation was that both adult males and females, when perinatally exposed to delta(9)-THC, showed a more marked AMPH-induced decrease in the production of L-3,4-dihydroxyphenylacetic acid (DOPAC), the main intraneuronal metabolite of dopamine (DA), although this did not affect the prolactin (PRL) release. In the second experiment, we evaluated the in vivo synthesis of DA by analyzing the magnitude of L-3,4-dihydroxyphenylalanine (L-DOPA) accumulation caused by the blockade of L-DOPA decarboxylase with NSD 1015. As expected, NSD 1015 increased L-DOPA accumulation and decreased DOPAC production, with a parallel increase in PRL release, all of similar magnitude in both delta(9)-THC- and oil-exposed adult animals. In the last experiment, we tested the magnitude of the increase in PRL release produced by the administration of either SKF 38393, a specific D1 agonist, or sulpiride, a specific D2 antagonist. Both compounds increased plasma PRL levels in adult animals of both sexes, the effects in females being significantly more marked. The perinatal exposure to delta(9)-THC also modified the degree of increase in plasma PRL levels induced by both compounds, with opposite responses as a function of sex. Thus, delta(9)-THC-exposed females responded more intensely to SKF 38393 and, particularly, to sulpiride than oil-exposed females, whereas delta(9)-THC-exposed males responded to SKF 38393 lesser than oil-exposed males, although both responded equally to sulpiride. In summary, our results are consistent with the possible existence of subtle changes in the activity of hypothalamic dopaminergic neurons in adulthood caused by the exposure to delta(9)-THC during perinatal development. These silent changes could be revealed after the administration of drugs such as: (i) AMPH, whose effect producing a decreased DOPAC accumulation was more marked in delta(9)-THC-exposed males and females; and (ii) SKF 38393 and sulpiride, whose stimulatory effects on PRL secretion were of different magnitude in delta(9)-THC-exposed animals, with an evident sexual dimorphism in the response. The neurochemical basis for these differences remains to be determined.
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Animales Recién Nacidos/fisiología , Dopaminérgicos/farmacología , Dopamina/fisiología , Dronabinol/toxicidad , Hipotálamo/efectos de los fármacos , Neuronas/efectos de los fármacos , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Anfetamina/farmacología , Animales , Cromatografía Líquida de Alta Presión , Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Inhibidores de Captación de Dopamina/farmacología , Femenino , Hidrazinas/farmacología , Hipotálamo/citología , Hipotálamo/metabolismo , Masculino , Prolactina/sangre , Ratas , Ratas Wistar , Sulpirida/farmacologíaRESUMEN
A brain constituent, the N-amide derivative of arachidonic acid, termed anandamide, has been recently proposed as a possible endogenous ligand for the cannabinoid receptor. The present study has been designed to examine whether the acute or chronic exposure to anandamide affected the binding of cannabinoid receptors in specific brain areas as occurred with the exogenous cannabinoid agonist, delta 9-tetrahydrocannabinol (THC). To this end, we measured the maximum binding capacity (Bmax) and the affinity (Kd) of cannabinoid receptors, by using [3H]CP-55,940 binding assays, in membranes obtained from several brain areas of male rats acutely or chronically treated with anandamide or THC. Results were as follows. The acute administration of either anandamide or THC increased the Bmax of cannabinoid receptors in the cerebellum and, particularly, in the hippocampus. This effect was also observed after 5 days of a daily exposure to either anandamide or THC. However, whereas the increase in the Bmax after the acute treatment seems to be caused by changes in the receptor affinity (high Kd), the increase after the chronic exposure may be attributed to an increase in the density of receptors. On the contrary, the [3H]CP-55,940 binding to cannabinoid receptors in the striatum, the limbic forebrain, the mesencephalon, and the medial basal hypothalamus was not altered after the acute exposure to anandamide or THC. However, the chronic exposure to THC significantly decreased the Bmax of these receptors in the striatum and nonsignificantly in the mesencephalon. This effect was not elicited after the chronic exposure to anandamide and was not accompanied by changes in the Kd.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácidos Araquidónicos/farmacología , Química Encefálica/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Cannabinoides/farmacología , Cannabis , Dronabinol/farmacología , Receptores de Droga/efectos de los fármacos , Analgésicos/farmacología , Animales , Ciclohexanoles , Endocannabinoides , Técnicas In Vitro , Cinética , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Alcamidas Poliinsaturadas , Ratas , Ratas Wistar , Receptores de CannabinoidesRESUMEN
The present study has been designed to test whether the recently described endogenous ligand for the cannabinoid receptor, arachidonylethanolamide, termed anandamide, can mimic the effects produced by exogenous cannabinoids on motor behavior and to test possible neurochemical substrates for this potential effect. To this end, adult male rats were submitted to an acute i.p. injection of anandamide, delta 9-tetrahydrocannabinol (THC) or vehicle. Animals were behaviorally tested ten minutes after injection of the drug and, then, sacrificed and their brains used for dopaminergic analyses. Ambulation was not significantly affected by the treatment with either THC or anandamide, but a very pronounced increase was observed in the time spent in inactivity in rats treated with either THC or anandamide. This was accompanied by a marked decrease in the frequency of spontaneous non-ambulatory activities, such as grooming and rearing, although only the administration of THC decreased shaking behavior. The anandamide-induced decrease in grooming was dose-dependent, but the decrease in rearing was higher with the dose of 3 mg/kg than with the dose of 10 mg/kg. The administration of anandamide also caused a dose-dependent decrease in the activity of tyrosine hydroxylase and in the ratio between the number of D1 and D2 receptors in the striatum. Moreover, the administration of 3 mg/kg of anandamide significantly decreased the contents of dopamine and L-3,4-dihydroxyphenylacetic acid in the striatum although lesser and higher doses were less effective. THC only tended to decrease these parameters. No changes were seen in dopaminergic activity in the limbic forebrain after either cannabimimetics. In summary, anandamide, as well as THC, decreases motor behavior. This effect was paralleled by reduction in the activity of nigrostriatal dopaminergic neurons. However, subtle differences in the behavioral and neurochemical effects between anandamide and THC could be observed.
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Ácidos Araquidónicos/fisiología , Cuerpo Estriado/fisiología , Actividad Motora/fisiología , Receptores Dopaminérgicos/fisiología , Sustancia Negra/fisiología , Animales , Ácidos Araquidónicos/farmacología , Cannabinoides/metabolismo , Cuerpo Estriado/citología , Dronabinol/farmacología , Endocannabinoides , Masculino , Actividad Motora/efectos de los fármacos , Alcamidas Poliinsaturadas , Ratas , Ratas Wistar , Receptores de Cannabinoides , Receptores de Droga/metabolismo , Sustancia Negra/citologíaRESUMEN
The administration of delta 9-tetrahydrocannabinol (THC) or related cannabinoids markedly affected neurobehavioral and neuroendocrine indices in male rodents but usually failed to affect those indices in females. We examined whether inhibition of the cytochrome P450-linked monooxygenase system in female rats is able to elicit the effects of THC on one of the most characteristic targets of cannabinoid action, tuberoinfundibular dopaminergic neurons, whose activity is known to increase after cannabinoid exposure in males. It was found that the administration of THC to ovariectomized rats acutely replaced with estradiol (to discard problems derived from differences in the estrogenic status) did not affect either dopamine and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents and tyrosine hydroxylase activity in the medial basal hypothalamus or the density of D2-dopaminergic receptors in the anterior pituitary. However, the administration of THC to estrogen-replaced ovariectomized rats that had been pretreated with two separately administered inhibitors of cytochrome P450, piperonyl butoxide or metyrapone, significantly increased DOPAC content in the medial basal hypothalamus, with no changes in the other parameters. Collectively, these results indicate that the metabolism of THC to inactive compounds might play a protective role in females, counteracting the effects of this cannabinoid on tuberoinfundibular dopaminergic activity because pharmacological inhibition of cytochrome P450-linked monooxygenase system elicited a significant stimulation of these neurons by THC.
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Inhibidores Enzimáticos del Citocromo P-450 , Dronabinol/farmacología , Hipotálamo Medio/efectos de los fármacos , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Dopamina/metabolismo , Dronabinol/sangre , Femenino , Hipotálamo Medio/metabolismo , Masculino , Metirapona/farmacología , Neuronas/efectos de los fármacos , Butóxido de Piperonilo/farmacología , Ratas , Ratas Wistar , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Cannabinoid effects on brain dopaminergic activity vary as a function of gonadal status. In this work, we examined whether these variations might be due to sex steroid-dependent differences in brain cannabinoid receptors (CNr). Four experiments were done: (i) male versus females; (ii) females at each stage of the ovarian cycle; (iii) estradiol (E2) and/or progesterone (P)-replaced ovariectomized (OVX) females; and (iv) testosterone (T)-replaced orchidectomized males. The density of CNr in the medial basal hypothalamus fluctuated in females during the estrous cycle. The density was higher in diestrus and decreased in estrus. This parameter did not change after ovariectomy and E2 replacement. However, P increased the density of CNr when administered to OVX rats acutely treated with E2, but not administered alone or after chronic E2 treatment. In the striatum, the affinity of CNr was slightly higher in males than females, with no changes in density. Ovariectomy increased the affinity of CNr, which normalized only after administration of acute E2. Interestingly, the high affinity values observed in this area after P alone or combined with E2, corresponded to low densities as compared with intact females. In the limbic forebrain, the affinity for the cannabinoid ligand was also higher in males than females with no changes in density. Affinity was also higher in diestrus and lower in estrus, whereas density was unchanged. Ovariectomy decreased CNr density. A normal situation was found after administration of acute E2 or P alone, whereas chronic E2 markedly increased the density of CNr as compared with both intact and OVX females. Interestingly, this latter increase was prevented by coadministration of P. Orchidectomy did not affect CNr density, but administration of T produced a marked decrease. In the mesencephalon, the density and affinity of CNr was higher in males than females. Administration of P to OVX rats produced opposite effects, increasing the density when administered alone and decreasing it when administered to acute E2-replaced OVX rats. In summary, these results reveal the existence of subtle, sometimes more pronounced, sex dimorphisms, fluctuations along the ovarian cycle and changes after gonadectomy and sex steroid replacement in CNr density and affinity in certain brain areas. This supports the hypothesis of possible sex steroid-dependent differences in the sensitivity of certain neuronal processes to cannabinoid treatment.
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Encéfalo/ultraestructura , Estradiol/farmacología , Estro/fisiología , Ovario/fisiología , Progesterona/farmacología , Receptores de Droga/metabolismo , Testículo/fisiología , Testosterona/farmacología , Animales , Encéfalo/metabolismo , Cannabinoides/metabolismo , Cannabinoides/farmacología , Ciclohexanoles/metabolismo , Ciclohexanoles/farmacología , Dopamina/farmacología , Dronabinol/metabolismo , Femenino , Cinética , Masculino , Neuronas/fisiología , Orquiectomía , Ovariectomía , Ovario/cirugía , Ratas , Ratas Wistar , Receptores de Cannabinoides , Caracteres Sexuales , Testículo/cirugía , TritioRESUMEN
In the present study we examined the effects of intracerebroventricular (i.c.v.) injections of prolactin (PRL) on the presynaptic activity and post-synaptic sensitivity of mesolimbic and nigrostriatal dopaminergic neurons. In addition, the effects of PRL on in vitro release of dopamine (DA) from perifused striatal fragments were examined. Tyrosine hydroxylase (TH) activity and D2 receptor density in the striatum decreased after i.c.v. PRL administration; this was accompanied by an increase in D2 receptor affinity. These effects occurred after i.c.v. administration of PRL to normoprolactinemic rats, although normally they did not appear after administration to animals with pituitary grafting-induced hyperprolactinemia. Thus, in these animals, i.c.v. PRL failed to decrease TH activity and D1 and D2 receptor densities to a significant extent. In the case of D2 receptors, this was probably due to the fact that pituitary grafting-induced hyperprolactinemia itself was able to reduce the density of this receptor. No changes were observed in DA or L-3, 4-dihydroxyphenylacetic acid (DOPAC) contents after i.c.v. administration of PRL to both normo-and hyperprolactinemic animals. Basal and K(+)-evoked DA release in vitro from perifused striatal fragments of normoprolactinemic rats were not affected by the addition of PRL, whereas this hormone enhanced K(+)-evoked DA release when added to perifused striatal fragments from hyperprolactinemic animals. In the limbic forebrain, i.c.v. administration of PRL to normoprolactinemic animals produced a decrease in DA and DOPAC contents and D1 receptor density. Interestingly, none of these effects appeared when PRL was injected to hyperprolactinemic animals. In summary, our results suggest a possible inhibitory role of PRL on the activity of both the nigrostriatal and mesolimbic dopaminergic neuronal systems. These inhibitory effects were reflected in the decreases elicited in a set of neurochemical parameters, indicating either presynaptic activity or postsynaptic sensitivity, after i.c.v.-administered PRL. This observation supports the hypothesis of a possible neuromodulatory role for an extrapituitary PRL on the activity of these neurons, although the fact that most of these effects did not appear when i.c.v. administration was performed in hyperprolactinemic rats also suggests that they are influenced by peripheral PRL levels.
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Dopamina/fisiología , Sistema Límbico/efectos de los fármacos , Mesencéfalo/efectos de los fármacos , Prolactina/farmacología , Ácido 3,4-Dihidroxifenilacético/análisis , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/fisiología , Femenino , Hiperprolactinemia/fisiopatología , Inyecciones Intraventriculares , Riñón , Sistema Límbico/fisiología , Masculino , Mesencéfalo/fisiología , Proteínas del Tejido Nervioso/análisis , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Adenohipófisis/metabolismo , Adenohipófisis/trasplante , Potasio/farmacología , Prolactina/administración & dosificación , Prolactina/metabolismo , Ratas , Ratas Wistar , Receptores Dopaminérgicos/análisis , Sustancia Negra/efectos de los fármacos , Sustancia Negra/fisiología , Trasplante Heterotópico , Tirosina 3-Monooxigenasa/análisisRESUMEN
Cannabinoid consumption has been reported to affect several neurotransmitter systems and their related behaviors. The present study has been designed to examine cannabinoid effects on certain behaviors, which have been currently located in the limbic forebrain, in parallel to their effects on mesolimbic dopaminergic neurons. To this end, male rats treated with an oral dose of delta 9-tetrahydrocannabinol (THC) or vehicle were used 1 h after treatment for two different behavioral tests or neurochemical analyses of mesolimbic dopaminergic activity. Treatments, behavioral tests and sacrifice were performed in the dark phase of photoperiod because it corresponds to the maximum behavioral expression in the rat. Behavioral tests were a dark-light emergence test, which allows measurements of emotional reactivity, and a socio-sexual approach behavior test, which allows measurements of sexual motivation and also of spontaneous and stereotypic activities. Neurochemical analyses consisted of measurements of dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents, tyrosine hydroxylase activity, in vitro DA release and number and affinity of D1 receptors in the limbic forebrain. Results were as follows. THC exposure markedly altered the pattern executed by the animals in both tests. Concretely, THC-exposed animals exhibited a low number of visits to an incentive female in addition to high time spent in the vicinity of an incentive male, both observed in the socio-sexual approach behavior test, and an increased emergence latency to go out of a dark compartment in the dark-light emergence test. However, the fact that THC also decreased spontaneous activity and the frequency of rearing and self-grooming behaviors, in addition to the observations of either low total number of visits to both incentive sexual areas or high escape latency to go out of a light compartment, when the animal is placed in this compartment, also suggest the possible existence of an accompanying motor deficit. These behavioral effects were accompanied by increases in DA and DOPAC contents and in D1 receptor density in the limbic forebrain and to a slight decrease in the pattern of K(+)-evoked DA release in vitro from perifused limbic fragments, with no changes in the remaining neurochemical parameters. Collectively, these results allow us to conclude that acute THC markedly altered the behavioral pattern executed by the animals in a socio-sexual approach behavior test and in a dark-light emergence test, presumably indicating loss of sexual motivation and increased emotionality, although also accompanied by motor deficiencies.(ABSTRACT TRUNCATED AT 400 WORDS)
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Nivel de Alerta/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Dronabinol/farmacología , Sistema Límbico/efectos de los fármacos , Mesencéfalo/efectos de los fármacos , Receptores Dopaminérgicos/efectos de los fármacos , Ácido 3,4-Dihidroxifenilacético/metabolismo , Administración Oral , Animales , Dopamina/metabolismo , Dronabinol/farmacocinética , Emociones/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Receptores de Dopamina D1/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
The effects of delta 9-tetrahydrocannabinol (THC) on the activity of brain dopaminergic neurons might be subject to gonadal influence. In this work, we tested this hypothesis in relation to the effects of THC on tuberoinfundibular dopaminergic (TIDA) activity, the anterior pituitary sensitivity to dopamine (DA) and prolactin (PRL) secretion. To this end, we examined the effects of an acute dose of this cannabinoid administered during different phases of the estrous cycle in the morning or afternoon. The results were as follows. THC, administered during the morning of estrus, stimulated TIDA activity as reflected by increases in DA and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents and tyrosine hydroxylase (TH) activity in the medial basal hypothalamus. This was accompanied by an increase in the responsiveness of the anterior pituitary to DA, as reflected by the increase in the density of D2 receptors and the corresponding decrease in PRL release. By contrast, plasma PRL levels increased when THC was administered on the afternoon of estrus, in parallel with a significant reduction in the number of D2 receptors in the anterior pituitary gland and no effects on TIDA activity. A similar decrease in the anterior pituitary density of D2 receptors, but with no changes in plasma PRL levels, was observed when THC was administered during the morning of diestrus. This effect was not accompanied by changes in TIDA activity either.(ABSTRACT TRUNCATED AT 250 WORDS)
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Dopamina/metabolismo , Dronabinol/farmacología , Estro/fisiología , Hipotálamo Medio/efectos de los fármacos , Adenohipófisis/efectos de los fármacos , Prolactina/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Diestro/fisiología , Dopamina/farmacología , Femenino , Hipotálamo Medio/fisiología , Adenohipófisis/fisiología , Proestro/fisiología , Ratas , Ratas Wistar , Receptores de Dopamina D2/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Exposure to cannabinoids has been reported to affect several neurotransmitter systems and their related behaviors. The present study has been designed to further explore the effects of cannabinoids on motor behavior and test the involvement of nigrostriatal dopaminergic neurotransmission and other neurotransmitters as possible neurochemical targets for these cannabinoid effects. Male rats treated with an oral dose of delta 9-tetrahydrocannabinol (THC), the main psychoactive ingredient of cannabinoid derivatives, or vehicle were used 1 h after treatment for analyses of spontaneous motor and stereotypic activities together with neurochemical analyses of the nigrostriatal dopaminergic activity. Treatments and analyses were performed in the dark phase of photoperiod because it corresponds to the maximum behavioral expression in the rat. Neurochemical analyses were measurements of presynaptic activity--dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents, tyrosine hydroxylase (TH) activity, and in vitro DA release--and postsynaptic sensitivity--number and affinity of D1 and D2 receptors--in the striatum. In addition, measurements of 5-hydroxytryptamine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) contents were also performed to evaluate serotoninergic activity in the striatum. An oral dose of THC produced a loss of spontaneous motor activity, measured in both actimeter and open-field test, and a decrease in the frequency of several stereotypic behaviors, such as rearing and self-grooming. This decrease was correlated to a low number of D1-dopaminergic receptors in the striatum, although neither DA and DOPAC contents nor TH activity and D2 receptors were altered.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cuerpo Estriado/efectos de los fármacos , Dronabinol/toxicidad , Actividad Motora/efectos de los fármacos , Animales , Cuerpo Estriado/fisiología , Dopamina/metabolismo , Masculino , Actividad Motora/fisiología , Ratas , Ratas Wistar , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D1/metabolismo , Serotonina/metabolismoRESUMEN
Alterations in the activity of striatal dopaminergic neurons have been implicated in the development of morphine tolerance-dependence in rodents. To further explore this possibility, we examined the activity of these neurons in mice exposed to morphine during 4 days (addiction group) and subsequently treated with naloxone (withdrawal group). The efficiency of opiate treatment was assessed behaviorally. Striatal dopaminergic activity was evaluated by measuring: a) the ratio between the amounts of L-3,4-dihydroxyphenylacetic acid (DOPAC), the main intraneuronal metabolite of dopamine (DA), and the neurotransmitter itself, as an index of presynaptic activity; and b) the number and affinity of D1 and D2 dopaminergic receptors, as well as the amount of their coupled second messenger, cyclic adenosine monophosphate (cAMP), as postsynaptic parameters. Spontaneous motor activity was decreased in chronically morphine-exposed mice. In these animals, the number of striatal D2 receptors also decreased, with no changes in their affinity, whereas the number and affinity of D1 receptors remained unchanged. This hyposensitivity of D2 receptors was paralleled by an increase in the amount of cAMP with a good statistical correlation between both parameters. Treatment with naloxone of morphine-exposed mice resulted in the typical jumping behavior indicative of opiate withdrawal. The differences in D2 receptors between placebo- and morphine-exposed mice disappeared after naloxone-induced opiate withdrawal, although this effect was due more to the inhibitory effect of naloxone on the density of these receptors in placebo-exposed mice rather than to a stimulatory effect in morphine-addicted mice. The morphine-induced increase in cAMP content also disappeared after naloxone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cuerpo Estriado/metabolismo , Dependencia de Morfina/psicología , Morfina/farmacología , Receptores de Dopamina D2/efectos de los fármacos , Ácido 3,4-Dihidroxifenilacético/farmacología , Adenilil Ciclasas/metabolismo , Animales , Química Encefálica/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , AMP Cíclico/metabolismo , Dopamina/metabolismo , Cinética , Masculino , Ratones , Dependencia de Morfina/metabolismo , Actividad Motora/efectos de los fármacos , Naloxona/farmacología , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D1/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
In this work, we examined the acute effects of two doses of delta-9-tetrahydrocannabinol (THC) on several pre- and postsynaptic biochemical measures of dopaminergic activity in the striatum, limbic forebrain, and hypothalamic-anterior pituitary area of adult male rats. The exposure to a low dose of THC (0.5 mg/kg bw) decreased the number of striatal D2 dopaminergic binding sites, but did not affect their affinity. Treatment with a higher dose of THC was ineffective. In addition, both doses decreased the number of D1 dopaminergic binding sites in the limbic forebrain without changing their affinity. We did not find any changes in the dopamine (DA) or L-3,4-dihydroxyphenylacetic acid (DOPAC) content, or in the DOPAC/DA ratio, in either the striatum or limbic forebrain. THC treatment produced a dose-related decline in plasma prolactin (PRL) levels. Furthermore, both the basal and DA-inhibited in vitro release of PRL were reduced in animals exposed to THC in a dose-dependent manner. This inhibitory effect of THC on PRL release was accompanied by a decreased DOPAC/DA ratio in medial basal hypothalamus that, in turn, may be a result of the fall in PRL levels rather than a direct action of the drug. These data show that acute exposure to THC can alter brain dopaminergic neurotransmission. Our results suggest that the reduction of PRL release following THC exposure, both in vivo and in vitro, might be elicited by a direct action of THC on the pituitary.
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Encéfalo/fisiología , Dopamina/fisiología , Dronabinol/farmacología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Encéfalo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Dronabinol/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Cinética , Sistema Límbico/efectos de los fármacos , Masculino , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Prolactina/metabolismo , Ratas , Ratas Endogámicas , Receptores Dopaminérgicos/efectos de los fármacos , Receptores de Dopamina D1 , Receptores de Dopamina D2 , Sinapsis/efectos de los fármacosRESUMEN
It is known that several aspects of dopaminergic neurotransmission deteriorate with advanced age. In the present report, we have studied the possible existence of sexual differences in these aging-induced changes. Thus, we measured several pre- and postsynaptic biochemical parameters, indicative of the activity of dopaminergic neurons, in striatum, limbic forebrain and hypothalamic-anterior pituitary area of aged (24-26 months) and young (2 months) rats of both sexes. Tyrosine hydroxylase (TH) activity, as well as the number of D2-dopaminergic receptors, decreased in the striatum of aged rats, especially in the males in which the decrease in the number of receptors was associated with an increase in their affinity. In addition, the ratio between dopamine (DA) and its intraneuronal metabolite, L-3,4-dihydroxyphenyl-acetic acid (DOPAC), which can be used as an index of neurotransmitter turnover, was increased in aged females in parallel with a decreased DA content. In the limbic forebrain, TH activity was also decreased during aging, but only in males, whereas the DOPAC/DA ratio was increased in females, although in parallel with an increased DOPAC production. Finally, in the hypothalamic-anterior pituitary area, aging only affected the females, in which increased plasma prolactin levels were observed. This effect might be the result of a low responsiveness of pituitary lactotrophs to DA released from hypothalamic neurons, in spite of high prolactin levels producing a constant, although ineffective, stimulation of the activity of these neurons, as reflected by the high DOPAC content and DOPAC/DA ratio observed in the medial basal hypothalamus. In summary, these data allow us to suggest that the activity of brain dopaminergic neurons is modified with aging and there are significant differences as a function of sex and brain area.
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Envejecimiento/metabolismo , Encéfalo/metabolismo , Dopamina/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Femenino , Masculino , Neuronas/metabolismo , Ratas , Ratas Endogámicas , Receptores Dopaminérgicos/metabolismo , Receptores de Dopamina D2 , Caracteres Sexuales , Distribución Tisular , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Although it had been suggested that prolactin (PRL) modulates the dopaminergic receptor sensitivity in extrahypothalamic areas, recent studies have questioned this role. We studied the effects of PRL on the receptor sensitivity in the striatum and the limbic forebrain, analyzing the number of D1 and D2 receptors and the amount of their second messenger, cyclic-adenosine monophosphate (cAMP). Tyrosine hydroxylase (TH) activity and dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) content were also measured as indices of presynaptic activity. The study was carried out in male rats submitted to either acute (PRL injection) or chronic (pituitary grafts or diethylstilbestrol (DES)-induced pituitary tumors) rises of plasma PRL levels. The results showed a common lack of effect of PRL on the dopaminergic receptor sensitivity in both brain areas and, only some few effects on presynaptic activity in the striatum. Thus, grafted rats showed a slight decrease in DA content in the striatum, but neither D1 and D2 receptor number and cAMP content nor DOPAC content and TH activity, were modified, whereas DES animals exhibited no changes in all the parameters studied. A single injection of ovine PRL caused a decrease in DOPAC content and an increase in TH activity in the striatum. In the case of the limbic area, both chronic and acute hyperprolactinemia failed to alter any of the indices studied. In summary, we cannot support the view that PRL plays a role as modulator of dopaminergic receptor sensitivity. The only effects were always produced at the presynaptic level on the striatum, and after acute treatment, which supports the possible development of tolerance after chronic changes in peripheral PRL levels.