Design and application of a 23-gene panel by next-generation sequencing for inherited coagulation bleeding disorders.

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.

Comparisons between brain dopaminergic neurons of juvenile and aged rats: sex-related differences.

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.

Effects of pre- and perinatal exposure to hashish extracts on the ontogeny of brain dopaminergic neurons.

The changes induced by maternal exposure to cannabinoids in the maturation of nigrostriatal, tuberoinfundibular and mesolimbic dopaminergic activities of rat offspring 15-40 days old were studied. In the striatum, tyrosine hydroxylase activity was constantly decreased during cannabinoid exposure in males. This decrease was correlative to increased number of D1 and D2 dopaminergic receptors. Both effects were also observed after the drug withdrawal caused by weaning on day 24. In females, the most consistent effect appeared on day 20, when decreased dopamine content and number of D1 receptors were observed. Both effects disappeared after drug withdrawal, but the reduction in the number of D1 receptors was again observed 40 days after birth. In the limbic area, cannabinoid exposure caused a decrease in the number of D1 receptors in 15-day-old females, along with decreases in the content of dopamine and its metabolite, L-3,4-dihydroxyphenylacetic acid. Changes in receptors disappeared on subsequent days, but increases in L-3,4-dihydroxyphenylacetic acid content and in its ratio with dopamine (L-3,4-dihydroxyphenylacetic acid/dopamine) were observed on day 20 followed by a decrease in the neurotransmitter content on day 30. In males, tyrosine hydroxylase activity increased on day 30, followed by an increase in L-3,4-dihydroxyphenylacetic acid content and L-3,4-dihydroxyphenylacetic acid/dopamine ratio on day 40. In the hypothalamus, the cannabinoid effects were always manifested after the cessation of drug exposure. Thus, a rise in L-3,4-dihydroxyphenylacetic acid/dopamine ratio was observed in 30-day-old females, and it was followed by a decrease on day 40, accompanied by a decrease in the anterior pituitary content of dopamine. Rise in prolactin release was not significant. In males, tyrosine hydroxylase activity was increased 30 days after birth, while L-3,4-dihydroxyphenylacetic acid content decreased. On day 40, L-3,4-dihydroxyphenylacetic acid content increased, paired to a rise in L-3,4-dihydroxyphenylacetic acid/dopamine ratio and anterior pituitary content of dopamine and to a decrease in the prolactin release. Perinatal exposure to cannabinoids altered the normal development of nigrostriatal, mesolimbic and tuberoinfundibular dopaminergic neurons, as reflected by changes in several indices of their activity. These changes were different regarding the sex and brain areas. Cannabinoid effects were more marked and constant in the striatum of males, while alterations in limbic neurons were mostly transient and those in hypothalamic neurons occurred after drug withdrawal. A long-term impact of these early changes on the neurological processes of adulthood is plausible.

Possible role of dopamine and noradrenaline in the regulation of prolactin secretion from an ectopic anterior pituitary gland in female rats.

It was recently reported that anterior pituitary tissue transplanted to an ectopic site contains measurable amounts of dopamine and noradrenaline. To examine the possibility of local catecholaminergic control of prolactin secretion from ectopic pituitaries, pituitary grafted and sham-operated female rats were submitted to several pharmacological treatments modifying catecholamine synthesis. Administration of a single dose of alpha-methyl-p-tyrosine (alpha-MPT) significantly reduced dopamine content in the graft, while noradrenaline content was not modified. Similar changes in the contents of dopamine and noradrenaline after alpha-MPT administration were observed in the hypothalamus and in the in-situ pituitary in both grafted and sham-operated rats. Plasma concentrations of prolactin were increased in both grafted and sham-operated rats after administration of alpha-MPT. A single injection of L-3,4-dihydroxyphenylalanine (L-DOPA) increased dopamine content in the ectopic pituitary gland without altering the noradrenaline content, and produced similar effects in the hypothalamus and in-situ pituitary of grafted and control rats. Plasma prolactin concentrations were decreased by L-DOPA in both pituitary grafted and control rats. Administration of DL-treo-dihydroxyphenylserine (DOPS) increased noradrenaline content in the ectopic pituitary and reduced plasma prolactin concentrations in pituitary grafted rats. In contrast, injection of DOPS to control rats increased both hypothalamic noradrenaline content and plasma prolactin concentrations. These results suggest that dopamine and noradrenaline present in the ectopic pituitary tissue have a role in mediating prolactin release from pituitary transplants.

Possible role of the cytochrome P450-linked monooxygenase system in preventing delta 9-tetrahydrocannabinol-induced stimulation of tuberoinfundibular dopaminergic activity in female rats.

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.

Changes in endocannabinoid transmission in the basal ganglia in a rat model of Huntington's disease.

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.

An acute dose of delta 9-tetrahydrocannabinol affects behavioral and neurochemical indices of mesolimbic dopaminergic activity.

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)

Cannabinoid CB(1) receptors colocalize with tyrosine hydroxylase in cultured fetal mesencephalic neurons and their activation increases the levels of this enzyme.

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.

Changes in brain dopaminergic indices induced by perinatal exposure to cannabinoids in rats.

Perinatal exposure to cannabinoid derivatives has been shown to produce effects on brain development. In this study, we evaluated the changes induced by maternal exposure to hashish crude extract (HCE) during gestation and lactation in several biochemical indices of dopamine activity in the striatum and the limbic forebrain of offspring. Studies were performed either during the HCE exposure or after drug withdrawal. Perinatal exposure to HCE reduced the number of striatal D1 binding sites in females and increased the L-3,4-dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratio, whereas an increase in the number of striatal D2 binding sites, with a reduction in their affinity, and a decrease in the activity of tyrosine hydroxylase (TH) were observed in males. The DOPAC/DA ratio was also increased in the limbic forebrain in HCE-exposed females, but there were no changes in binding site parameters. Most of these effects disappeared after cessation of cannabinoid treatment, but the decrease in striatal TH activity in males was maintained during drug withdrawal. Interestingly, the affinity of D2 receptors in the striatum of females, the number of striatal D1 receptors in males, and the limbic TH activity in males increased after the cessation of drug treatment. These results allow us to conclude that: (1) the effects of perinatal exposure to HCE were different depending on the sex and the specific brain area studied; and (2) most of the effects disappeared after cessation of cannabinoid treatment, although some new changes then appeared.

The endogenous cannabinoid receptor ligand, anandamide, inhibits the motor behavior: role of nigrostriatal dopaminergic neurons.

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.

Cannabinoid receptors in rat brain areas: sexual differences, fluctuations during estrous cycle and changes after gonadectomy and sex steroid replacement.

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.

Lack of effect of prolactin on the dopaminergic receptor sensitivity in striatal and limbic areas after experimentally-induced alterations in its peripheral levels.

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.

Catecholaminergic control of plasma growth hormone and thyrotropin levels in hypophysectomized rats bearing ectopic pituitary transplants.

Transplantation of the anterior pituitary to an ectopic site leads to stimulation of PRL secretion and suppression of the release of other adenohypophyseal hormones. We have previously reported that precursors and blockers of catecholamine synthesis can affect PRL release from the ectopic pituitary. In the present study we have measured the effects of L-3,4-dihydroxyphenylalanine (DOPA), DL-threo-dihydroxyphenylserine (DOPS), alpha-methyl-para-tyrosine (alpha-mpt) and diethyldithiocarbamate (ddc) on plasma growth hormone (GH) and thyrotropin (TSH) levels in hypophysectomized rats with pituitary transplants under the renal capsule. In these animals, peripheral plasma GH levels were elevated by a precursor (DOPA) and reduced by a blocker (alpha-mpt) of catecholamine synthesis. Plasma TSH levels were increased by a precursor (DOPS) and reduced by a blocker (ddc) of norepinephrine synthesis. We suspect that GH and TSH present in the circulation of pituitary-grafted animals were derived, in part, from the ectopic pituitary tissue and suggest that the small but detectable secretion of hormones other than PRL in this animal model is under the control of endogenous catecholamines.

Changes in rat brain cannabinoid binding sites after acute or chronic exposure to their endogenous agonist, anandamide, or to delta 9-tetrahydrocannabinol.

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)

Acute effects of delta-9-tetrahydrocannabinol on dopaminergic activity in several rat brain areas.

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.

Modifications of striatal D2 dopaminergic postsynaptic sensitivity during development of morphine tolerance-dependence in mice.

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)

Motor disturbances induced by an acute dose of delta 9-tetrahydrocannabinol: possible involvement of nigrostriatal dopaminergic alterations.

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)

Perinatal delta(9)-tetrahydrocannabinol exposure alters the responsiveness of hypothalamic dopaminergic neurons to dopamine-acting drugs in adult rats.

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.

Role of estrogens on striatal dopaminergic activity.

Studies were undertaken to evaluate the effects of estradiol and prolactin on striatal dopamine receptor activity. Dopamine receptors were quantified in partially purified striatal membranes by equilibrium binding using [3H]spiroperidol. When we investigated whether the D-2 dopamine receptor activity changes during the estrous cycle, the results suggest an increase in dopamine receptor density in diestrous, without modifications in the affinity. The finding that in ovariectomized rats the dopamine receptor binding parameters remained unchanged, suggests that gonadal steroids are not essential in the mechanism of action of this receptor. Results of activity of D-2 dopamine receptors showing that hyperprolactinemia fails to increase the number of these receptors do not support the hypothesis that circulating prolactin regulates the activity of these striatal dopamine receptors. Administration of estradiol benzoate (250 micrograms/kg per day) to hyperprolactinemic rats, by s.c. injection, significantly decreased both the density and the affinity of the striatal dopamine receptors. The present data indicate that, although prolactin does not seem to modify the activity of striatal dopamine receptors, it could modulate the estrogen-induced hypersensitivity of these receptors.

Effects of estradiol on circulating levels of prolactin in female rats bearing ectopic pituitaries.

The existence of local mechanisms controlling the prolactin (PRL) release from anterior pituitaries (AP) grafted to an ectopic location has been recently described. To study if these mechanisms are affected by estrogens, pituitary-grafted (GRAFT) and sham-operated (SHAM) rats were injected with a single dose of estradiol benzoate (EB), their plasma PRL levels as well as their hypothalamic and AP contents of norepinephrine (NE) and dopamine (DA) being analyzed. Administration of EB to GRAFT animals produced a small increase in their previously high plasma PRL levels, with both an increased NE and a decreased DA content in the ectopic AP. Since NE enhances the PRL release from ectopic AP and DA partially inhibits this secretion these changes may explain such a small increase in PRL levels. However, an additional increase in the decreased PRL release from the in situ AP of these animals cannot be discarded since EB produced also a decrease of the DA content in this tissue with an unaltered hypothalamic content. Finally, administration of this steroid to SHAM animals produced an important increase in plasma PRL levels. Since this increase was correlative to a decrease in DA and NE hypothalamic contents and unaltered AP contents. EB may be supposed to be able to reduce the DA synthesis in the tuberoinfundibular neurons, while the changes in noradrenergic inputs could be more related to the feedback effects of estrogens on the gonadotrophin release.