R. de Miguel

THE ACTIVATION OF CANNABINOID RECEPTORS IN STRIATONIGRAL GABAERGIC NEURONS INHIBITED GABA UPTAKE

Cannabinoid receptors (CNRs) in basal ganglia are located on striatal efferent neurons which are gamma-aminobutiric acid (GABA)-containing neurons. Recently, we have demonstrated that CN-induced motor inhibition is reversed by GABA-B, but not GABA-A, receptor antagonists, presumably indicating that the activation of CNRs in striatal outflow nuclei, mainly in the substantia nigra, should be followed by an increase of GABA concentrations into the synaptic cleft of GABA-B receptor synapses. The present study was designed to examine whether this was originated by increasing GABA synthesis and/or release or by decreasing GABA uptake. We analyzed: (i) GABA synthesis, by measuring the activity of glutamic acid decarboxylase (GAD) and GABA contents in brain regions that contain striatonigral GABAergic neurons, after in vivo administration of CNs and/or the CNR antagonist SR141716; (ii) [3H]GABA release in vitro in the presence or the absence of a synthetic CN agonist, HU-210, by using perifusion of small fragments of substantia nigra; and (iii) [3H]GABA uptake in vitro in the presence or the absence of WIN-55,212-2, by using synaptosomes obtained from either globus pallidus or substantia nigra. Results were as follows. Delta9-tetrahydrocannabinol (delta9-THC) and HU-210, did not alter neither GAD activity nor GABA contents in both the striatum and the ventral midbrain at any of the two times tested, thus suggesting that CNs apparently failed to change GABA synthesis in striatonigral GABAergic neurons. A similar lack of effect of HU-210 on in vitro [3H]GABA release, both basal and K+-evoked, was seen when this CN was added to perifused substantia nigra fragments, also suggesting no changes at the level of GABA release. However, when synaptosome preparations obtained from the substantia nigra were incubated in the presence of WIN-55,212-2, a decrease in [3H]GABA uptake could be measured. This lowering effect was specific of striatonigral GABAergic neurons since it was not observed in synaptosome preparations obtained from the globus pallidus. In summary, the activation of CNRs located on striatonigral GABAergic neurons, which primarily access to GABA-B receptor synapses, was accompanied by a reduction in neurotransmitter uptake, thus prolonging the presence of GABA into the synaptic cleft. This mechanism might underly the CN-induced motor inhibition through the potentiation of the inhibitory effect of GABA on neuronal activity, in particular of nigrostriatal dopaminergic neurons.

Time-course of the effects of anandamide, the putative endogenous cannabinoid receptor ligand, on extrapyramidal function

We have recently described the dose-response effect of anandamide (AEA), the N-amide derivative of arachidonic acid that acts as an endogenous ligand for the cannabinoid receptor, on extrapyramidal function. The present study has been designed to examine the time-course of this effect. To this end, adult male rats were submitted to an acute i.p. injection of AEA, delta9-tetrahydrocannabinol (THC) or vehicle and examined at different times after drug administration. Animals were tested in an open-field test, then sacrificed and their striata used for analyses of dopaminergic indices. Results were as follows. The administration of AEA or THC produced the expected inhibition of motor behavior. Thus, the administration of AEA decreased the ambulation and the frequency of stereotypic movements (in particular, the number of rears) and increased the time spent by the rats in inactivity. These effects were evident at 10 and 30 min after the administration of the cannabinoid agonist, but mostly disappeared at 60 min. Interestingly, motor inhibition was observed again around 2 or 3 h after the administration of AEA. This was a small but persistent effect (decreased ambulation followed by increased inactivity), because it was observed until at least 6 h after AEA administration. The other cannabimimetic, THC, was always able of decreasing the ambulation and the frequency of rearing and grooming behavior, and of increasing the time spent in inactivity. This effect was usually something more marked than the effect of AEA, but the most characteristic fact was its persistence at all times studies, even at 6 h after administration. These motor disturbances were accompanied by changes in the activity of nigrostriatal dopaminergic neurons. Thus, the administration of AEA decreased the activity of tyrosine hydroxylase (TH) in the striatum at 10 and 30 min after treatment, suggesting a decreased nigrostriatal activity parallel to the motor deficit observed at these times. This was followed by an increase in TH activity and dopamine and L-3,4-dihydroxyphenylacetic acid contents at 60 min after treatment, which would likely reflect a compensatory stimulation of these neurons, whereas restoration of control values was found at 180 min after AEA administration, suggesting that the motor deficit observed at this time was not dependent on dopaminergic influence. Paradoxically, the administration of THC only produced changes in dopaminergic activity at 60 min after treatment, similar to those seen with AEA, but was ineffective at the other times. In summary, A-EA inhibits motor behavior in parallel to reductions in the activity of nigrostriatal dopaminergic neurons. However, this effect was of short duration, disappearing at 60 min after treatment, as compared with the inhibitory effect of THC on motor behavior which was observed at all times studied. Interestingly, a new AEA-induced inhibition of motor behavior, which was not accompanied by dopaminergic changes, appeared at longer times although its meaning remains to be determined.

Motor disturbances induced by an acute dose of Δ9-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)

An acute dose of δ9-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)

Extrapyramidal and neuroendocrine effects of AM404, an inhibitor of the carrier-mediated transport of anandamide.

A selective inhibitor of the carrier-mediated transport of endogenous cannabinoids, N-(4-hydroxyphenyl)-arachidonylethanolamide (AM404), has been recently synthesized and proposed as a useful tool for studying the physiological effects of endogenous cannabinoids and as a potential therapeutic agent in a variety of diseases. In the present study, we have examined the effects of this compound in two important brain processes in which a role for anandamide and other endogenous cannabinoids has been claimed: neuroendocrine regulation and extrapyramidal motor activity. A single and well-characterized dose of AM404, which presumably resulted in a significant elevation of the levels of endogenous cannabinoids, produced a marked decrease in plasma prolactin (PRL) levels, with no changes in luteinizing hormone (LH) levels. This decrease in PRL levels was accompanied by an increase in the activity of tyrosine hydroxylase (TH) in the medial basal hypothalamus. Both decreased PRL secretion and increased hypothalamic TH activity have been reported to occur after the administration of anandamide. Administration of AM404 also produced a marked motor inhibition in the open-field test, as also reported for anandamide, with a decrease in ambulatory and exploratory activities and an increase in the time spent in inactivity. This was accompanied by a decrease in the activity of TH in the substantia nigra, an effect also previously observed for anandamide.

Perinatal cannabinoid exposure modifies the sociosexual approach behavior and the mesolimbic dopaminergic activity of adult male rats

In the present work, we attempted to study whether hashish exposure during perinatal development affects sociosexual approach behavior in adult rats. To this end, we subjected adult female and male rats that had been perinatally exposed to hashish extracts to a sociosexual approach behavior test, completed with a dark-light emergence test and with a social interaction test. It was found that adult males perinatally exposed to hashish extracts exhibited marked changes in the behavioral patterns executed in the sociosexual approach behavior test; these changes did not exists in females. Thus, control males first visited the incentive male and took longer to visit the incentive female, whereas hashish-exposed males followed the opposite pattern. Moreover, hashish-exposed males spent more time in the vicinity of the incentive female, whereas they decreased their frequency of visits to, and the time spent in, the male incentive area. This behavior was observed early on, during the first third of the test, but became normalized and even inverted later on during the last two-thirds. Additionally, in the social interaction test, the normal reduction in the time spent in active social interaction following the exposure to a neophobic situation (high light levels) in controls did not occur in hashish-exposed males, although these exhibited a response in the dark-light emergence test similar to that of their corresponding controls. No changes were seen in spontaneous locomotor activity in both tests. These behavioral alterations observed in hashish-exposed males were paralleled by a significant decrease in L-3,4-dihydroxyphenylacetic acid contents in the limbic forebrain; this suggests a decreased activity of mesolimbic dopaminergic neurons. No effects were seen in females. Collectively, these results show that in the rat, perinatal cannabinoid exposure affects the sociosexual approach behavior and the mesolimbic dopaminergic activity in adulthood, although the effects were sexually dimorphic because they only appeared in the males.

Time-course of the effects of ovarian steroids on the activity of limbic and striatal dopaminergic neurons in female rat brain

This paper studies the time-course of the effects of pharmacological administrations of ovarian steroids on the functional state of dopaminergic terminals in the striatum and the limbic forebrain, using the ratio between the contents of dopamine (DA) and its metabolite, L-3,4 dihydroxyphenylacetic acid (DOPAC), as an index of nerve activity. Estradiol produced an increase in the dopaminergic activity of both limbic and striatal neurons, reflected in the high DOPAC/DA ratio observed in both areas. This estrogenic effect was only observed at 4 hours, disappearing in the subsequent times studied. The effect was antagonized by progesterone in both tissues, since a single injection of this steroid to estrogen-pretreated rats restored to control values the estradiol-induced increase, suggesting the existence of negative interactions between both steroids. Furthermore, treatment with progesterone produced also a late decrease of the DOPAC/DA ratio in the striatum, which was observed only in the animals nonpretreated with estrogens.

The prenatal exposure to δ9-tetrahydrocannabinol affects the gene expression and the activity of tyrosine hydroxylase during early brain development

We have previously reported that the exposure of pregnant female rats to delta 9-tetrahydrocannabinol (THC) during the perinatal period affected the gene expression and the activity of tyrosine hydroxylase (TH) in the brain of their male offspring. Those studies were done in animals perinatally exposed to THC but tested at peripubertal and adult ages. In the present work, we explored whether these effects also appear during early fetal brain development, when TH expression plays an important role in neuronal development. To this end, TH-mRNA concentrations were measured by Northern blot analysis with a specific TH probe in the brain of fetuses at gestational days 14 and 16 which had been prenatally exposed to THC or vehicle from day 5 of gestation. In parallel, measurements of TH activity and catecholamine contents by HPLC were also done. The results obtained were as follows. The prenatal exposure to THC markedly affected the expression of the TH gene in the brain of fetuses at gestational day 14. Thus, the amounts of TH-mRNA at this age were higher (2-fold) in THC-exposed fetuses than in controls. This corresponded with a marked increase in the activity of this enzyme (3-fold) at this age. Normalization was found in both parameters at gestational day 16. In summary, the prenatal exposure to THC affected the expression of the TH gene and the activity of this enzyme in brain catecholaminergic neurons during early fetal brain development. These results support the notion that cannabinoids are able to act at the level of the gene expression of specific key proteins for brain development.

Nigrostriatal and mesolimbic dopaminergic activities were modified throughout the ovarian cycle of female rats

In this work, we have studied the changes in the functional state of nigrostriatal (NSDA) and mesolimbic (MLDA) dopaminergic neurons during the estrous cycle of the female rat. The activity of tyrosine hydroxylase (TH), the turnover rate (Kt) after inhibition of dopamine (DA) synthesis and the ratio between the contents of this amine and its metabolite, L-3,4 dihydroxyphenylacetic acid (DOPAC), were used as indices of neuronal activity. The neuronal activity of NSDA neurons rose during estrous and declined during proestrous, as reflected by the values of Kt and DOPAC/DA ratio measured during both phases. Interestingly, the course of variations in striatal TH activity was similar, although retarded in relation to the changes in neuronal activity. Thus, TH activity was high during diestrous, whereas it was low during estrous. The activity of MLDA neurons was reduced during proestrous. This can be concluded from the decreased Kt and DOPAC/DA ratio measured in this phase and it was accompanied by a low TH activity. Thereupon, both Kt and TH activity increased during estrous. These results indicate the existence of physiological changes in the functional state of both dopaminergic systems during the ovarian cycle, which are partially different for each neuronal pathway. This supports the existence of a specific regulation, and not indiscriminate effects, by the hormones involved in this cycle, mainly estradiol and progesterone.

Cannabinoid receptor binding did not vary in several hypothalamic nuclei after hypothalamic deafferentation

Cannabinoid receptors are sparsely distributed in the hypothalamic nuclei, although they seem to be located on key neurons because their activation produces important neuroendocrine effects. In the present study, we have examined whether cannabinoid receptor-containing hypothalamic neurons are intrinsic or extrinsic to this brain region. To this end, we have examined whether the hypothalamic deafferentation was followed by a general loss of cannabinoid receptor binding, thus suggesting that cannabinoid receptor-containing neurons would have their cell bodies outside the hypothalamus, or whether this was followed by no changes in binding, thus suggesting that cannabinoid receptors would be located on intrinsic neurons. Three experimental groups were then analyzed: (i) animals with complete hypothalamic deafferentation in both sides; (ii) hemideafferentated animals; and (iii) sham-operated animals. In the three cases, cannabinoid receptor binding did not vary among these three groups in any of the hypothalamic nuclei analyzed. These were the arcuate nucleus, ventromedial hypothalamic nucleus, lateral and dorsal hypothalamic areas, paraventricular nucleus and medial preoptic area. This clearly supports the view that cannabinoid receptor-containing neurons into the hypothalamus are all intrinsic to this brain region.