Effects of a short-term exposure to alcohol in rats on FAAH enzyme and CB1 receptor in different brain areas.

Acute alcohol exposure in rats (8% ethanol in the liquid diet for a period of 24 h) is associated with a decrease in the levels of endocannabinoids (anandamide and 2-arachidonoyl-glycerol) as well as in various N-acylethanolamines, in different brain regions. In the present study, we wanted to further explore: (i) whether these decreases might be caused by an increase in fatty acid amide hydrolase (FAAH), the enzyme involved in the degradation of N-acylethanolamines including anandamide, and (ii) whether the changes in endocannabinoid levels are accompanied by parallel changes in the major cannabinoid receptor type, the CB(1) receptor, activated by these ligands in the brain. Our data proved that FAAH activity did not increase in any of the four regions analyzed, even it was reduced in the hypothalamus and the prefrontal cortex. Paradoxically, FAAH levels increased in the hypothalamus and, to a lesser extent, in the prefrontal cortex and the amygdala, but not in the caudate-putamen. By contrast, the levels of CB(1) receptors were markedly reduced in the amygdala and prefrontal cortex of these rats, although no changes were seen in the hypothalamus and the caudate-putamen. These results suggest that reductions in the levels of endocannabinoids and related N-acylethanolamines caused by acute alcohol exposure are not originated by an enhanced degradation by FAAH enzyme, but they are associated with low levels of the receptors activated by these ligands, although this parallelism did not occur in all brain regions analyzed. In any case, these observations would support the notion of a general reduction in the activity of the cannabinoid signaling system by acute alcohol exposure.

CB1 receptor blockade reduces the anxiogenic-like response and ameliorates the neurochemical imbalances associated with alcohol withdrawal in rats.

There is strong evidence that blocking CB1 receptors may reduce alcohol intake in alcohol-dependent individuals. However, there is still limited evidence that CB1 receptor antagonists may also be beneficial in the attenuation of alcohol withdrawal syndrome, even though alcohol withdrawal appears to be milder in CB1 receptor knockout mice. Here we have examined whether the CB1 receptor antagonist rimonabant (SR141716) can alleviate the behavioral symptoms and revert the neurochemical imbalance elicited by a 3-h interruption of chronic alcohol exposure (7.2% in the drinking water for 10 days) in male Wistar rats. Administration of rimonabant attenuated the strong anxiogenic traits of the animals that developed when regular alcohol intake was interrupted. This may reflect the correction of the GABA/glutamate imbalances developed by the animals that received rimonabant in various brain regions involved in emotional (e.g. prefrontal cortex) and motor (e.g. caudate-putamen and globus pallidus) responses. In addition, rimonabant also affected the dopamine deficits generated by alcohol abstinence in the amygdala and ventral-tegmental area, albeit to a lesser extent. However, this antagonist was unable to correct the impairment caused by alcohol abstinence in serotonin and neuropeptide Y. The endocannabinoid activity in the brain of alcohol-abstinent rats indicated that the behavioral and neurochemical improvements caused by rimonabant were not related to the attenuation of a possible increase in this activity generated by alcohol withdrawal. Conversely, the density of CB1 receptors was reduced in alcohol-abstinent animals (e.g. globus pallidus, substantia nigra), as were the levels of endocannabinoids and related N-acylethanolamines (e.g. amygdala, caudate-putamen). Thus, rimonabant possibly enhances an endogenous response generated by interrupting the regular use of alcohol. In summary, rimonabant might attenuate withdrawal symptoms associated with alcohol abstinence, an effect that was presumably due to the normalization of GABA and glutamate, and to a lesser extent, dopamine transmission in emotion- and motor-related areas.

An overview on the biochemistry of the cannabinoid system.

About 40 years ago, cannabinoids were considered as the substances responsible for the psychoactive properties of marijuana and other derivatives of Cannabis sativa, whereas their medicinal use remained unexplored. However, with the discovery of the endocannabinoid system 20 years later, the compounds able to modify this system are being reconsidered for their therapeutic potential. Thus, the term "cannabinoid" includes now much more compounds than those present in C. sativa derivatives, for instance, numerous synthetic cannabinoids obtained by modifications from plant-derived cannabinoids or from the compounds that behave as endogenous ligands for the different cannabinoid receptor types. The term "cannabinoid" should also refer to some prototypes of selective antagonists for these receptors. The explanation for this exponential growth in cannabinoid pharmacology is the discovery and characterization of the endocannabinoid signaling system (receptors, ligands, and inactivation system) which plays a modulatory role mainly in the brain but also in the periphery. The objective of the present review article was to give an overview of the present state-of-the-art of biochemistry of the endocannabinoid system. Other authors in this volume will review their functions in the brain, their alterations in a variety of neurological and psychiatric pathologies, and the proposed therapeutic benefits in these diseases of new cannabinoid-related compounds that improve the pharmacological properties of classic cannabinoids.

Effects of rimonabant, a selective cannabinoid CB1 receptor antagonist, in a rat model of Parkinson's disease.

Recent evidence suggest that the blockade of cannabinoid CB1 receptors might be beneficial to alleviate motor inhibition typical of Parkinson's disease (PD). In the present study, we have explored the motor effects of rimonabant, a selective antagonist of CB1 receptors, in a rat model of PD generated by an intracerebroventricular injection of 6-hydroxydopamine. Compared with rats subjected to unilateral injection of this toxin in the medial forebrain bundle, this model allows nigral dopaminergic neurons be symmetrically affected. Dose-response studies with 6-hydroxydopamine revealed that the application of 200 microg per animal caused hypokinetic signs (decreased ambulatory activity, increased inactivity, and reduced motor coordination), which paralleled several signs of degeneration of nigrostriatal dopaminergic neurons (dopamine depletion in the caudate-putamen, and decreased mRNA levels for tyrosine hydroxylase and superoxide dismutase-1 and -2 in the substantia nigra). In these conditions, the degree of hypokinesia and dopaminergic degeneration may be considered moderate, comparable to the disturbances occurring in early and middle stages of PD in humans, a period that might be appropriate to test the effects of rimonabant. There is also degeneration of other dopaminergic pathways out of the basal ganglia, but this does not appear to interfere significantly with the hypokinetic profile of these rats. Higher doses of 6-hydroxydopamine elevated significantly animal mortality and lower doses failed in general to reproduce motor inhibition. Like other animal models of PD, these rats exhibited an increase in the density of CB(1) receptors in the substantia nigra, which is indicative of the expected overactivity of the cannabinoid transmission in this disease and supports the potential of CB1 receptor blockade to attenuate hypokinesia associated with nigral cell death. Thus, the injection of 0.1 mg/kg of rimonabant partially attenuated the hypokinesia shown by these animals with no effects in control rats, whereas higher doses (0.5-1.0 mg/kg) were not effective. We also found that the antihypokinetic effects of low doses of rimonabant did not influence the dopamine deficits of these animals, as well as it did not modify GABA or glutamate transmission in the caudate-putamen. In summary, rimonabant may have potential antihypokinetic activity in moderate parkinsonism at low doses, but this effect is not related to changes in dopaminergic, GABAergic, or glutamatergic transmission in the striatum. Therefore, the elucidation of the neurochemical substrate involved in this effect remains a major challenge for the future.

Chronic Delta9-tetrahydrocannabinol administration affects serotonin levels in the rat frontal cortex.

Adult rats were subjected to chronic treatment with the cannabinoid agonist, Delta9-tetrahydrocannabinol, or with vehicle, and their brains used to analyze the contents of serotonin (5HT) and of its intraneuronal metabolite, 5-hydroxyindolacetic acid (5HIAA). 5HT and 5HIAA contents were not affected by chronic cannabinoid administration in most of the brain regions analyzed. We found a marked increase in 5HT contents in the frontal cortex that was accompanied by no changes in 5HIAA contents. This originated a decrease in 5HIAA/5HT ratio, which suggests a possible reduction in the activity of serotoninergic terminals reaching this cortical area. This effect was not seen after an acute injection of this cannabinoid. The relevance of these observations was that they occurred in a region where changes in serotoninergic transmission have been implicated in the development of depression; therefore, our data support the theory that the cannabinoid system might be a potential target for the treatment of this neuropsychiatric disease.

Effects of neonatal exposure to methamphetamine: catecholamine levels in brain areas of the developing rat.

Neonatal exposure to moderate doses of methamphetamine during the first month of life in the rat affects tyrosine hydroxylase gene expression in the substantia nigra and nigrostriatal tyrosine hydroxylase activity. The main goal of this work was to evaluate the ontogeny of the neurochemical effects of repeated exposure to moderate doses of methamphetamine during the first month of life in the rat. Norepinephrine, dopamine, and dihydroxyphenylacetic acid levels were measured in target areas of methamphetamine: the substantia nigra, ventral tegmental area, caudate-putamen, nucleus accumbens, and medial prefrontal cortex. On postnatal day 1 (PND1), Wistar rat litters, culled to eight pups, sex balanced, were randomly attributed to either methamphetamine or control groups. Methamphetamine groups were administered 10 mg of (+/-)-methamphetamine/kg body weight/day, subcutaneously, from PND1 until the day prior to sacrifice; control groups received isovolumetric saline. Groups were sacrificed on PND7, PND14, and PND30. Neonatal methamphetamine exposure increased norepinephrine levels in the substantia nigra of PND30 rats; on PND14, this variation was evident only in male pups. In the substantia nigra, the dihydroxyphenylacetic/dopamine ratio was also affected in PND30 males. In the ventral tegmental area, catecholamine levels were not affected by methamphetamine. Norepinephrine levels were also increased in the caudate-putamen of PND7 male and PND14 female methamphetamine-exposed pups and in the nucleus accumbens of PND14 female and PND30 male and female pups. Catecholamine levels in the medial prefrontal cortex were not affected by neonatal methamphetamine administration.

Cannabinoids and gene expression during brain development.

Cannabis is the most commonly used illicit drug in western societies, in particular among young people. It is consumed even by women during pregnancy and lactation, which result in a variety of disturbances in the development of their offspring, because, like other habit-forming drugs, cannabinoids, the psychoactive ingredients of marijuana, can cross the placental barrier and be secreted in the maternal milk. Through this way, cannabinoids affect the ontogeny of various neurotransmitter systems leading to changes in different behavioral patterns. Dopamine and endogenous opioids are among the neurotransmitters that result more affected by perinatal cannabinoid exposure, which, when animals mature, produce changes in motor activity, drug-seeking behavior, nociception and other processes. These disturbances are likely originated by the capability of cannabinoids to influence the expression of key genes for both neurotransmitters, in particular, the enzyme tyrosine hydroxylase and the opioid precursor proenkephalin. In addition, cannabinoids seem to be also able to influence the expression of genes encoding for neuron-glia cell adhesion molecules, which supports a potential influence of cannabinoids on the processes of cell proliferation, neuronal migration or axonal elongation in which these proteins are involved. In support of this possibility, CB1 receptors, which represent the major targets for the action of cannabinoids, are abundantly expressed in certain brain regions, such as the subventricular areas, which have been involved in these processes during brain development. Finally, cannabinoids might also be involved in the apoptotic death that occurs during brain development, possibly by influencing the expression of Bcl-2/Bax system. Also in support of this option, CB1 receptors are transiently expressed during brain development in different group of neurons which do not contain these receptors in the adult brain. This paper will review all evidence relating cannabinoids to the expression of key genes for neural development, trying to establish the future research addressed to elucidate the mechanisms involved in the epigenetic action of cannabinoids during brain development.

Changes in endocannabinoid contents in reward-related brain regions of alcohol-exposed rats, and their possible relevance to alcohol relapse.

1. Chronic alcohol exposure modifies endocannabinoid levels in different brain regions, while pharmacological targeting of the endocannabinoid system has been reported to influence ethanol intake in laboratory animals. 2. The present study was aimed at evaluating the pattern of changes of endocannabinoids and their receptors, with emphasis on reward-related brain areas, in Wistar rats subjected to consecutive phases of alcoholization, alcohol deprivation (abstinence), and voluntary consumption of alcohol (relapse). 3. We observed that, in the limbic forebrain, anandamide (AEA) and 2-arachidonoylglycerol (2-AG) contents increased after 7 days of alcoholization, then to dramatically decrease after 48 h of alcohol deprivation and, in the case of 2-AG, to further decrease when rats were allowed to relapse to alcohol consumption. By contrast, in the midbrain, there was a marked reduction in AEA, but not 2-AG, content, after alcoholization. This decrease was not affected during alcohol abstinence, but both AEA and 2-AG contents were then significantly reduced when rats were allowed to relapse to alcohol consumption. 4. Based on these data, we examined whether pharmacological activation/blockade of endocannabinoid transmission might influence ethanol intake in rats allowed to relapse to alcohol consumption after subsequent periods of alcoholization and alcohol deprivation. 5. Treatment with either Delta(9)-tetrahydrocannabinol or CP55,940, two cannabinoid agonists, reduced both total liquid and ethanol intake but did not affect ethanol preference. Treatment with SR141716, a selective cannabinoid CB(1) receptor antagonist, also produced a significant reduction in both total liquid and ethanol intake without affecting ethanol preference. Accordingly, none of these effects on ethanol intake were accompanied by changes in dopamine and GABA in limbic structures. 6. In summary, the levels of endocannabinoids underwent significant changes in reward-related areas during alcoholization, alcohol deprivation, and relapse, showing the lowest values in this latter phase. Treatment with cannabinoid agonists or a selective CB(1) receptor antagonist resulted in a reduction of ethanol intake by rats allowed to relapse to alcohol consumption after periods of alcoholization and alcohol deprivation, but these effects did not appear to be due to changes in neurobiological substrates currently involved in alcohol reinforcement/relapse.

Involvement of vanilloid-like receptors in the effects of anandamide on motor behavior and nigrostriatal dopaminergic activity: in vivo and in vitro evidence.

The administration of the endocannabinoid anandamide to rats produces hypokinesia in parallel to a decrease in the activity of nigrostriatal dopaminergic neurons. It was earlier hypothesized that this effect was mediated through the activation of CB(1) receptors, although these receptors have not been found in dopaminergic neurons, but in striatal projection neurons connected with them. However, two recent discoveries: (i) that anandamide is also able to activate vanilloid VR(1) receptors, and (ii) that VR(1) receptors are located on nigrostriatal dopaminergic neurons, allow to re-evaluate this hypothesis and suggest that the activation of vanilloid-like receptors rather than CB(1) receptors might be responsible of anandamide-induced hypokinesia and decreased nigrostriatal dopaminergic activity. To validate this new hypothesis, we carried out two different experiments. First, we explored whether the inhibitory effects of anandamide on motor activity and dopaminergic transmission were reversed by capsazepine, an antagonist for vanilloid-like receptors. Our data demonstrated that anandamide reduced ambulation, stereotypies and exploration, measured in the open-field test, whereas it increased the time spent in inactivity. All these effects were completely reversed by capsazepine, which had no effect by itself. Anandamide also caused a significant decrease in nigrostriatal dopaminergic activity, reflected by a reduction in DOPAC contents in the caudate-putamen, which was also reversed by capsazepine. As a second objective, we explored whether anandamide is able to directly influence nigrostriatal dopaminergic function by examining its effects on in vitro dopamine (DA) release using perifused striatal fragments. Our data confirmed that anandamide significantly decreased K(+)-stimulated dopamine release from nigrostriatal terminals and that this effect was vanilloid-like receptor-mediated since it was prevented by capsazepine. This in vitro inhibitory effect was not seen with a classic cannabinoid agonist that does not bind vanilloid-like receptors. In summary, anandamide behaves as a hypokinetic substance, thus producing motor depression in the open-field test, presumably related to a decrease in nigrostriatal dopaminergic activity. These effects were completely reversed by the vanilloid-like receptor antagonist capsazepine, thus indicating a role of these receptors, which are located on dopaminergic neurons, in mediating hypokinetic effects of anandamide. In vitro studies, using perifused striatal fragments, support this vanilloid-like receptor-mediated direct action, which would not be available for classic cannabinoid agonists.

Transthyretin is involved in depression-like behaviour and exploratory activity.

Transthyretin (TTR), the major transporter of thyroid hormones and vitamin A in cerebrospinal fluid (CSF), binds the Alzheimer beta-peptide and thus might confer protection against neurodegeneration. In addition, altered TTR levels have been described in the CSF of patients with psychiatric disorders, yet its function in the CNS is far from understood. To determine the role of TTR in behaviour we evaluated the performance of TTR-null mice in standardized tasks described to assess depression, exploratory activity and anxiety. We show that the absence of TTR is associated with increased exploratory activity and reduced signs of depressive-like behaviour. In order to investigate the mechanism underlying these alterations, we measured the levels of catecholamines. We found that the levels of noradrenaline were significantly increased in the limbic forebrain of TTR-null mice. This report represents the first clear indication that TTR plays a role in behaviour, probably by modulation of the noradrenergic system.

Effects of perinatal exposure to delta 9-tetrahydrocannabinol on operant morphine-reinforced behavior.

The present study examined the effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) when administered during the perinatal period on morphine self-administration in adulthood. To this end, pregnant Wistar rats were daily exposed to Delta(9)-THC from the fifth day of gestation up to pup weaning, when they were separated by gender and left to mature to be used for analyses of operant food- and morphine-reinforced behavior in a progressive ratio (PR) schedule. We also analyzed dopaminergic activity (DOPAC/DA) in reward-related structures during specific phases of the behavioral study. In both reinforcement paradigms, food and morphine, females always reached higher patterns of self-administration than males, but this occurred for the two treatment groups, Delta(9)-THC or vehicle. These higher patterns measured in females corresponded with a higher DOPAC/DA in the nucleus accumbens prior to the onset of morphine self-administration in comparison to males. Interestingly, DOPAC/DA was lower in Delta(9)-THC-exposed females compared to oil-exposed females and similar to oil- and Delta(9)-THC-exposed males. In addition, Delta(9)-THC-exposed females also exhibited a reduction in DOPAC/DA in the ventral tegmental area, which did not exist in males. All these changes, however, disappeared after 15 days of morphine self-administration and they did not reappear after 15 additional days of extinction of this response. Our data suggest that females are more vulnerable than males in a PR schedule for operant food and morphine self-administration; perinatal Delta(9)-THC exposure is not a factor influencing this vulnerability. The neurochemical analysis revealed that the activity of limbic dopaminergic neurons prior to morphine self-administration was higher in females than males, as well as that the perinatal Delta(9)-THC treatment reduced the activity of these neurons only in females, although this had no influence on morphine vulnerability in these animals.

Compounds acting at the endocannabinoid and/or endovanilloid systems reduce hyperkinesia in a rat model of Huntington's disease.

We have recently reported that the administration of AM404, an inhibitor of the endocannabinoid re-uptake process, which also has affinity for the vanilloid VR1 receptors, is able to reduce hyperkinesia, and causes recovery from neurochemical deficits, in a rat model of Huntington's disease (HD) generated by bilateral intrastriatal injections of 3-nitropropionic acid (3NP). In the present study, we wanted to explore the mechanism(s) by which AM404 produces its antihyperkinetic effect in 3NP-lesioned rats by employing several experimental approaches. First, we tried to block the effects of AM404 with selective antagonists for the CB1 or VR1 receptors, i.e. SR141716A and capsazepine, respectively. We found that the reduction caused by AM404 of the increased ambulation exhibited by 3NP-lesioned rats in the open-field test was reversed when the animals had been pre-treated with capsazepine but not with SR141716A, thus suggesting a major role of VR1 receptors in the antihyperkinetic effects of AM404. However, despite the lack of behavioral effects of the CB1 receptor antagonist, the pretreatment with this compound abolished the recovery of neurochemical [gamma-aminobutyric acid (GABA) and dopamine] deficits in the caudate- putamen caused by AM404, as also did capsazepine. In a second group of studies, we wanted to explore the potential antihyperkinetic effects of various compounds which, compared to AM404, exhibit more selectivity for either the endovanilloid or the endocannabinoid systems. First, we tested VDM11 or AM374, two selective inhibitors or the endocannabinoid re-uptake or hydrolysis, respectively. Both compounds were mostly unable to reduce hyperkinesia in 3NP-lesioned rats, although VDM11 produced a certain motor depression, and AM374 exhibited a trend to stimulate ambulation, in control rats. We also tested the effects of selective direct agonists for VR1 (capsaicin) or CB1 (CP55,940) receptors. Capsaicin exhibited a strong antihyperkinetic activity and, moreover, was able to attenuate the reductions in dopamine and GABA transmission provoked by the 3NP lesion, whereas CP55,940 had also antihyperkinetic activity but was unable to cause recovery of either dopamine or GABA deficits in the basal ganglia. In summary, our data indicate a major role for VR1 receptors, as compared to CB1 receptors, in the antihyperkinetic effects and the recovery of neurochemical deficits caused in 3NP-lesioned rats by compounds that activate both CB1 and VR1 receptors, either directly or via manipulation of the levels of endogenous agonists.

Prenatal cannabinoid and gene expression for neural adhesion molecule L1 in the fetal rat brain.

The consumption by women of cannabis derivatives during pregnancy and/or lactation affects the development of their offspring because like other psychoactive drugs, cannabinoids, the psychoactive ingredients of marijuana, can cross the placental barrier and be secreted into the maternal milk. Through this way, cannabinoids are able to affect the expression of key genes for neural developmental leading to neurotransmitter and behavioral disturbances. In this present study, we wanted to explore the influence of prenatal cannabinoid exposure on the gene expression of a key protein for brain development, the neural adhesion molecule L1, which plays an important role in processes of cell proliferation and migration, neuritic elongation and guidance, and synaptogenesis. To this end, pregnant rats were daily treated with delta9-tetrahydrocannabinol (delta9-THC) since the 5th day of gestation up to the day before birth (GD21), day at which rats were killed and their pups removed for analysis of L1-mRNA levels in different brain structures. Our results confirmed that the levels of L1 transcripts were significantly increased after prenatal delta9-THC exposure in several regions such as the fimbria, stria terminalis, stria medullaris and corpus callosum, which share the properties of being white matter regions and containing, exclusively during development, an abundant population of cannabinoid CB1 receptors, the major targets for the action of plant-derived cannabinoids. L1-mRNA levels were also increased in grey matter structures such as the septum nuclei and the habenula, but remained unchanged in most of the grey matter structures analyzed (cerebral cortex, basolateral amygdaloid nucleus, hippocampus, thalamic and hypothalamic nuclei, basal ganglia and subventricular zones) and also in a few white matter structures (fornix and fasciculus retroflexus). An important aspect of these observations is that the increase in L1-mRNA levels reached statistical significance only in the case of delta9-THC-exposed males but not in the case of delta9-THC-exposed females where only trends or no effects were detected, this supporting previous evidence on a sexual dimorphism, with greater effects in male fetuses, for the action of cannabinoids in the developing brain. In summary, cannabinoids seem to influence the expression of L1 in specific brain structures during the prenatal period, which, considering the role played by this protein in different events related to neural development, might explain the neurotransmitter and behavioral disturbances reported after prenatal consumption of marijuana.

The endogenous cannabinoid system and the basal ganglia. biochemical, pharmacological, and therapeutic aspects.

New data strengthen the idea of a prominent role for endocannabinoids in the modulation of a wide variety of neurobiological functions. Among these, one of the most important is the control of movement. This finding is supported by 3 lines of evidence: (1) the demonstration of a powerful action, mostly inhibitory in nature, of synthetic and plant-derived cannabinoids and, more recently, of endocannabinoids on motor activity; (2) the presence of the cannabinoid CB(1) receptor subtype and the recent description of endocannabinoids in the basal ganglia and the cerebellum, the areas that control movement; and (3) the fact that CB(1) receptor binding was altered in the basal ganglia of humans affected by several neurological diseases and also of rodents with experimentally induced motor disorders. Based on this evidence, it has been suggested that new synthetic compounds that act at key steps of endocannabinoid activity (i.e., more-stable analogs of endocannabinoids, inhibitors of endocannabinoid reuptake or metabolism, antagonists of CB(1) receptors) might be of interest for their potential use as therapeutic agents in a variety of pathologies affecting extrapyramidal structures, such as Parkinson's and Huntington's diseases. Currently, only a few data exist in the literature studying such relationships in humans, but an increasing number of journal articles are revealing the importance of this new neuromodulatory system and arguing in favour of the funding of more extensive research in this field. The present article will review the current knowledge of this neuromodulatory system, trying to establish the future lines for research on the therapeutic potential of the endocannabinoid system in motor disorders.

Antinociceptive, behavioural and neuroendocrine effects of CP 55,940 in young rats.

The peripubertal period appears to be critical in relation to the abuse of cannabinoids and opioids in humans. However there is little information about the acute effects of cannabinoids and their interactions with opioids in young experimental animals. We have studied the effects of the cannabinoid agonist CP 55,940 (0.1, 0.2, 0.4 and 0.6 mg/kg) on the nociceptive responses (tail immersion test) and holeboard activity of 40-day-old rats, and the involvement of the CB(1) receptor (antagonism by SR 141716A, 3 mg/kg). The implication of the opioid system was evaluated using the opioid antagonist naloxone (1 mg/kg) and a combined treatment with subeffective doses of CP 55,940 (0.1 mg/kg) and morphine (1 mg/kg). The effects of CP 55,940 on the serum corticosterone levels (radioimmunoassay) and on the dopamine and DOPAC contents of discrete brain regions (high-performance liquid chromatography) were also assessed. The antinociceptive effect of CP 55,940 was of a similar magnitude at all the doses used. The results show the involvement of the CB(1) receptor. The cannabinoid agonist significantly depressed the holeboard activity in a dose-dependent manner. The results indicate that the CB(1) receptor is involved in the effects on motor activity but not in the effects on the exploratory activity. The behavioural effects of CP 55,940 were modulated by morphine. The cannabinoid agonist (0.6 mg/kg) induced a CB(1)-mediated increase in the serum corticosterone levels, but no effect on the dopaminergic systems of either the striatum or the limbic forebrain was found.

Prenatal exposure to methamphetamine in the rat: ontogeny of tyrosine hydroxylase mRNA expression in mesencephalic dopaminergic neurons.

Methamphetamine (Meth) is an illicit substance known to interfere with catecholaminergic systems and a popular recreational drug among young adult women, that is, in gestational age. Tyrosine hydroxylase (TH), the rate-limiting enzyme of the synthetic pathway of catecholamines, is a good marker to assess potential effects of Meth in catecholaminergic (particularly in dopaminergic) systems. In the rat, prolonged neonatal Meth exposure altered several dopaminergic markers (TH activity and gene expression) in substantia nigra pars compacta (SN) and in caudate-putamen (TH activity) when animals matured. However, it was never verified whether gestational exposure to Meth might compromise TH enzyme in the pups during the neonatal immature periods. The present study was designed to address this issue by analyzing TH gene expression, measured by in situ hybridization in SN and ventral tegmental area (VTA), dopaminergic areas that are well characterized as target areas for Meth, and in rats prenatally exposed to this psychostimulant. To this end, dated pregnant Wistar rat dams received 5 mg Meth hydrochloride/kg body weight/day. It was administered subcutaneously from gestational day 8 until 22. The control group was pair-fed and saline injected, using the same experimental protocol as for Meth-treated dams. On the day of birth (postnatal day 0, PND 0), litters were culled to 8 pups, sex-balanced whenever possible, and were followed until the day of sacrifice (PND 7, 14, or 30). Meth treatment differentially affected TH mRNA levels in VTA and SN, in an age- and gender-dependent manner. Thus, TH mRNA levels were decreased in the VTA of PND 7 and PND 14 females gestationally exposed to Meth; this effect was not evident in males or on PND 30. TH mRNA levels also tend to decrease in SN of PND 14 females gestationally exposed to Meth. Collectively, the present results indicated that gestational Meth exposure affects TH gene expression in the postnatal life, a phenomenon that appears to be transient, since it is no longer evident by the end of the first month of life in the rat.

Alleviation of motor hyperactivity and neurochemical deficits by endocannabinoid uptake inhibition in a rat model of Huntington's disease.

Recent studies have demonstrated a loss of cannabinoid CB1 receptors in the postmortem basal ganglia of patients affected by Huntington's disease (HD) and in transgenic mouse models for this disease. These studies have led to the notion that substances that increase the endocannabinoid activity, such as receptor agonists or inhibitors of endocannabinoid uptake and/or metabolism, might be useful in the treatment of hyperkinetic symptoms of this disease. In the present study, we employed a rat model of HD generated by bilateral intrastriatal injections of 3-nitropropionic acid (3-NP), a toxin that selectively damages striatal GABAergic efferent neurons. These rats exhibited biphasic motor disturbances, with an early (1-2 weeks) hyperactivity followed by a late (3-4 weeks) motor depression. Analysis of GABA, dopamine, and their related enzymes, glutamic acid decarboxylase and tyrosine hydroxylase, in the basal ganglia proved marked decreases compatible with the motor hyperkinesia. In addition, mRNA levels for CB1 receptor, neuronal-specific enolase, proenkephalin, and substance P decreased in the caudate-putamen of 3-NP-injected rats. There were also reductions in CB1 receptor binding in the caudate putamen, the globus pallidus, and, to a lesser extent, the substantia nigra. By contrast, mRNA levels for tyrosine hydroxylase in the substantia nigra remained unaffected. Interestingly, the administration of AM404, an inhibitor of endocannabinoid uptake, to 3-NP-injected rats attenuated motor disturbances observed in the early phase of hyperactivity. Administration of AM404 also tended to induce recovery from the neurochemical deficits caused by the toxin in GABA and dopamine indices in the basal ganglia. In summary, morphological, behavioral, and biochemical changes observed in rats intrastriatally lesioned with 3-NP acid were compatible with a profound degeneration of striatal efferent GABAergic neurons, similar to that occurring in the brain of HD patients. As expected, a loss of CB1 receptors was evident in the basal ganglia of these rats. However, the administration of substances that increase endocannabinoid activity, by inhibiting the uptake process, allowed an activation of the remaining population of CB1 receptors, resulting in a significant improvement of motor disturbances and neurochemical deficits. These observations might be relevant to the treatment of hyperkinetic symptoms in HD, a human disorder with unsatisfactory symptomatic treatment for most patients.

Exposure to cannabinoids in the development of endogenous cannabinoid system.

New data strengthen the idea of a prominent role for endocannabinoids in the modulation of a wide variety of neurobiological functions. Among these, two functions, control of movement and antinociception, have attracted the maximal interest because of the possibility that cannabinoids and related compounds might be used with a therapeutic purpose. However, the functions of endocannabinoids in the brain, and also in the periphery, are large and involve, not only the adulthood, but also the period of prenatal and postnatal development, when endocannabinoids have been reported to be significantly present and to play a role in processes of brain development as neuronal proliferation and migration, axonal elongation, synaptogenesis and/or myelinogenesis. The present review article will summarize the different studies carried out on this topic and will suggest future lines of research to clarify the role of endocannabinoids and their receptors in the development.

Loss of cannabinoid CB(1) receptors in the basal ganglia in the late akinetic phase of rats with experimental Huntington's disease.

We have recently examined the status of the endocannabinoid transmission in the basal ganglia in Huntington's disease (HD) using a rat model generated by bilateral intrastriatal injections of 3-nitropropionic acid (3-NP). In these previous studies, we focused on the early phase of hyperactivity that occurs 1-2 weeks after the lesion, comparable to early grades of the human disease, while in the present study, we wanted to explore the late akinetic phase observed 3-4 weeks after the lesion (similar to advanced grades). First, we confirmed that 3-NP-lesioned rats exhibited a marked akinesia tested at 4 weeks post-lesion. We observed a marked reduction in ambulatory and exploratory activities and a trend towards a decrease in stereotypies, paralleled by a strong increase in the time spent in inactivity. There was also a profound reduction in GABA contents and glutamic acid decarboxylase activity, particularly in the caudate-putamen and the globus pallidus. Dopamine and DOPAC contents, as well as the activity of tyrosine hydroxylase, were also reduced, particularly in the caudate-putamen. mRNA levels for neuronal-specific enolase, proenkephalin and substance P were also dramatically reduced in the caudate-putamen, thus indicating a death of both the direct (striatonigral) and the indirect (striatopallidal) GABAergic projection pathways, which corresponded with a marked loss of CB(1) receptor-mRNA levels observed in both parts, lateral and medial, of the caudate-putamen. However, losses of CB(1) receptor binding were confined to the globus pallidus and the caudate-putamen, whereas there were no changes in the substantia nigra and the entopeduncular nucleus. Finally, we failed to reduce the marked akinesia found in these animals by administering SR141716A, a selective antagonist of CB(1) receptors, which had exhibited hyperlocomotor effects in previous studies with naive animals. In summary, behavioral and biochemical changes observed in rats intrastriatally lesioned with 3- NP were compatible with a profound degeneration of striatal efferent GABAergic neurons, similar to those occurring in advances stages of the human disease. As expected, a loss of CB(1) receptors was evident in the basal ganglia of these rats during the late akinetic stage of the disease. Further studies should demonstrate whether these receptors might be a target for a new therapy in HD, a disease with a poor pharmacological outcome.