Biomedical subjects

C Ledent

Publications and source records attributed to C Ledent.

At least 19 recordsLinked to original sources

PubMed2006

Genetic interdependence of adenosine and dopamine receptors: evidence from receptor knockout mice.

Dopamine and adenosine receptors are known to share a considerable overlap in their regional distribution, being especially rich in the basal ganglia. Dopamine and adenosine receptors have been demonstrated to exhibit a parallel distribution on certain neuronal populations, and even when not directly co-localized, relationships (both antagonistic and synergistic) have been described. This study was designed to investigate dopaminergic and purinergic systems in mice with ablations of individual dopamine or adenosine receptors. In situ hybridization histochemistry and autoradiography was used to examine the level of mRNA and protein expression of specific receptors and transporters in dopaminergic pathways. Expression of the mRNA encoding the dopamine D2 receptor was elevated in the caudate putamen of D1, D3 and A2A receptor knockout mice; this was mirrored by an increase in D2 receptor protein in D1 and D3 receptor knockout mice, but not in A2A knockout mice. Dopamine D1 receptor binding was decreased in the caudate putamen, nucleus accumbens, olfactory tubercle and ventral pallidum of D2 receptor knockout mice. In substantia nigra pars compacta, dopamine transporter mRNA expression was dramatically decreased in D3 receptor knockout mice, but elevated in A2A receptor knockout mice. All dopamine receptor knockout mice examined exhibited increased A2A receptor binding in the caudate putamen, nucleus accumbens and olfactory tubercle. These data are consistent with the existence of functional interactions between dopaminergic and purinergic systems in these reward and motor-related brain regions.

Affinity Labels↗

PubMed2005

Cannabinoid CB1 receptor dependent effects of the NMDA antagonist phencyclidine in the social withdrawal model of schizophrenia.

Clinical and laboratory findings suggest that cannabinoid signalling is implicated in schizophrenia. However, the interaction remains poorly understood, as data are often contradictory. Here we investigated wild-type (WT) and cannabinoid CB1 receptor-knockout (CB1-KO) mice in the phencyclidine-induced social withdrawal model of schizophrenia. N-methyl-D-aspartate (NMDA) antagonists (including phencyclidine) induce psychotic symptoms in humans, and are used to model schizophrenia in a variety of experimental conditions. In WTs, 5 mg/kg phencyclidine increased locomotion and stereotyped behaviours, and decreased social interactions. These changes are consistent with a schizophrenia-like effect. In CB1-KOs, phencyclidine decreased locomotion, enhanced ataxia and stereotypy more markedly than in WTs, but did not affect social interactions. Locomotion showed a significant negative correlation with both ataxia and stereotypy, suggesting that in CB1-KOs, the locomotor suppressive effect of phencyclidine was secondary to changes in these variables. Our findings demonstrate that CB1 gene disruption dramatically alters the behavioural effects of the NMDA antagonist phencyclidine, suggesting that the CB1 receptor is involved in schizophrenia. As social disruption and stereotypy respectively are believed to model negative and positive symptoms of schizophrenia, our findings tentatively suggest that cannabinoids are differentially involved in these two symptom categories. These findings require verification by experiments involving CB1 receptor blockers, as the genetic and pharmacological blockade of receptors may not always provide similar results.

Animals↗

PubMed2004

The role of endogenous cannabinoids in the hypothalamo-pituitary-adrenal axis regulation: in vivo and in vitro studies in CB1 receptor knockout mice.

Exogenous cannabinoids affect multiple hormonal systems including the hypothalamo-pituitary-adrenocortical (HPA) axis. These data suggest that endogenous cannabinoids are also involved in the HPA control; however, the mechanisms underlying this control are poorly understood. We assessed the role of endogenous cannabinoids in the regulation of the HPA-axis by studying CB1 receptor knockout (KO) and wild type (WT) mice. Basal and novelty stress-induced plasma levels of adrenocorticotropin (ACTH) and corticosterone were higher in CB1-KO than in WT mice. We investigated the involvement of the pituitary in the hormonal effects of CB1 gene disruption by studying the in vitro release of ACTH from anterior pituitary fragments using a perifusion system. Both the basal and corticotropin releasing hormone (CRH)-induced ACTH secretion were similar in CB1-KO and WT mice. The synthetic glucocorticoid, dexamethasone suppressed the CRH-induced ACTH secretion in both genotypes; thus, the negative feedback of ACTH secretion was not affected by CB1 gene disruption. The cannabinoid agonist, WIN 55,212-2 had no effects on basal and CRH-stimulated ACTH secretion by anterior pituitary slices. In our hands, the disruption of the CB1 gene lead to HPA axis hyperactivity, but the pituitary seems not to be involved in this effect. Our data are consistent with the assumption that endogenous cannabinoids inhibit the HPA-axis via centrally located CB1 receptors, however the understanding of the exact underlying mechanism needs further investigation.

Adrenocorticotropic Hormone↗

PubMed2004

CB1 cannabinoid receptors mediate anxiolytic effects: convergent genetic and pharmacological evidence with CB1-specific agents.

Cannabinoids are known to modulate GABAergic and glutamatergic transmission in cortical areas, the former via CB1 and the latter via a novel receptor. Pharmacological data demonstrate that several widely used cannabinoid ligands bind to both receptors, which may explain the inconsistencies in their behavioural effects. Earlier we showed that the cannabinoid antagonist SR-141716A affected behaviour in both CB1 knockout and wild-type animals, and its effect (anxiolysis) was different from that of CB1 gene disruption (anxiogenesis). In the present experiments, we studied the effects of the CB1 antagonist AM-251, and the cannabinoid agonist WIN-55,212-2 in wild-type as well as in CB1 knockout mice. CB1 knockout mice showed higher scores of anxiety-like behaviour than the wild-type animals in the elevated plus-maze. Selective blockade of CB1 receptors by AM-251 (0.3, 1 and 3 mg/kg) increased anxiety-like behaviour dose-dependently in the wild-type mice but had no effect in the knockouts. In wild types, the cannabinoid agonist WIN-55,212-2 (1 and 3 mg/kg) caused a decrease in anxiety-like behaviour, which was abolished by the CB1-selective antagonist AM-251 (3 mg/kg). The same agonist did not change plus-maze behaviour in CB1 knockout animals. These data demonstrate at the behavioural level that AM-251 and, at low concentrations, WIN-55,212-2, are selective ligands of the CB1 cannabinoid receptor in mice. Our studies on the behavioural effects of the cannabinoid antagonist SR-141716A and the CB1 antagonist AM-251 show that the CB1 and the novel cannabinoid receptor mediate anxiolytic and anxiogenic effects, respectively. This suggests that agonists of the former, or antagonists of the latter, are promising new compounds in the pharmacotherapy of anxiety.

Animals↗

PubMed2004

Context-dependent effects of CB1 cannabinoid gene disruption on anxiety-like and social behaviour in mice.

Contrasting data were reported regarding the effects of cannabinoids on anxiety and social behaviour in both animals and humans. The cognitive effects of cannabinoids and their interactions with the HPA-axis raise the possibility that cannabinoid effects are context but not behaviour specific. To assess this hypothesis, we submitted CB1 receptor knock-out (CB1-KO) and wild-type (WT) mice to tests, which involved similar behaviours, but the behavioural context was different. The elevated plus-maze test was performed under less and more anxiogenic conditions, i.e. under low and high light, respectively. We also compared the social behaviour of the two genotypes in the resident/intruder and social interaction tests. Both tests represent a social challenge and induce similar behaviours, but involve different contexts. The behaviour of CB1-KO and WT mice was similar under low light, but CB1 gene disruption increased anxiety-like behaviour under the high light condition. CB1 gene disruption promoted aggressive behaviour in the home-cage, whereas it inhibited social behaviour in the unfamiliar cage. Thus, the anxiogenic-like effect was restricted to the more stressful unfamiliar environment. These data suggest that the effects of CB1 gene disruption were context and not behaviour specific. Novelty stress resulted in higher ACTH levels in CB1-KOs than in WTs, which suggests that context dependency occurred in conjunction with an altered HPA axis function. The present data at least partly explain contrasting effects of cannabinoids in different contexts as well as in different species and strains that show differential stress responses and coping strategies.

Adrenocorticotropic Hormone↗

PubMed2003

A2A receptor and striatal cellular functions: regulation of gene expression, currents, and synaptic transmission.

A2A receptor is highly coexpressed with enkephalin and D2 receptor in striatopallidal neurons. A2A antagonists acutely enhance motor behavior in animal models of Parkinson's disease (PD) and are therefore considered potential PD therapeutic agents. Analysis of gene expression regulation using pharmacologic tools or A2A receptor-deficient mice (A2A-/-) shows that the A2A receptor positively and tonically controls the expression of enkephalin and immediate early genes in striatopallidal neurons. Because this regulation strictly mirrors the effect of D2 receptor, these data strongly support the hypothesis that A2A antagonists reduce the activity of striatopallidal neurons in models of PD. However, analysis of A2A-/- mice suggests additional effects of A2A receptor in the control of striatal physiology. Unexpectedly, these animals exhibited a reduction in exploratory activity and a 50% reduction in substance P expression. This was associated with a 45% decrease in the striatal extracellular dopamine concentration, suggesting that chronic absence of A2A receptor results in a functional hypodopaminergic state in the striatum. The A2A receptor controls inhibitory synaptic transmission negatively in the striatum and positively in the globus pallidus; this further supports the efficacy of A2A antagonists in reducing the activity of striatopallidal neurons in PD. The A2A receptor does not modulate basal alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-mediated excitatory corticoaccumbal synaptic transmission during normal physiologic conditions. However, genetic inactivation or pharmacologic blockade of the A2A receptor significantly reduced long-term potentiation (LTP) at this synapse. Therefore, this receptor is implicated in the induction of corticoaccumbal LTP, an effect that could be related to its involvement in long-term behavioral sensitization to repeated dopaminergic treatment.

Animals↗

PubMed2003

Caffeine reduces hypnotic effects of alcohol through adenosine A2A receptor blockade.

The role of the adenosine A(2A) receptor in the hypnotic effects of ethanol was assessed in mice. The duration of the loss of righting reflex following acute ethanol administration was shorter for A(2A) receptor-deficient mice (A(2A)R KO) than for wild-type mice (A(2A)R WT), whereas the fall in body temperature was not different between the two phenotypes. In contrast, the duration of the loss of righting reflex was increased in A(2A)R KO mice versus controls after administration of pentobarbital. Dipyridamole, an inhibitor of adenosine uptake, increased the sleep time observed following administration of ethanol in CD1 mice and in A(2A)R WT but not in A(2A)R KO mice. SCH 58261, a selective A(2A) receptor antagonist, unlike DPCPX, a selective A(1) receptor antagonist, shortened the duration of the loss of righting reflex induced by ethanol, thus mimicking the lack of receptor in deficient mice. Finally, the non-selective adenosine receptor antagonist caffeine (25 mg/kg) reduced ethanol-induced hypnotic effects. These results indicate that the activation of A(2A) receptors that follows an increase in extracellular adenosine levels caused by the administration of high doses of ethanol plays a role in its hypnotic effects. Thus, A(2A) receptor antagonists may be useful therapeutic agents for alleviating ethylic coma.

Adenosine A1 Receptor Antagonists↗

PubMed2003

Adenosine A2A receptor gene disruption provokes marked changes in melanocortin content and pro-opiomelanocortin gene expression.

A2A receptor knockout (A2AR-/-) mice are more anxious and aggressive, and exhibit reduced exploratory activity than their wild-type littermates (A2AR+/+). Because alpha-melanocyte-stimulating hormone (alpha-MSH) influences anxiety, aggressiveness and motor activity, we investigated the effect of A2AR gene disruption on alpha-MSH content in discrete brain regions and pro-opiomelanocortin (POMC) expression in the hypothalamus and pituitary. No modification in alpha-MSH content was observed in the hypothalamus and medulla oblongata where POMC-expressing perikarya are located. In the arcuate nucleus of the hypothalamus, POMC mRNA levels were not affected by A2AR disruption. Conversely, in A2AR-/- mice, a significant increase in alpha-MSH content was observed in the amygdala and cerebral cortex, two regions that are innervated by POMC terminals. In the pars intermedia of the pituitary, A2AR disruption provoked a significant reduction of POMC mRNA expression associated with a decrease in alpha-MSH content. By contrast, in the anterior lobe of the pituitary, a substantial increase in POMC mRNA and adrenocorticotropin hormone concentrations was observed, and plasma corticosterone concentration was significantly higher in A2AR-/- mice, revealing hyperactivity of their pituitary-adrenocortical axis. Together, these results suggest that adenosine, acting through A2A receptors, may modulate the release of alpha-MSH in the cerebral cortex and amygdala. The data also indicate that A2A receptors are involved in the control of POMC gene expression and biosynthesis of POMC-derived peptides in pituitary melanotrophs and corticotrophs.

Animals↗

PubMed2003

Relationships between cell division, expression of growth factors and microcirculation in the thyroids of Tg-A2aR transgenic mice and patients with Graves' disease.

Tissue heterogeneity and nodule formation are hallmarks of thyroid growth. This is accounted for by the clonality theory that acknowledges different individual cellular abilities to respond to trophic stimuli. In order to test the hypothesis that functional and mitotic properties of thyrocytes could be influenced by paracrine interactions with neighbour endothelial cells, studies were conducted in both mouse and human goitre models. In the first part of the study, homogenous goitres in C57 black mice were compared with heterogeneous goitres in transgenic hyperthyroid mice expressing the A2 adenosine receptor (Tg-A2aR). The second part of the study concentrated on comparing human thyroid tIssue of control individuals and of patients with Graves' disease. The rate of cell division was evaluated by immunohistochemical detection of cells positive for proliferating cell nuclear antigen (PCNA). Their spatial distribution was then correlated with immunohistochemical cellular expression of growth- and vasoactive-related factors (fibroblast growth factor-2, transforming growth factor-beta, endothelin-1, vascular endothelial growth factor, nitric oxide synthase III), and with microcirculation expansion. Observations were made on digitalised images of histological serial sections. The nearest-neighbour method was used to distinguish between random or clustered distribution. PCNA-positive cells were both randomly and uniformly distributed in homogenous goitres from C57 black mice, and were clustered in tIssue areas identified as papillary and hyperplastic zones in heterogeneous goitres from Tg-A2aR mice. However, they were absent in the so-called compact cellular zones featuring resting cells. Moreover, whereas papillary and hyperplastic zones were highly vascularised, compact zones were nearly free of microvessels. Spatial distribution of dividing cells was positively correlated with the expression of growth-related factors. A similar pattern was observed in the thyroids of patients with Graves' disease. In accordance with the recent demonstration of the presence of angiofollicular units in the thyroid, these data strongly support the hypothesis that functional and mitotic properties of each single thyrocyte, likely to be responsible for growth heterogeneity of hyperplastic glands, may be adjusted at tIssue level by specific interactions with neighbour endothelial cells that, in turn, could alter the mitotic rate of thyrocytes through paracrine signals.

Animals↗

PubMed2002

Relaxation of the mouse isolated aorta and carotid artery in response to adenosine analogues in genetically-modified mice lacking the adenosine A(2A) receptor.

The aim of this study was to characterise the receptor(s) mediating responses to adenosine and/or adenosine analogues in mouse isolated aorta and carotid artery. In addition, since mice lacking the A(2A) adenosine receptor are reported to be hypertensive, the possibility that this gene deletion or the altered phenotype results in alteration of responses mediated via adenosine analogues was investigated. This was achieved by comparing results obtained in parallel within single experiments using tissues from A(2A) knock-out animals and their wild-type littermates.In aortic rings, adenosine and 5'- N-ethylcarboxamidoadenosine (NECA) caused relaxations above 10 microM and 30 microM, respectively, which were unaffected by either 8-sulphophenyltheophylline (8-SPT, 100 microM) or A(2A) receptor knockout. 2-[ p-(2-Carbonylethyl)phenylethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) was virtually inactive. R- N(6)-Phenylisopropyladenosine (R-PIA) induced relaxations which were not inhibited by 8-SPT (100 microM) or altered by A(2A) receptor knockout. No A(1)-mediated contractile responses were observed in wild-type or knock-out tissues in contrast with results in mice of the same strain obtained commercially rather than from our breeding programme. In carotid artery rings NECA contracted at low concentrations (0.1-1 microM) and relaxed at higher concentrations. Curves to NECA were not different in tissues from wild-type and A(2A) receptor knock-out mice and both the contractile and relaxant phases were right-shifted by 8-SPT (100 microM) in tissues from animals of both genotype. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX, 3 nM) attenuated contractile NECA responses but did not affect relaxant responses. CGS 21680 was inactive in carotid artery rings from both wild-type and A(2A) receptor knock-out mice. In the presence of DPCPX (30 nM) to abolish contractions, R-PIA induced relaxant curves which were not different in tissues from wild-type and A(2A) knock-out mice and were not inhibited by 8-SPT (100 microM). These results confirm the absence of A(2A) or A(2B) receptors in murine aorta and suggest that relaxations to NECA in carotid artery are A(2B) receptor-mediated whilst contractions are A(1) receptor-mediated. They also indicate the presence of an antagonist-resistant site activated by R-PIA in both vascular preparations. There is no evidence for compensatory changes in responses mediated by adenosine and its analogues due to the gene deletion or the reported resulting hypertensive phenotype in either aortic or carotid arterial rings obtained from A(2A) knock-out mice.

Adenosine↗

PubMed2002

Lack of CB1 cannabinoid receptors modifies nicotine behavioural responses, but not nicotine abstinence.

Cannabis is the most widely consumed illicit drug and its consumption is currently associated with tobacco, which contains another psychoactive compound, namely nicotine. Interactions between cannabinoids and other drugs of abuse, such as opioids, have been previously reported. The aim of the present study was to evaluate the possible role of CB1 cannabinoid receptor in responses induced by acute and repeated nicotine administration by using knockout mice lacking the CB1 cannabinoid receptor and their wild-type littermates. Acute nicotine (0.5, 1, 3 and 6 mg/kg, sc) administration decreased locomotor activity and induced antinociceptive responses in the tail-immersion and the hot-plate test, in wild-type animals. The antinociceptive effects in the tail-immersion test were significantly enhanced in CB1 knockout mice. In wild-type mice nicotine (0.5 mg/kg, sc) produced a significant rewarding effect, as measured by a conditioned place preference paradigm. This response was absent in CB1 knockout mice. Finally, a model of mecamylamine-induced abstinence in chronic nicotine-treated mice (10 mg/kg/day, sc) was developed. Mecamylamine (1 and 2 mg/kg, sc) precipitated several somatic signs of nicotine withdrawal in wild-type dependent mice. However, no difference in the severity of nicotine withdrawal was observed in CB1 knockout mice. These results demonstrate that some acute effects and motivational responses elicited by nicotine can be modulated by the endogenous cannabinoid system and support the existence of a physiological interaction between these two systems.

Analgesics↗

PubMed2002

The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety.

The aim of this study was to compare the effects of the genetic and pharmacological disruption of CB1 cannabinoid receptors on the elevated plus-maze test of anxiety. In the first experiment, the behaviour of CB1-knockout mice and wild-type mice was compared. In the second experiment, the cannabinoid antagonist SR141716A (0, 1, and 3 mg/kg) was administered to both CB1-knockout and wild type mice. Untreated CB1-knockout mice showed a reduced exploration of the open arms of the plus-maze apparatus, thus appearing more anxious than the wild-type animals, however no changes in locomotion were noticed. The vehicle-injected CB1-knockout mice from the second experiment also showed increased anxiety as compared with wild types. Surprisingly, the cannabinoid antagonist SR141716A reduced anxiety in both wild type and CB1 knockout mice. Locomotor behaviour was only marginally affected. Recent evidence suggests the existence of a novel cannabinoid receptor in the brain. It has also been shown that SR141716A binds to both the CB1 and the putative novel receptor. The data presented here supports these findings, as the cannabinoid receptor antagonist affected anxiety in both wild type and CB1-knockout mice. Tentatively, it may be suggested that the discrepancy between the effects of the genetic and pharmacological blockade of the CB1 receptor suggests that the novel receptor plays a role in anxiety.

Animals↗

PubMed2002

The role of cyclic AMP and its effect on protein kinase A in the mitogenic action of thyrotropin on the thyroid cell.

Cyclic AMP has been shown to inhibit cell proliferation in many cell types and to activate it in some. The latter has been recognized only lately, thanks in large part to studies on the regulation of thyroid cell proliferation in dog thyroid cells. The steps that led to this conclusion are outlined. Thyrotropin activates cyclic accumulation in thyroid cells of all the studied species and also phospholipase C in human cells. It activates directly cell proliferation in rat cell lines, dog, and human thyroid cells but not in bovine or pig cells. The action of cyclic AMP is responsible for the proliferative effect of TSH. It accounts for several human diseases: congenital hyperthyroidism, autonomous adenomas, and Graves' disease; and, by default, for hypothyroidism by TSH receptor defect. Cyclic AMP proliferative action requires the activation of protein kinase A, but this effect is not sufficient to explain it. Cyclic AMP action also requires the permissive effect of IGF-1 or insulin through their receptors, mostly as a consequence of PI3 kinase activation. The mechanism of these effects at the level of cyclin and cyclin-dependent protein kinases involves an induction of cyclin D3 by IGF-1 and the cyclic AMP-elicited generation and activation of the cyclin D3-CDK4 complex.

Animals↗

PubMed2001

Metabotropic glutamate receptors drive the endocannabinoid system in hippocampus.

Endocannabinoids are key intercellular signaling molecules in the brain, but the physiological regulation of the endocannabinoid system is not understood. We used the retrograde signal process called depolarization-induced suppression of inhibition (DSI) to study the regulation of this system. DSI is produced when an endocannabinoid released from pyramidal cells suppresses IPSCs by activating CB1R cannabinoid receptors located on inhibitory interneurons. We now report that activation of group I metabotropic glutamate receptors (mGluRs) enhances DSI and that this effect is blocked by antagonists of both mGluRs and of CB1R. We also found that DSI is absent in CB1R knock-out (CB1R(-/-)) mice, and, strikingly, that mGluR agonists have no effect on IPSCs in these mice. We conclude that group I mGluR-induced enhancement of DSI, and suppression of IPSCs, is actually mediated by endocannabinoids. This surprising result opens up new approaches to the investigation of cannabinoid actions in the brain.

Animals↗

PubMed2001

Distribution of CB1 cannabinoid receptors in the amygdala and their role in the control of GABAergic transmission.

Cannabinoids are the most popular illicit drugs used for recreational purposes worldwide. However, the neurobiological substrate of their mood-altering capacity has not been elucidated so far. Here we report that CB1 cannabinoid receptors are expressed at high levels in certain amygdala nuclei, especially in the lateral and basal nuclei, but are absent in other nuclei (e.g., in the central nucleus and in the medial nucleus). Expression of the CB1 protein was restricted to a distinct subpopulation of GABAergic interneurons corresponding to large cholecystokinin-positive cells. Detailed electron microscopic investigation revealed that CB1 receptors are located presynaptically on cholecystokinin-positive axon terminals, which establish symmetrical GABAergic synapses with their postsynaptic targets. The physiological consequence of this particular anatomical localization was investigated by whole-cell patch-clamp recordings in principal cells of the lateral and basal nuclei. CB1 receptor agonists WIN 55,212-2 and CP 55,940 reduced the amplitude of GABA(A) receptor-mediated evoked and spontaneous IPSCs, whereas the action potential-independent miniature IPSCs were not significantly affected. In contrast, CB1 receptor agonists were ineffective in changing the amplitude of IPSCs in the rat central nucleus and in the basal nucleus of CB1 knock-out mice. These results suggest that cannabinoids target specific elements in neuronal networks of given amygdala nuclei, where they presynaptically modulate GABAergic synaptic transmission. We propose that these anatomical and physiological features, characteristic of CB1 receptors in several forebrain regions, represent the neuronal substrate for endocannabinoids involved in retrograde synaptic signaling and may explain some of the emotionally relevant behavioral effects of cannabinoid exposure.

Amygdala↗

PubMed2001

Dual role of Fyn in the regulation of FAK+6,7 by cannabinoids in hippocampus.

In hippocampus endocannabinoids modulate synaptic function and plasticity and increase tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK). Autophosphorylation of FAK on Tyr-397 is generally a critical step for its activation, allowing the recruitment of Src family kinases, and phosphorylation of FAK and associated proteins. We have examined the mechanisms of the regulation of FAK by cannabinoids in rat and mouse hippocampal slices. Anandamide and 2-arachidonoylglycerol, two endocannabinoids, and Delta9-tetrahydrocannabinol, stimulated tyrosine phosphorylation of FAK+6,7, a neuronal splice isoform of FAK, on several residues including Tyr-397. Cannabinoids increased phosphorylation of p130-Cas, a protein associated with FAK, but had no effect on PYK2, a tyrosine kinase related to FAK and enriched in hippocampus. Pharmacological experiments and the use of knockout mice demonstrated that the effects of cannabinoids were mediated through CB1 receptors. These effects were sensitive to manipulation of cAMP-dependent protein kinase, suggesting that they were mediated by inhibition of a cAMP pathway. PP2, an Src family kinase inhibitor, prevented the effects of cannabinoids on p130-Cas and on FAK+6,7 tyrosines 577 and 925, but not 397, indicating that FAK autophosphorylation was upstream of Src family kinases in response to CB1-R stimulation. Endocannabinoids increased the association of Fyn, but not Src, with FAK+6,7. In hippocampal slices from Fyn -/- mice, the levels of p130-Cas were increased, and the effects of endocannabinoids on tyrosine phosphorylation, including of Tyr-397, were completely abolished. These results demonstrate the specific functional association of Fyn with FAK+6,7 in a pathway regulated by endocannabinoids, in which Fyn may play roles dependent and independent of its catalytic activity.

Animals↗

PubMed2001

Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum.

In the striatum, dopamine D(1) and adenosine A(2A) receptors stimulate the production of cAMP, which is involved in neuromodulation and long-lasting changes in gene expression and synaptic function. Positive coupling of receptors to adenylyl cyclase can be mediated through the ubiquitous GTP-binding protein Galpha(S) subunit or through the olfactory isoform, Galpha(olf), which predominates in the striatum. In this study, using double in situ hybridization, we show that virtually all striatal efferent neurons, identified by the expression of preproenkephalin A, substance P, or D(1) receptor mRNA, contained high amounts of Galpha(olf) mRNA and undetectable levels of Galpha(s) mRNA. In contrast, the large cholinergic interneurons contained both Galpha(olf) and Galpha(s) transcripts. To assess the functional relationship between dopamine or adenosine receptors and G-proteins, we examined G-protein levels in the striatum of D(1) and A(2A) receptor knock-out mice. A selective increase in Galpha(olf) protein was observed in these animals, without change in mRNA levels. Conversely, Galpha(olf) levels were decreased in animals lacking a functional dopamine transporter. These results indicate that Galpha(olf) protein levels are regulated through D(1) and A(2A) receptor usage. To determine the functional consequences of changes in Galpha(olf) levels, we used heterozygous Galpha(olf) knock-out mice, which possess half of the normal Galpha(olf) levels. In these animals, the locomotor effects of amphetamine and caffeine, two psychostimulant drugs that affect dopamine and adenosine signaling, respectively, were markedly reduced. Together, these results identify Galpha(olf) as a critical and regulated component of both dopamine and adenosine signaling.

Adenosine↗

PubMed2001

The central cannabinoid receptor inactivation suppresses endocrine reproductive functions.

The function of central cannabinoid (CB1) receptor was investigated in the regulation of the pituitary-gonad axis in CB1 receptor knockout male mouse. Serum luteinizing hormone (LH) and testosterone (T) levels and basal T secretion in vitro of testes were significantly decreased in mutant (CB1-/-) mice. The receptor agonist, anandamide (ANA), suppressed LH and T secretion in wild type (CB1+/+) mice but had no effect in receptor inactivated animals. The results are the first descriptions indicating the direct action of CB1 receptors on LH and T secretion and the immunohistological demonstration of CB1 receptors in the Leydig cells. The results also indicate that CB1 receptors are responsible for the effects of exogenous cannabinoids on reproductive functions.

Animals↗