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The primary cilium is a central signaling component during embryonic development. Here we focus on CROCCP2, a hominid-specific gene duplicate from ciliary rootlet coiled coil (CROCC), also known as rootletin, that encodes the major component of the ciliary rootlet. We find that CROCCP2 is highly expressed in the human fetal brain and not in other primate species. CROCCP2 gain of function in the mouse embryonic cortex and human cortical cells and organoids results in decreased ciliogenesis and increased cortical progenitor amplification, particularly basal progenitors. CROCCP2 decreases ciliary dynamics by inhibition of the IFT20 ciliary trafficking protein, which then impacts neurogenesis through increased mTOR signaling. Loss of function of CROCCP2 in human cortical cells and organoids leads to increased ciliogenesis, decreased mTOR signaling, and impaired basal progenitor amplification. These data identify CROCCP2 as a human-specific modifier of cortical neurogenesis that acts through modulation of ciliary dynamics and mTOR signaling. ispartof: NEURON vol:111 issue:1 pages:65-+ ispartof: location:United States status: published

Corticosteroid and endocannabinoid actions converge on prefrontocortical circuits associated with neuropsychiatric illnesses. Corticosteroids can also modulate forebrain synapses by using endocannabinoid effector systems. Here, we determined whether corticosteroids can modulate transmitter release directly in the frontal cortex and, in doing so, whether they affect presynaptic CB1 cannabinoid receptor- (CB1R) mediated neuromodulation. By Western blotting of purified subcellular fractions of the rat frontal cortex, we found glucocorticoid receptors (GcRs) and CB1Rs enriched in isolated frontocortical nerve terminals (synaptosomes). CB1Rs were predominantly presynaptically located while GcRs showed preference for the post-synaptic fraction. Additional confocal microscopy analysis of cortical and hippocampal regions revealed vesicular GABA transporter-positive and vesicular glutamate transporter 1-positive nerve terminals endowed with CB1R immunoreactivity, apposing GcR-positive post-synaptic compartments. In functional transmitter release assay, corticosteroids, corticosterone (0.1–10 microM) and dexamethasone (0.1–10 microM) did not significantly affect the evoked release of [3H]GABA and [14C]glutamate in superfused synaptosomes, isolated from both rats and mice. In contrast, the synthetic cannabinoid, WIN55212-2 (1 microM) diminished the release of both [3H]GABA and [14C]glutamate, evoked with various depolarization paradigms. This effect of WIN55212-2 was abolished by the CB1R neutral antagonist, O-2050 (1 microM), and was absent in the CB1R KO mice. CB2R-selective agonists did not affect the release of either neurotransmitter. The lack of robust presynaptic neuromodulation by corticosteroids was unchanged upon either CB1R activation or genetic inactivation. Altogether, corticosteroids are unlikely to exert direct non-genomic presynaptic neuromodulation in the frontal cortex, but they may do so indirectly, via the stimulation of trans-synaptic endocannabinoid signaling.

There is mounting evidence that the neuropeptide oxytocin is a possible candidate for the treatment of drug addiction. Oxytocin was shown to reduce methamphetamine self-administration, conditioned place-preference, hyperactivity and reinstatement in rodents, highlighting its potential for the management of methamphetamine addiction. Thus, we hypothesised that the central endogenous oxytocinergic system is dysregulated following chronic methamphetamine administration. We tested this hypothesis by examining the effect of chronic methamphetamine administration on oxytocin receptor density in mice brains with the use of quantitative receptor autoradiographic binding. Saline (4 ml/kg/day, i.p.) or methamphetamine (1 mg/kg/day, i.p.) was administered daily for 10 days to male, CD1 mice. Quantitative autoradiographic mapping of oxytocin receptors was carried out with the use of [I]-vasotocin in brain sections of these animals. Chronic methamphetamine administration induced a region specific upregulation of oxytocin receptor density in the amygdala and hypothalamus, but not in the nucleus accumbens and caudate putamen. As there is evidence suggesting an involvement of central adenosine A receptors on central endogenous oxytocinergic function, we investigated whether these methamphetamine-induced oxytocinergic neuroadaptations are mediated via an A receptor-dependent mechanism. To test this hypothesis, autoradiographic oxytocin receptor binding was carried out in brain sections of male CD1 mice lacking A receptors which were chronically treated with methamphetamine (1 mg/kg/day, i.p. for 10 days) or saline. Similar to wild-type animals, chronic methamphetamine administration induced a region-specific upregulation of oxytocin receptor binding in the amygdala and hypothalamus of A receptor knockout mice and no genotype effect was observed. These results indicate that chronic methamphetamine use can induce profound neuroadaptations of the oxytocinergic receptor system in brain regions associated with stress, emotionality and social bonding and that these neuroadaptations are independent on the presence of A receptors. These results may at least partly explain some of the behavioural consequences of chronic methamphetamine use. © 2013 Elsevier Inc. All rights reserved.

Neocortical and striatal TRPV1 (vanilloid or capsaicin) receptors (TRPV1Rs) are excitatory ligand-gated ion channels, and are implicated in psychiatric disorders. However, the purported presynaptic neuromodulator role of TRPV1Rs in glutamatergic, serotonergic or dopaminergic terminals of the rodent forebrain remains little understood. With the help of patch-clamp electrophysiology and neurochemical approaches, we mapped the age-dependence of presynaptic TRPV1R function, and furthermore, we aimed at exploring whether the presence of CB1 cannabinoid receptors (CB1Rs) influences the function of the TRPV1Rs, as both receptor types share endogenous ligands. We found that the major factor which affects presynaptic TRPV1R function is age: by post-natal day 13, the amplitude of capsaicin-induced release of dopamine and glutamate is halved in the rat striatum, and two weeks later, capsaicin already loses its effect. However, TRPV1R receptor function is not enhanced by chemical or genetic ablation of the CB1Rs in dopaminergic, glutamatergic and serotonergic terminals of the mouse brain. Altogether, our data indicate a possible neurodevelopmental role for presynaptic TRPV1Rs in the rodent brain, but we found no cross-talk between TRPV1Rs and CB1Rs in the same nerve terminal.

Cardiac injury and dysfunction are contributors to disease progression and mortality in sepsis. This study evaluated the cardiovascular role of intrinsic A₂A adenosine receptor (A₂AAR) activity during lipopolysaccharide (LPS)-induced inflammation.We assessed the impact of 24 h of LPS challenge (20 mg/kg, IP) on cardiac injury, coronary function and inflammatory mediator levels in Wild-Type (WT) mice and mice lacking functional A₂AARs (A₂AAR KO).Cardiac injury was evident in LPS-treated WTs, with ~7-fold elevation in serum cardiac troponin I (cTnI), and significant ventricular and coronary dysfunction. Absence of A₂AARs increased LPS-provoked cTnI release at 24 h by 3-fold without additional demise of contraction function. Importantly, A₂AAR deletion per se emulated detrimental effects of LPS on coronary function, and LPS was without effect in coronary vessels lacking A₂AARs. Effects of A₂AAR KO were independent of major shifts in circulating C-reactive protein (CRP) and haptoglobin. Cytokine responses were largely insensitive to A₂AAR deletion; substantial LPS-induced elevations (up to 100-fold) in IFN-γ and IL-10 were unaltered in A₂AAR KO mice, as were levels of IL-4 and TNF-α. However, late elevations in IL-2 and IL-5 were differentially modulated by A₂AAR KO (IL-2 reduced, IL-5 increased). Data demonstrate that in the context of LPS-triggered cardiac and coronary injury, A₂AAR activity protects myocardial viability without modifying contractile dysfunction, and selectively modulates cytokine (IL-2, IL-5) release. A₂AARs also appear to be targeted by LPS in the coronary vasculature.These experimental data suggest that preservation of A₂AAR functionality might provide therapeutic benefit in human sepsis.

Both the serotonergic and endocannabinoid systems modulate frontocortical glutamate release; thus they are well positioned to participate in the pathogenesis of psychiatric disorders. With the help of fluorescent and confocal microscopy, we localized the CB(1) cannabinoid receptor (CB(1)R) in VGLUT1- and 2- (i.e. glutamatergic) and serotonin transporter- (i.e. serotonergic) -positive fibers and nerve terminals in the mouse and rat frontal cortex. CB(1)R activation by the synthetic agonists, WIN55212-2 (1 μM) and R-methanandamide (1 μM) inhibited the simultaneously measured evoked Ca(2+)-dependent release of [(14)C]glutamate and [(3)H]serotonin from frontocortical nerve terminals of Wistar rats, in a fashion sensitive to the CB(1)R antagonists, O-2050 (1 μM) and LY320135 (5 μM). CB(1)R agonists also inhibited the evoked release of [(14)C]glutamate in C57BL/6J mice in a reversible fashion upon washout. Interestingly, the evoked release of [(14)C]glutamate and [(3)H]serotonin was significantly greater in the CB(1)R knockout CD-1 mice. Furthermore, CB(1)R binding experiments revealed similar frontocortical CB(1)R density in the rat and the CD-1 mouse. Still, the evoked release of [(3)H]serotonin was modulated by neither CB(1)R agonists nor antagonists in wild-type CD-1 or C57BL/6J mice. Altogether, this is the first study to demonstrate functional presynaptic CB(1)Rs in frontocortical glutamatergic and serotonergic terminals, revealing species differences.

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.

GPR3 is an orphan G-protein coupled receptor broadly expressed in brain structures controlling emotional-like behaviors and pain. GPR3 receptor up-regulates cAMP and promotes neurite outgrowth in mammalian neurons, being a good candidate to participate in the pathophysiology of neurodegenerative diseases as well as brain and spinal cord injuries. In this study, we evaluated the role of GPR3 receptor in the development and expression of neuropathic pain after sciatic nerve ligature, and the inflammatory reaction in the dorsal horn of the spinal cord in both Gpr3−/− and Gpr3+/+ mice. Hyperalgesia to noxious thermal stimulus and allodynia to cold and mechanical stimuli were evaluated using the plantar test, the cold-plate test and the Von Frey filament model, respectively. Additionally, we evaluated the involvement of GPR3 receptors in morphine-induced antinociception using the tail immersion test. After nerve injury, Gpr3−/− mice showed a higher sensitivity to thermal non-noxious and noxious stimuli than Gpr3+/+ mice, whereas no differences were observed between genotypes in mechanical allodynia. In addition, no differences in microglia and astrocytes activation were found when compared the ipsilateral dorsal horn of Gpr3−/− and Gpr3+/+ mice exposed to nerve ligature. On the other hand, the genetic deletion of GPR3 receptors reduced morphine antinociception in the tail immersion test in mice without any changes in basal thermal threshold. Taken together, our results demonstrate, for the first time, the involvement of the orphan GPR3 receptor in the expression and development of neuropathic pain and in the analgesia induced by morphine. The lack of GPR3 receptors produced hypersensitivity to thermal non-noxious and noxious stimuli without affecting the spinal inflammatory response associated to sciatic nerve injury and reduced morphine antinociception in the tail immersion test. Our findings propose GPR3 receptors as a new molecular target in neuropathic pain therapy as well as a new component of a pro-opioid receptor system.

Resume Les relations intimes peuvent entrainer des manifestations allergiques ou intolerances variees, souvent meconnues, potentiellement graves et probablement sous-estimees. Les allergies au latex et au liquide seminal constituent deux causes classiques, mais une serie de materiaux ou de substances peuvent etre impliques. Par cette revue, les auteurs souhaitent attirer l’attention sur l’importance de l’anamnese qui doit etre soigneuse, policiere et parfois meme indiscrete. Malgre une evolution des mœurs en matiere de sexualite, il semble que la communaute medicale apporte peu de cas aux problemes qui lui sont lies. Une evaluation systematique en pratique allergologique permettrait sans doute une meilleure prise en charge de plaintes inhabituelles et parfois inexpliquees.

The CB(1) cannabinoid receptor has been implicated in the control of fear and anxiety. We investigated the effects of genetic and pharmacological blockade of the CB(1) cannabinoid receptor on the behaviour of CD1 mice using three different ethological models of fear and anxiety (elevated T-maze and plus-maze and open field test of emotionality). Furthermore, we measured tissue levels of noradrenalin (NA), dopamine (DA), serotonin (5-HT) and their metabolites in several forebrain regions, i.e. prefrontal cortex, hippocampus, septum, dorsal and ventral striatum to examine the relationship between CB(1) receptor manipulation and monoaminergic neurotransmission. The major findings can be summarized as follows: the CB(1) receptor antagonist SR141617A (rimonabant) modulated anxiety in a dose-dependent manner. At a dose of 3 mg/kg i.p., the compound consistently increased anxiety parameters in all of the three different anxiety tests applied, while a lower dosage of 1mg/kg had no such effect. The neurochemical evaluation of the mice administered 3mg/kg SR141617A revealed increases in the concentrations of DOPAC and 5-HIAA in the dorsal striatum, elevated DA levels in the hippocampus and reduced dopamine turnover in the septum. Furthermore, these animals had a higher HVA/DA turnover in the frontal cortex. CB(1) receptor knockout mice as well as mice treated with the selective CB(1) receptor antagonist AM251 (3 mg/kg; i.p.) did not display any significant alterations in anxiety-related behaviour as measured with the elevated plus-maze and open field test of emotionality, respectively. Our findings support the general idea of a SR141617A-sensitive receptive site that is different from the 'classical' CB(1) receptor and that has a pivotal role in the regulation of different psychological functions. However, with regard to its functional significance in terms of anxiety our findings suggest that under physiological conditions this receptive site seems to be involved in the control of anxiolysis rather than anxiogenesis as suggested previously.

Prostatic A 2A adenosine receptors mediate varied effects. This study aimed to test whether genetic disruption of this receptor affects prostate contractility. Prostates taken from mice which were homozygous (A 2A R−/−) and heterozygous (A 2A R+/−) for the disrupted A 2A adenosine receptor gene and wild-type littermates (A 2A R +/+) were mounted in organ baths. Contractile responses to nerve stimulation and noradrenaline were measured in the presence of various pharmacological tools. Electrical field stimulation (0.5 ms pulse duration, 60 V, 0.1–20 Hz) yielded frequency-dependent contractions while exogenous administration of noradrenaline (10 nM–1 mM) or tyramine (1 μM–1 mM) produced concentration-dependent responses. Contractile responses to electrical field stimulation from A 2A R−/− and A 2A R+/− prostates were reduced when compared to A 2A +/+ prostates ( P = 0.013, n = 33–36). Prazosin (0.3 μM) inhibited electrical field stimulation-induced responses in prostates from A 2A R+/+ and A 2A R+/− mice ( P ≤ 0.016, n = 5–7) but not A 2A R−/− mice ( P = 0.400, n = 6). Tetrodotoxin abolished electrical field stimulation-induced responses in all prostates ( P n = 5–7). NF 449 and ZM 241385 were without effect ( P ≤ 0.421, n = 4–6). There were no genotype differences in noradrenaline or tyramine concentration–response curves ( P ≥ 0.180, n = 10–13). Prazosin (0.3 μM) and cocaine (10 μM) attenuated the responses induced by noradrenaline ( P n = 6–7) and tyramine ( P n = 5–6), respectively, in all genotypes. Disruption of the A 2A adenosine receptor leads to reduced nerve mediated contractile responses of the prostate in mature mice.

Cardiac tissues express constitutively an NADPH oxidase, which generates reactive oxygen species (ROS) and is involved in redox signaling. Myocardial metabolism generates abundant adenosine, which binds to its receptors and plays important roles in cardiac function. The adenosine A2A receptor (A2AR) has been found to be expressed in cardiac myocytes and coronary endothelial cells. However, the role of the A2AR in the regulation of cardiac ROS production remains unknown. We found that knockout of A2AR significantly decreased (39+/-8%) NADPH-dependent O2- production in mouse hearts compared to age (10 weeks)-matched wild-type controls. This was accompanied by a significant decrease in Nox2 (a catalytic subunit of NADPH oxidase) protein expression, and down-regulation of ERK1/2, p38MAPK, and JNK phosphorylation (all P0.05). In wild-type mice, intraperitoneal injection of the selective A2AR antagonist SCH58261 (3-10 mg/kg body weight for 90 min) inhibited phosphorylation of p47phox (a regulatory subunit of Nox2), which was accompanied by a down-regulated cardiac ROS production (48+/-8%), and decreased JNK and ERK1/2 activation by 54+/-28% (all P0.05). In conclusion, A2AR through MAPK signaling regulates p47phox phosphorylation and cardiac ROS production by NADPH oxidase. Modulation of A2AR activity may have potential therapeutic applications in controlling ROS production by NADPH oxidase in the heart.

10 pages, 5 figures.-- PMID: 17467187 [PubMed].-- Printed version published Jun 8, 2007.-- Supporting information (Suppl. tables S1-S3) available at: http://dx.doi.org/10.1016/j.neuroscience.2007.03.017, The CB1 cannabinoid receptors modulate the addictive processes associated with different drugs of abuse, including psychostimulants. Mice lacking CB1 receptors exhibit an important attenuation of the reinforcing responses produced by cocaine in an operant self-administration paradigm. We have investigated the effect of chronic cocaine treatment on dendrite structure and spine density of the principal cortical neuron, the pyramidal neuron, in CB1 knockout mice and wild type littermates. Layer III pyramidal cells of the motor cortex were injected intracellularly in fixed cortical slices and their morphometric parameters analyzed. Under basal conditions, the field area of the dendritic arbors was more extensive and dendritic spine density was higher in wild type mice than in CB1 knockout mice. Chronic treatment of cocaine diminished the size and length of the basal dendrites and spine density on pyramidal cells from wild type mice. However, the total number of spines in the pyramidal cells of CB1 knockout mice augmented slightly following chronic cocaine treatment, although no changes in the morphology of the dendritic arbor were observed. Our data demonstrate that microanatomy and synaptic connectivity are affected by cocaine, the magnitude and nature of these changes depend on the presence of CB1 receptors., This work was supported by the "Ministerio de Ciencia y Tecnología" (BFI 2003–02745 and BFU2006-13395 to J.D., SAF2004/0568 to O.V.; BFU2004/00920BFI to R.M., and GEN2003-20651-C06-06 to J.D. and R.M.), the European Community (GENADDICT OJ2004/C164 No. 005166 and NEWMOOD LSHM-CT-2003 5003474), and the National Institutes of Health (grant 1RO1DA016768). I.B.-Y. was supported by the MEC (AP2001-0671).

A self-made electronic nose consisting in a sensor array of six commercial tin oxide gas sensors is used to monitor the odour emission from a compost facility. Supervised data processing tools, such as discriminant analysis, are able to recognize, in real time, the odour of compost with respect to other possible sources in the hall. The paper shows that with unsupervised methods, such as principal component analysis, it is not essential to identify all the possible odour sources during the learning phase. The closeness to the compost group centroid could be used as an indicator of the compost odour level. Alternatively, by a suitable calibration from olfactory measurements, the signals generated by the sensor array can be used to estimate the odour emission rate from the compost hall. Such real time monitoring should allow to assess and to anticipate the annoyance in the surrounding.

Neuropathic pain is a clinical manifestation characterized by the presence of spontaneous pain, allodynia and hyperalgesia. Here, we have evaluated the involvement of CB1 cannabinoid receptors in the development and expression of neuropathic pain. For this purpose, partial ligation of the sciatic nerve was performed in CB1 cannabinoid receptor knockout mice and their wild-type littermates. The development of mechanical and thermal allodynia, and thermal hyperalgesia was evaluated by using the von Frey filaments, cold-plate and plantar tests, respectively. Pre-surgical tactile and thermal withdrawal thresholds were similar in both genotypes. In wild-type mice, sciatic nerve injury led to a neuropathic pain syndrome characterized by a marked and long-lasting reduction of the paw withdrawal thresholds to mechanical and thermal stimuli. These manifestations developed similarly in mice lacking CB1 cannabinoid receptors. We have also investigated the consequences of gabapentin administration in these animals. Gabapentin (50 mg/kg/day, i.p.) induced a similar suppression of mechanical and thermal allodynia in both wild-type and CB1 knockout mice. Mild differences between genotypes were observed concerning the effect of gabapentin in the expression of thermal hyperalgesia. Taken together, our results indicate that CB1 cannabinoid receptors are not critically implicated in the development of neuropathic pain nor in the anti-allodynic and anti-hyperalgesic effects of gabapentin in this model.

Behavioral and biochemical studies have suggested a functional link between the endogenous cannabinoid and opioid systems. Different hypotheses have been proposed to explain the interactions between opioid and cannabinoid systems such as a common pathway stimulating the dopaminergic system, a facilitation of signal-transduction- and/or a cannabinoid-induced enhancement of opioid peptide release. However, at this time, all the studies have been performed with exogenous agonists (delta-9-tetrahydrocannabinol or morphine), leading to a generally excessive stimulation of receptors normally stimulated by endogenous effectors (anandamide or opioid peptides) in various brain structures. To overcome this problem, we have measured various behavioral responses induced by the stimulation of the endogenous opioid system using the dual inhibitor of enkephalin-degrading enzymes, RB101, in CB1 receptor knockout mice. Thus, analgesia, locomotor activity, anxiety and antidepressant-like effects were measured after RB101 administration (80 and 120 mg/kg i.p. or 10 mg/kg, i.v.) in CB1 receptor knockout mice and their wild-type littermates. In all the experiments, inhibition of enkephalin catabolism produced similar modifications in behavior observed in CB1 knockout and wild-type mice. These results suggest limited physiological interaction between cannabinoid and opioid systems.

The binding of the adenosine A(2A) receptor antagonist [3H] 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol ([3H]ZM 241385) to mouse brain and spinal cord was investigated. In brain homogenates, single-site binding was observed with a Bmax of 299+/-28 fmol mg(-1) protein and a Kd of 0.75+/-0.08 nM. In autoradiographic studies, there was a high density of specific binding of [3H]ZM 241385 in the striatum, with a very low density in the cortex and no binding elsewhere in the brain or in the spinal cord. All specific binding of [3H]ZM 241385 was lost in genetically modified mice lacking the adenosine A(2A) receptor, confirming the selectivity of this radioligand.

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.

Caffeine has biphasic effects on locomotion, and blockade of the adenosine A(2A) receptor (A2AR) is necessary for the stimulatory effect of low doses of caffeine, but not for the locomotor depressant effect observed at high doses. We wanted to elucidate the role of the adenosine A(1) receptor (A1R) in mediating the locomotor effects of increasing doses of caffeine using wild-type mice (A1R(WT)), mice heterozygous for (A1R(HET)), and mice lacking the adenosine A(1) receptor (A1R(KO)). Caffeine had the typical biphasic dose-effect relationship in all three genotypes, but the stimulatory action of caffeine was facilitated in the A1R(KO) mice. In order to investigate the interaction between blockade of A1Rs and A2ARs, mice lacking both receptors (A1R(KO)/A2AR(KO)) were tested. Regardless of A1R genotype, animals lacking A2AR were not stimulated by caffeine, whereas animals heterozygous for A2AR were. As expected, the A1R is not crucial for the stimulatory effect of caffeine, but seems to modulate the effect of caffeine exerted via A2AR blockade. Furthermore, these results suggest that the inhibitory effect of high doses of caffeine is due neither to blockade of the A1R, nor of the A2AR, and an effect independent of these adenosine receptors is likely.

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.

dInhibition of prostaglandins synthesis does not completely explain non-steroidal anti-inflammatory drug-induced spinal antinociception. Among other mediators, endocannabinoids are involved in pain modulation. Indomethacin-induced antinociception, in the formalin test performed in spinally microdialysed mice, was reversed by co-administration of the cannabinoid 1 (CB1) antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide (AM-251), but not by co-infusion of prostaglandin E-2. Indomethacin was ineffective in CB1 knockout mice. AM-251 also reversed the indomethacin-induced antinociception in a test of inflammatory hyperalgesia to heat. Furthermore, during the formalin test, indomethacin lowered the levels of spinal nitric oxide (NO), which activates cellular reuptake and thus breakdown of endocannabinoids. The pronociceptive effect of an NO donor, 3-methyl-N-nitrososydnone-5-imine (RE-2047), was abolished by co-administration of the endocannabinoid transporter blocker N-(4-hydroxyphenyl) arachidonoyl amide (AM-404). Moreover, the antinociceptive activity of the NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME), was reversed by AM-251. Thus we propose that at the spinal level, indomethacin induces a shift of arachidonic acid metabolism towards endocannabinoids synthesis secondary to cyclooxygenase inhibition. In addition, it lowers NO levels with subsequent higher levels of endocannabinoids. (C) 2002 Elsevier Science B.V. All rights reserved.

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.

In this study we report data suggesting the presence of a non-CB1, non-CB2 cannabinoid site in the cerebellum of CB1-/- mice. We have carried out [(35)S]GTPgammaS binding experiments in striata, hippocampi, and cerebella of CB1-/- and CB1(+/+) mice with Delta(9)-THC, WIN55,212-2, HU-210, SR141716A, and SR144528. In CB1-/- mice Delta(9)-THC and HU-210 did not stimulate [(35)S]GTPgammaS binding. However, WIN55,212-2 was able to stimulate [(35)S]GTPgammaS binding in cerebella of CB1-/- mice. The maximal effect of this stimulation was 31% that of wild type animals. This effect was reversible neither by CB1 nor CB2 receptor antagonists. Similar results were obtained with the endogenous cannabinoid, anandamide. However, adenylyl cyclase was not inhibited by WIN55,212-2 or anandamide in the CB1(minus sign/minus sign) animals. In striata and hippocampi of CB1-/- mice no [(35)S]GTPgammaS stimulation curve could be obtained with WIN55,212. Our findings suggest that there is a non-CB1 non-CB2 receptor present in the cerebellum of CB1-/- mice.

Psychoactive effects of cannabinoids are thought to be mediated, at least in part, by suppression of both glutamate and GABA release via CB1 cannabinoid receptor. Two types of cannabinoid receptor (CB1 and CB2) have been cloned so far. The CB1 receptors are abundantly expressed in the nervous system, whereas CB2 receptors are limited to lymphoid organs (Matsuda et al., 1990; Munro et al., 1993). Immunocytochemical and electrophysiological studies revealed that in the hippocampus CB1 receptors are expressed on axon terminals of GABAergic inhibitory interneurons (Tsou et al., 1999; Katona et al., 1999) and activation of these receptors decreases GABA release (Hájos et al., 2000). Other physiological studies pointed out the involvement of CB1 receptors in the modulation of hippocampal glutamatergic synaptic transmission and long-term potentiation (Stella et al., 1997; Misner and Sullivan, 1999), but anatomical studies could not confirm the existence of CB1 receptors on glutamatergic terminals. Here we examined cannabinoid actions on both glutamatergic and GABAergic synaptic transmission in the hippocampus of wild type (CB1+/+) and CB1 receptor knockout mice (CB1-/-). The synthetic cannabinoid agonist WIN55,212-2 reduced the amplitudes of excitatory postsynaptic currents in both wild type and CB1-/- mice, while inhibitory postsynaptic currents were decreased only in wild type mice, but not in CB1-/- animals. Our findings are consistent with a CB1 cannabinoid receptor-dependent modulation of GABAergic postsynaptic currents, but a novel cannabinoid-sensitive receptor must be responsible for the inhibition of glutamatergic neurotransmission.

The rewarding effects of morphine, cocaine, amphetamine and nicotine were evaluated in CB1 receptor knockout mice by means of an intravenous self-administration model. Experiments were carried out on drug-naive animals using a nose-poking response (NPR)-like as operandum. The results of the present study indicate that morphine did not induce intravenous self-administration in mutant CB1 receptor knockout mice, whereas it was significantly self-administered by the corresponding wild type mice. On the contrary, cocaine, amphetamine and nicotine were self-administered to the same extent by both wild type and CB1 receptor knockout mice. These data clearly indicate that the CB1 cannabinoid receptor is essential not only for the expression of cannabinoid reinforcing effects but also for the modulation of morphine rewarding effects. The specificity of such interaction is supported by the finding that contrary to morphine, cocaine, d-amphetamine and nicotine were self-administered by mice at the same extent either in presence or in absence of the CB1 receptor.

The present study was designed to assess whether adenosine A(2a) receptor knockout mice exhibit altered purine utilisation in brain nuclei. Specifically, the properties of adenosine transporters and adenosine A(1) receptors were characterised in brain membranes and on slide-mounted sections. The B(MAX) for [(3)H]nitrobenzylthioinosine ([(3)H]NBTI) binding (adenosine transporter density) was significantly reduced in brainstem membranes of homozygotes (560+/-52 fmol/mg protein, n=5, P0.05, Kruskal-Wallis ANOVA) compared to wildtype (1239+/-213 fmol/mg protein) and heterozygous mice (1300+/-558 fmol/mg protein). Quantitative autoradiography data indicated that [(3)H]NBTI binding in the medulla oblongata of heterozygous mice was seen to decrease significantly (P0.05) in the subpostremal nucleus tractus solitarius (NTS), medial NTS, inferior olive and area postrema (AP). On the other hand, in the homozygous mice a decrease was seen in the medial NTS and AP. In the pons, [(3)H]1, 3-dipropyl-8-cyclopentylxanthine ([(3)H]DPCPX) (adenosine A(1) receptor density) binding increased significantly (P0.05, Kruskal-Wallis ANOVA) in the lateral parabrachial nucleus, caudal pontine reticular nucleus and locus coeruleus of homozygotes compared to wildtype. In higher brain centres, [(3)H]NBTI binding was reduced in the paraventricular thalamic nucleus of both heterozygous and homozygous mice, whereas [(3)H]DPCPX binding was reduced in the hippocampus and lateral hypothalamus of heterozygotes. In homozygotes, [(3)H]DPCPX binding in the hippocampus increased compared to wildtype mice. The present study indicates that deletion of the A(2a) receptor may have contributed to region-specific compensatory changes in purine utilisation in brain nuclei associated with autonomic, neuroendocrine and behavioural regulation.

Adenosine A(2a) receptor knockout mice (A(2a)R k/o) have been characterised as hypertensive, aggressive, anxious and hypoalgesic [12]. Thus, the present study was designed to investigate markers of glutamatergic transmission in specific brain nuclei associated with autonomic regulation. Visualisation of alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid binding sites in the brains of A(2a)R k/o mice was achieved by utilising quantitative receptor autoradiography with (S)-[(3)H]-5-fluorowillardiine ([(3)H]FW:10 nM) on slide-mounted sections of mouse-brain. [(3)H]FW binding significantly increased in the nucleus tractus solitarius and area postrema of A(2a)R k/o mice compared with wildtype CD-1 mice. In other autonomic nuclei examined, binding was unchanged between wildtype and knockout mice. The present study suggests that glutamatergic neurotransmission within certain neural pathways involved in autonomic and motor control is altered in the brains of A(2a)R k/o mice.

We have previously reported that, in addition to modifying IgG levels and subclass distributions, wasp venom immunotherapy (VIT) rapidly changes IgG antibody specificity.We investigated whether such a change can be documented in the IgG response to the major bee venom allergen, phospholipase A2 (PLA2), from patients allergic to bees treated with VIT; whether it is coupled to the shift in IgG subclass distribution (IgG4 predominance) usually observed during VIT; and whether it restores the specificity displayed by IgG antibodies from nonallergic individuals.Antibody specificity was evaluated in 17 patients allergic to bee venom in competitive ELISAs by using streptavidin biotin technology. Patients were tested before and during specific immunotherapy (at 15 days and 6 months) and compared with another group of 17 patients treated with venom injections for at least 2 years (VIT patients) and 30 healthy individuals.The capacity of individual sera to prevent PLA2 binding of pooled IgG from allergic patients changed rapidly with mean percentage inhibitions falling from 84% +/- 14% before starting VIT to 27% +/- 13% and 28% +/- 7% after 15 days and 6 months of treatment, respectively (p0.001 by one-way analysis of variance [ANOVA]). IgG titers were only slightly increased. The capacity of individual sera to prevent the binding of pooled IgG from patients receiving VIT changed rapidly with mean percentage inhibition increasing from 60% +/- 12% before starting VIT to 85% +/- 6% and 82% +/- 6% after 15 days and 6 months of treatment, respectively (p0.001 by one-way ANOVA). Similar results were found regardless of whether pooled IgG1 or pooled IgG4 were used.VIT results in a rapid change in the antigenic reactivity of anti-PLA2 IgG antibody of human allergic sera, restoring, although not completely, the specificity peculiar to lgG from healthy individuals. This suggests that allergic status and immunoprotection correlate with the preferential expression of distinct IgG specificities, which appear equally distributed over the IgG1 and IgG4 antibody subclasses. It is, however, not known whether the shift in IgG specificity is one of the operative mechanisms of VIT.

International audience; Adenosine A(2A) receptor knockout mice (A(2A)R KO) were compared to wild-type controls (A(2A)R WT) in a caffeine intake paradigm. When mice had ad libitum access to caffeine (0.3 g/l) and water in a two-bottle paradigm for 12 consecutive days, adenosine A(2A)R KO mice drank less caffeinated solution, demonstrating a reduced appetite for caffeine as compared to adenosine A(2A)R WT mice. These data reveal an important role for the adenosine A(2A) receptor in the appetitive properties of caffeine.

The DNA sequences involved in transcription control by a cAMP-dependent mechanism have been localized in the thyroglobulin gene promoter region by a functional assay. The proximal 5'-flanking sequences from the bovine thyroglobulin gene were linked to the bacterial chloramphenicol acetyl-transferase gene. Transient expression of this reporter gene was studied in dog thyrocytes in primary culture in the presence, or absence, of cAMP stimulation. Deletion analysis showed that the cAMP-responsive region is contained within the first 250 base-pairs of the promoter, and suggests that it could correspond to a sequence conserved between species. These DNA sequences do not bear significant homology with cAMP-responsive elements (CRE) described previously. By contrast, some similarities were found with the fat-specific element (FSE2) of genes under cAMP control in adipocytes and with DNA elements mediating cAMP-dependent regulation of expression of two different genes in the lower eukaryote Dictyostelium discoideum. This suggests that control of Tg gene transcription by cAMP could involve a mechanism different from the one mediated by a classical CRE.