Liste des publications

Team publications

IF du Neurocentre

165 publications

* equal contribution
The indicated IF have been collected by the Web of Sciences in June 2020

12/2009 | Nat Neurosci
Self-modulation of neocortical pyramidal neurons by endocannabinoids.
Marinelli S, Pacioni S, Cannich A, Marsicano G, Bacci A

Control of pyramidal neuron excitability is vital for the functioning of the neocortex. Somatodendritic slow self-inhibition (SSI) allows inhibitory neurons to regulate their own activity, but the existence of similar mechanisms in excitatory cells has not been shown. We found that in rodents endocannabinoids mediated SSI and long-term modulation of inhibitory connections in layer 2/3 pyramidal neurons with a distinct dendritic morphology, suggesting that a glutamatergic network in cortical circuits is self-regulated.

10/2009 | Genes Brain Behav
Endocannabinoids render exploratory behaviour largely independent of the test aversiveness: role of glutamatergic transmission.
Jacob W, Yassouridis A, Marsicano G, Monory K, Lutz B, Wotjak CT

To investigate the impact of averseness, controllability and familiarity of a test situation on the involvement of the endocannabinoid system in the regulation of exploratory behaviour, we tested conventional and conditional cannabinoid receptor type 1 (CB1)-deficient mice in behavioural paradigms with different emotional load, which depended on the strength of illumination and the ability of the animals to avoid the light stimulus. Complete CB1 null-mutant mice (Total-CB1-KO) showed an anxiogenic-like phenotype under circumstances where they were able to avoid the bright light such as the elevated plus-maze and the light/dark avoidance task. Conditional mutant mice lacking CB1 expression specifically in cortical glutamatergic neurons (Glu-CB1-KO), in contrast, failed to show a similar phenotype under the same experimental conditions. However, both mutant lines showed increased avoidance of open arm exploration during a second exposure to the elevated plus-maze. If tested in situations where the fear eliciting light could not be avoided, Total-CB1-KO mice showed increased thigmotaxis in an open field, decreased social investigation and decreased novel object exploration under aversive light conditions, but not under non-aversive low light. This time, Glu-CB1-KO also showed decreased exploratory behaviour towards objects and conspecific juveniles and increased thigmotaxis in the open field. Taking into consideration that the behavioural performance of wild-type mice was only marginally affected by changes in light intensities, these data indicate that the endocannabinoid system renders exploratory behaviour largely independent of the test averseness. This process differentially involves endocannabinoid-controlled glutamatergic transmission, depending on the controllability of the test situation.

09/2009 | Nat Neurosci
Cannabinoid modulation of hippocampal long-term memory is mediated by mTOR signaling.
Puighermanal E, Marsicano G, Busquets-Garcia A, Lutz B, Maldonado R, Ozaita A

Cognitive impairment is one of the most important negative consequences associated with cannabis consumption. We found that CB1 cannabinoid receptor (CB1R) activation transiently modulated the mammalian target of rapamycin (mTOR)/p70S6K pathway and the protein synthesis machinery in the mouse hippocampus, which correlated with the amnesic properties of delta9-tetrahydrocannabinol (THC). In addition, non-amnesic doses of either the mTOR blocker rapamycin or the protein synthesis inhibitor anisomycin abrogated the amnesic-like effects of THC, pointing to a mechanism involving new protein synthesis. Moreover, using pharmacological and genetic tools, we found that THC long-term memory deficits were mediated by CB1Rs expressed on GABAergic interneurons through a glutamatergic mechanism, as both the amnesic-like effects and p70S6K phosphorylation were reduced in GABA-CB1R knockout mice and by NMDA blockade.

07/08/2009 | Science
Spinal endocannabinoids and CB1 receptors mediate C-fiber-induced heterosynaptic pain sensitization.
Pernia-Andrade AJ, Kato A, Witschi R, Nyilas R, Katona I, Freund TF, Watanabe M, Filitz J, Koppert W, Schuttler J, Ji G, Neugebauer V, Marsicano G, Lutz B, Vanegas H, Zeilhofer HU

Diminished synaptic inhibition in the spinal dorsal horn is a major contributor to chronic pain. Pathways that reduce synaptic inhibition in inflammatory and neuropathic pain states have been identified, but central hyperalgesia and diminished dorsal horn synaptic inhibition also occur in the absence of inflammation or neuropathy, solely triggered by intense nociceptive (C-fiber) input to the spinal dorsal horn. We found that endocannabinoids, produced upon strong nociceptive stimulation, activated type 1 cannabinoid (CB1) receptors on inhibitory dorsal horn neurons to reduce the synaptic release of gamma-aminobutyric acid and glycine and thus rendered nociceptive neurons excitable by nonpainful stimuli. Our results suggest that spinal endocannabinoids and CB1 receptors on inhibitory dorsal horn interneurons act as mediators of heterosynaptic pain sensitization and play an unexpected role in dorsal horn pain-controlling circuits.

06/2009 | Eur J Neurosci
WIN55,212-2, a cannabinoid receptor agonist, protects against nigrostriatal cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.
Price DA, Martinez AA, Seillier A, Koek W, Acosta Y, Fernandez E, Strong R, Lutz B, Marsicano G, Roberts JL, Giuffrida A

Parkinson's disease (PD) is characterized by the progressive loss of nigrostriatal dopamine neurons leading to motor disturbances and cognitive impairment. Current pharmacotherapies relieve PD symptoms temporarily but fail to prevent or slow down the disease progression. In this study, we investigated the molecular mechanisms by which the non-selective cannabinoid receptor agonist WIN55,212-2 (WIN) protects mouse nigrostriatal neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity and neuroinflammation. Stereological analyses showed that chronic treatment with WIN (4 mg/kg, intraperitoneal), initiated 24 h after MPTP administration, protected against MPTP-induced loss of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta independently of CB1 cannabinoid receptor activation. The neuroprotective effect of WIN was accompanied by increased dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra pars compacta and dorsal striatum of MPTP-treated mice. At 3 days post-MPTP, we found significant microglial activation and up-regulation of CB2 cannabinoid receptors in the ventral midbrain. Treatment with WIN or the CB2 receptor agonist JWH015 (4 mg/kg, intraperitoneal) reduced MPTP-induced microglial activation, whereas genetic ablation of CB2 receptors exacerbated MPTP systemic toxicity. Furthermore, chronic WIN reversed MPTP-associated motor deficits, as revealed by the analysis of forepaw step width and percentage of faults using the inverted grid test. In conclusion, our data indicate that agonism at CB2 cannabinoid receptors protects against MPTP-induced nigrostriatal degeneration by inhibiting microglial activation/infiltration and suggest that CB2 receptors represent a new therapeutic target to slow the degenerative process occurring in PD.

03/2009 | Genes Brain Behav
Endocannabinoids mediate acute fear adaptation via glutamatergic neurons independently of corticotropin-releasing hormone signaling
Kamprath K, Plendl W, Marsicano G, Deussing J M, Wurst W, Lutz B, Wotjak C T

Recent evidence showed that the endocannabinoid system plays an important role in the behavioral adaptation of stress and fear responses. In this study, we chose a behavioral paradigm that includes criteria of both fear and stress responses to assess whether the involvement of endocannabinoids in these two processes rely on common mechanisms. To this end, we delivered a footshock and measured the fear response to a subsequently presented novel tone stimulus. First, we exposed different groups of cannabinoid receptor type 1 (CB(1))-deficient mice (CB(1) (-/-)) and their wild-type littermates (CB(1) (+/+)) to footshocks of different intensities. Only application of an intense footshock resulted in a sustained fear response to the tone in CB(1) (-/-). Using the intense protocol, we next investigated whether endocannabinoids mediate their effects via an interplay with corticotropin-releasing hormone (CRH) signaling. Pharmacological blockade of CB(1) receptors by rimonabant in mice deficient for the CRH receptor type 1 (CRHR1(-/-)) or type 2 (CRHR2(-/-)), and in respective wild-type littermates, resulted in a sustained fear response in all genotypes. This suggests that CRH is not involved in the fear-alleviating effects of CB(1). As CRHR1(-/-) are known to be severely impaired in stress-induced corticosterone secretion, our observation also implicates that corticosterone is dispensable for CB(1)-mediated acute fear adaptation. Instead, conditional mutants with a specific deletion of CB(1) in principal neurons of the forebrain (CaMK-CB(1) (-/-)), or in cortical glutamatergic neurons (Glu-CB(1) (-/-)), showed a similar phenotype as CB(1) (-/-), thus indicating that endocannabinoid-controlled glutamatergic transmission plays an essential role in acute fear adaptation.

2009 | Science
Spinal endocannabinoids mediate activity-dependent central sensitization in pain pathways
Pernia-Andrade AJ, Witschi R, Nyials R, Katona I, Freund TF, Watanabe M, Filitz J, Koppert W, Schüttler J, Ji G, Neugebauer V, Marsicano G, Lutz B, Vanegas H, Zeilhofer HU

2009 | Eur J Neurosci
WO'55,212,2, a cannabinoid receptor agonist, protects against nigrostriatal cell loss in the 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine mouse model of Parkinson's disease.
Price DA, Martinez AA, Seillier A, Koek W, Acosta Y, Fernandez H, Strong R, Lutz B, Marsicano G, Roberts JL, Giuffrida A

2009 | Curr Top Behav Neurosci
Roles of the endocannabinoid system in learning and memory.
Marsicano G, Lafenetre P

The endocannabinoid system (ECS) plays a central role in the regulation of learning and memory processes. The fine-tuned regulation of neural transmission by the system is likely to be the mechanism underlying this important function. In this chapter, we review the data in the literature showing the direct involvement of the physiological activation of cannabinoid receptors in the modulation of different forms of learning and memory. When possible, we also address the likely mechanisms of this involvement. Finally, given the apparent special role of the ECS in the extinction of fear, we propose a reasonable model to assess how neuronal networks could be influenced by the endocannabinoids in these processes. Overall, the data reviewed indicate that, despite the enormous progress of recent years, much is still to be done to fully elucidate the mechanisms of the ECS influence on learning and memory processes.

09/2008 | J Clin Invest
Hepatic CB1 receptor is required for development of diet-induced steatosis, dyslipidemia, and insulin and leptin resistance in mice
Osei-Hyiaman D, Liu J, Zhou L, Godlewski G, Harvey-White J, Jeong W I, Batkai S, Marsicano G, Lutz B, Buettner C, Kunos G

Diet-induced obesity is associated with fatty liver, insulin resistance, leptin resistance, and changes in plasma lipid profile. Endocannabinoids have been implicated in the development of these associated phenotypes, because mice deficient for the cannabinoid receptor CB1 (CB1-/-) do not display these changes in association with diet-induced obesity. The target tissues that mediate these effects, however, remain unknown. We therefore investigated the relative role of hepatic versus extrahepatic CB1 receptors in the metabolic consequences of a high-fat diet, using liver-specific CB1 knockout (LCB1-/-) mice. LCB1(-/-) mice fed a high-fat diet developed a similar degree of obesity as that of wild-type mice, but, similar to CB1(-/-) mice, had less steatosis, hyperglycemia, dyslipidemia, and insulin and leptin resistance than did wild-type mice fed a high-fat diet. CB1 agonist-induced increase in de novo hepatic lipogenesis and decrease in the activity of carnitine palmitoyltransferase-1 and total energy expenditure were absent in both CB1(-/-) and LCB1(-/-) mice. We conclude that endocannabinoid activation of hepatic CB1 receptors contributes to the diet-induced steatosis and associated hormonal and metabolic changes, but not to the increase in adiposity, observed with high-fat diet feeding. Theses studies suggest that peripheral CB1 receptors could be selectively targeted for the treatment of fatty liver, impaired glucose homeostasis, and dyslipidemia in order to minimize the neuropsychiatric side effects of nonselective CB1 blockade during treatment of obesity-associated conditions.

The complete genetic loss or pharmacological blockade of cannabinoid receptor type 1 (CB1) in mice results in both altered behavioral performance and increased stress hormone secretion in response to stressful encounters such as forced swim test (FST) exposure. CB1 is expressed on nerve terminals belonging to different neurotransmitter systems, including the glutamatergic and GABAergic system, where it is able to suppress excitatory and inhibitory neurotransmission, respectively. In the current study, we used the conditional mutagenesis approach in mice to investigate the neurotransmitter systems involved in these behavioral and neuroendocrine phenotypes in regard to CB1 signaling. Mice lacking CB1 in cortical glutamatergic neurons (Glu-CB1(-/-)) showed decreased passive stress coping (decreased immobility) in the FST, whereas mice lacking CB1 in principal forebrain neurons (CaMK-CB1(-/-)) and GABAergic neurons (GABA-CB1(-/-)), respectively, behaved as littermate controls. However, we found increased FST-induced corticosterone secretion only in CaMK-CB1(-/-) mice, whereas Glu-CB1(-/-) and GABA-CB1(-/-) mice exhibited normal corticosterone release as compared to controls. Thus, behavioral and neuroendocrine acute stress coping in response to the FST is mainly influenced by CB1 signaling on different glutamatergic neuronal subpopulations, but not by CB1 on GABAergic neurons.

08/2008 | Diabetes
Cannabinoid type 1 receptor blockade promotes mitochondrial biogenesis through endothelial nitric oxide synthase expression in white adipocytes
Tedesco L, Valerio A, Cervino C, Cardile A, Pagano C, Vettor R, Pasquali R, Carruba M O, Marsicano G, Lutz B, Pagotto U, Nisoli E

OBJECTIVE: Cannabinoid type 1 (CB1) receptor blockade decreases body weight and adiposity in obese subjects; however, the underlying mechanism is not yet fully understood. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) induces mitochondrial biogenesis and function in adipocytes. This study was undertaken to test whether CB1 receptor blockade increases the espression of eNOS and mitochondrial biogenesis in white adipocytes. RESEARCH DESIGN AND METHODS: We examined the effects on eNOS and mitochondrial biogenesis of selective pharmacological blockade of CB1 receptors by SR141716 (rimonabant) in mouse primary white adipocytes. We also examined eNOS expression and mitochondrial biogenesis in white adipose tissue (WAT) and isolated mature white adipocytes of CB1 receptor-deficient (CB1(-/-)) and chronically SR141716-treated mice on either a standard or high-fat diet. RESULTS: SR141716 treatment increased eNOS expression in cultured white adipocytes. Moreover, SR141716 increased mitochondrial DNA amount, mRNA levels of genes involved in mitochondrial biogenesis, and mitochondrial mass and function through eNOS induction, as demonstrated by reversal of SR141716 effects by small interfering RNA-mediated decrease in eNOS. While high-fat diet-fed wild-type mice showed reduced eNOS expression and mitochondrial biogenesis in WAT and isolated mature white adipocytes, genetic CB1 receptor deletion or chronic treatment with SR141716 restored these parameters to the levels observed in wild-type mice on the standard diet, an effect linked to the prevention of adiposity and body weight increase. CONCLUSIONS: CB1 receptor blockade increases mitochondrial biogenesis in white adipocytes by inducing the expression of eNOS. This is linked to the prevention of high-fat diet-induced fat accumulation, without concomitant changes in food intake.

07/2008 | Ann Neurol
Cannabinoids enhance susceptibility of immature brain to ethanol neurotoxicity
Hansen H H, Krutz B, Sifringer M, Stefovska V, Bittigau P, Pragst F, Marsicano G, Lutz B, Ikonomidou C

OBJECTIVE: Marijuana and alcohol are most widely abused drugs among women of reproductive age. Neurocognitive deficits have been reported in children whose mothers used marijuana during pregnancy. Maternal consumption of ethanol is known to cause serious developmental deficits METHODS: Infant rats and mice received systemic injections of Delta(9)-tetrahydrocannabinol (THC; 1-10mg/kg) or the synthetic cannabinoid WIN 55,212-2 (1-10mg/kg), alone or in combination with subtoxic and toxic ethanol doses, and apoptotic neurodegeneration was studied in the brains RESULTS: Acute administration of THC (1-10mg/kg), the principal psychoactive cannabinoid of marijuana, markedly enhanced proapoptotic properties of ethanol in the neonatal rat brain. THC did not induce neurodegeneration when administered alone. Neuronal degeneration became disseminated and severe when THC was combined with a mildly intoxicating ethanol dose (3gm/kg), with the effect of this drug combination resembling the massive apoptotic death observed when administering ethanol alone at much higher doses. The detrimental effect of THC was mimicked by the synthetic cannabinoid WIN 55,212-2 (1-10mg/kg) and counteracted by the CB(1) receptor antagonist SR141716A (0.4mg/kg). THC enhanced the proapoptotic effect of the GABA(A) agonist phenobarbital and the N-methyl-D-aspartate receptor antagonist dizocilpine. Interestingly, infant CB(1) receptor knock-out mice were less susceptible to the neurotoxic effect of ethanol. Furthermore, the CB(1) receptor antagonist SR141716A ameliorated neurotoxicity of ethanol INTERPRETATION: These observations indicate that CB(1) receptor activation modulates GABAergic and glutamatergic neurotransmission and primes the developing brain to suffer apoptotic neuronal death.

24/06/2008 | Proc Natl Acad Sci U S A
Endocannabinoid signaling controls pyramidal cell specification and long-range axon patterning
Mulder J, Aguado T, Keimpema E, Barabas K, Ballester Rosado C J, Nguyen L, Monory K, Marsicano G, Di Marzo V, Hurd Y L, Guillemot F, Mackie K, Lutz B, Guzman M, Lu H C, Galve-Roperh I, Harkany T

Endocannabinoids (eCBs) have recently been identified as axon guidance cues shaping the connectivity of local GABAergic interneurons in the developing cerebrum. However, eCB functions during pyramidal cell specification and establishment of long-range axonal connections are unknown. Here, we show that eCB signaling is operational in subcortical proliferative zones from embryonic day 12 in the mouse telencephalon and controls the proliferation of pyramidal cell progenitors and radial migration of immature pyramidal cells. When layer patterning is accomplished, developing pyramidal cells rely on eCB signaling to initiate the elongation and fasciculation of their long-range axons. Accordingly, CB(1) cannabinoid receptor (CB(1)R) null and pyramidal cell-specific conditional mutant (CB(1)R(f/f,NEX-Cre)) mice develop deficits in neuronal progenitor proliferation and axon fasciculation. Likewise, axonal pathfinding becomes impaired after in utero pharmacological blockade of CB(1)Rs. Overall, eCBs are fundamental developmental cues controlling pyramidal cell development during corticogenesis.

06/2008 | Pharmacogenomics J
Impaired cannabinoid receptor type 1 signaling interferes with stress-coping behavior in mice
Steiner M A, Wanisch K, Monory K, Marsicano G, Borroni E, Bachli H, Holsboer F, Lutz B, Wotjak C T

Dysregulation of the endocannabinoid system is known to interfere with emotional processing of stressful events. Here, we studied the role of cannabinoid receptor type 1 (CB1) signaling in stress-coping behaviors using the forced swim test (FST) with repeated exposures. We compared effects of genetic inactivation with pharmacological blockade of CB1 receptors both in male and female mice. In addition, we investigated potential interactions of the endocannabinoid system with monoaminergic and neurotrophin systems of the brain. Naive CB1 receptor-deficient mice (CB1-/-) showed increased passive stress-coping behaviors as compared to wild-type littermates (CB1+/+) in the FST, independent of sex. These findings were partially reproduced in C57BL/6N animals and fully reproduced in female CB1+/+ mice by pharmacological blockade of CB1 receptors with the CB1 receptor antagonist SR141716. The specificity of SR141716 was confirmed in female CB1-/- mice, where it failed to affect behavioral performance. Sensitivity to the antidepressants desipramine and paroxetine was preserved, but slightly altered in female CB1-/- mice. There were no genotype differences between CB1+/+ and CB1-/- mice in monoamine oxidase A and B activities under basal conditions, nor in monoamine content of hippocampal tissue after FST exposure. mRNA expression of vesicular glutamate transporter type 1 was unaffected in CB1-/- mice, but mRNA expression of brain-derived neurotrophic factor (BDNF) was reduced in the hippocampus. Our results suggest that impaired CB1 receptor function promotes passive stress-coping behavior, which, at least in part, might relate to alterations in BDNF function.

03/2008 | Learn Mem
Activation of CB1 specifically located on GABAergic interneurons inhibits LTD in the lateral amygdala
Azad S C, Kurz J, Marsicano G, Lutz B, Zieglgansberger W, Rammes G

Previously, we found that in the lateral amygdala (LA) of the mouse, WIN55,212-2 decreases both glutamatergic and GABAergic synaptic transmission via activation of the cannabinoid receptor type 1 (CB1), yet produces an overall reduction of neuronal excitability. This suggests that the effects on excitatory transmission override those on inhibitory transmission. Here we show that CB1 activation by WIN55,212-2 and Delta(9)-THC inhibits long-term depression (LTD) of basal synaptic transmission in the LA, induced by low-frequency stimulation (LFS; 900 pulses/1 Hz). The CB1 agonist WIN55,212-2 blocked LTD via G(i/o) proteins, activation of inwardly rectifying K+ channels (K(ir)s), inhibition of the adenylate cyclase-protein kinase A (PKA) pathway, and PKA-dependent inhibition of voltage-gated N-type Ca2+ channels (N-type VGCCs). Interestingly, WIN55,212-2 effects on LTD were abolished in CB1 knock-out mice (CB1-KO), and in conditional mutants lacking CB1 expression only in GABAergic interneurons, but were still present in mutants lacking CB1 in principal forebrain neurons. LTD induction per se was unaffected by the CB1 antagonist SR141716A and was normally expressed in CB1-KO as well as in both conditional CB1 mutants. Our data demonstrate that activation of CB1 specifically located on GABAergic interneurons inhibits LTD in the LA. These findings suggest that CB1 expressed on either glutamatergic or GABAergic neurons play a differential role in the control of synaptic transmission and plasticity.

03/2008 | Cell Metab
Paracrine activation of hepatic CB1 receptors by stellate cell-derived endocannabinoids mediates alcoholic fatty liver
Jeong W I, Osei-Hyiaman D, Park O, Liu J, Batkai S, Mukhopadhyay P, Horiguchi N, Harvey-White J, Marsicano G, Lutz B, Gao B, Kunos G

Alcohol-induced fatty liver, a major cause of morbidity, has been attributed to enhanced hepatic lipogenesis and decreased fat clearance of unknown mechanism. Here we report that the steatosis induced in mice by a low-fat, liquid ethanol diet is attenuated by concurrent blockade of cannabinoid CB1 receptors. Global or hepatocyte-specific CB1 knockout mice are resistant to ethanol-induced steatosis and increases in lipogenic gene expression and have increased carnitine palmitoyltransferase 1 activity, which, unlike in controls, is not reduced by ethanol treatment. Ethanol feeding increases the hepatic expression of CB1 receptors and upregulates the endocannabinoid 2-arachidonoylglycerol (2-AG) and its biosynthetic enzyme diacylglycerol lipase beta selectively in hepatic stellate cells. In control but not CB1 receptor-deficient hepatocytes, coculture with stellate cells from ethanol-fed mice results in upregulation of CB1 receptors and lipogenic gene expression. We conclude that paracrine activation of hepatic CB1 receptors by stellate cell-derived 2-AG mediates ethanol-induced steatosis through increasing lipogenesis and decreasing fatty acid oxidation.

01/2008 | J Mol Med
Increased endocannabinoid levels reduce the development of precancerous lesions in the mouse colon
Izzo A A, Aviello G, Petrosino S, Orlando P, Marsicano G, Lutz B, Borrelli F, Capasso R, Nigam S, Capasso F, Di Marzo V

Colorectal cancer is an increasingly important cause of death in Western countries. Endocannabinoids inhibit colorectal carcinoma cell proliferation in vitro. In this paper, we investigated the involvement of endocannabinoids on the formation of aberrant crypt foci (ACF, earliest preneoplastic lesions) in the colon mouse in vivo. ACF were induced by azoxymethane (AOM); fatty acid amide hydrolase (FAAH) and cannabinoid receptor messenger ribonucleic acid (mRNA) levels were analyzed by the quantitative reverse transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by liquid chromatography-mass spectrometry; caspase-3 and caspase-9 expressions were measured by Western blot analysis. Colonic ACF formation after AOM administration was associated with increased levels of 2-arachidonoylglycerol (with no changes in FAAH and cannabinoid receptor mRNA levels) and reduction in cleaved caspase-3 and caspase-9 expression. The FAAH inhibitor N-arachidonoylserotonin increased colon endocannabinoid levels, reduced ACF formation, and partially normalized cleaved caspase-3 (but not caspase-9) expression. Notably, N-arachidonoylserotonin completely prevented the formation of ACF with four or more crypts, which have been show to be best correlated with final tumor incidence. The effect of N-arachidonoylserotonin on ACF formation was mimicked by the cannabinoid receptor agonist HU-210. No differences in ACF formation were observed between CB(1) receptor-deficient and wild-type mice. It is concluded that pharmacological enhancement of endocannabinoid levels (through inhibition of endocannabinoid hydrolysis) reduces the development of precancerous lesions in the mouse colon. The protective effect appears to involve caspase-3 (but not caspase-9) activation.

01/2008 | Psychoneuroendocrinology
Antidepressant-like behavioral effects of impaired cannabinoid receptor type 1 signaling coincide with exaggerated corticosterone secretion in mice
Steiner M A, Marsicano G, Nestler E J, Holsboer F, Lutz B, Wotjak C T

Hypothalamic-pituitary-adrenocortical (HPA) axis hyperactivity is associated with major depressive disorders, and treatment with classical antidepressants ameliorates not only psychopathological symptoms, but also the dysregulation of the HPA axis. Here, we further elucidated the role of impaired cannabinoid type 1 receptor (CB1) signaling for neuroendocrine and behavioral stress coping in the mouse forced swim test (FST). We demonstrate that the genetic inactivation of CB1 is accompanied by increased plasma corticosterone levels both under basal conditions and at different time points following exposure to the FST. The latter effect could be mimicked in C57BL/6N mice by acute, subchronic, and chronic administration of the selective CB1 antagonist SR141716. Further experiments confirmed the specificity of corticosterone-elevating SR141716 actions for CB1 in CB1-deficient mice. Subchronic and chronic pharmacological blockade of CB1, but not its genetic deletion, induced antidepressant-like behavioral responses in the FST that were characterized by decreased floating and/or increased struggling behavior. The antidepressant-like behavioral effects of acute desipramine treatment in the FST were absent in CB1-deficient mice, but the dampening effects of desipramine on FST stress-induced corticosterone secretion were not compromised by CB1 deficiency. However, antidepressant-like behavioral desipramine effects were intact in C57BL/6N mice pre-treated with SR141716, indicating potential developmental deficits in CB1-deficient mice. We conclude that pharmacological blockade of CB1 signaling shares antidepressant-like behavioral effects with desipramine, but reveals opposite effects on HPA axis activity.

The endocannabinoid system recently emerged as an important modulator of many neuronal functions. Among them, the control of anxiety and acquired fear represents nowadays one of the most interesting fields of research. Despite contrasting results obtained by the use of cannabinoid receptor agonists in experimental animals, there is growing evidence that the physiological activation of the endocannabinoid system plays a central role in the control of basal anxiety levels and in the modulation of fear responses. This review will summarise recent data on the role of the endocannabinoid system in most commonly used tests of anxiety and in the processing of acquired fear, with particular attention to its involvement in fear extinction. Finally, a neurobiological model possibly able to implement the role of the endocannabinoid system in these processes will be proposed.

10/2007 | PLoS Biol
Genetic dissection of behavioural and autonomic effects of Delta(9)-tetrahydrocannabinol in mice
Monory K, Blaudzun H, Massa F, Kaiser N, Lemberger T, Schutz G, Wotjak C T, Lutz B, Marsicano G

Marijuana and its main psychotropic ingredient Delta(9)-tetrahydrocannabinol (THC) exert a plethora of psychoactive effects through the activation of the neuronal cannabinoid receptor type 1 (CB1), which is expressed by different neuronal subpopulations in the central nervous system. The exact neuroanatomical substrates underlying each effect of THC are, however, not known. We tested locomotor, hypothermic, analgesic, and cataleptic effects of THC in conditional knockout mouse lines, which lack the expression of CB1 in different neuronal subpopulations, including principal brain neurons, GABAergic neurons (those that release gamma aminobutyric acid), cortical glutamatergic neurons, and neurons expressing the dopamine receptor D1, respectively. Surprisingly, mice lacking CB1 in GABAergic neurons responded to THC similarly as wild-type littermates did, whereas deletion of the receptor in all principal neurons abolished or strongly reduced the behavioural and autonomic responses to the drug. Moreover, locomotor and hypothermic effects of THC depend on cortical glutamatergic neurons, whereas the deletion of CB1 from the majority of striatal neurons and a subpopulation of cortical glutamatergic neurons blocked the cataleptic effect of the drug. These data show that several important pharmacological actions of THC do not depend on functional expression of CB1 on GABAergic interneurons, but on other neuronal populations, and pave the way to a refined interpretation of the pharmacological effects of cannabinoids on neuronal functions.

09/2007 | Neurogastroenterol Motil
Cannabinoid type 1 receptor modulates intestinal propulsion by an attenuation of intestinal motor responses within the myenteric part of the peristaltic reflex
Yuece B, Sibaev A, Broedl U C, Marsicano G, Goke B, Lutz B, Allescher H D, Storr M

Cannabinoid-1 (CB1) receptor activation affects gastrointestinal propulsion in vivo. It was our aim to further characterize the involved myenteric mechanisms in vivo and in vitro. In CB1(-/-) mice and wild-type littermates we performed in vivo transit experiments by charcoal feeding and in vitro electrophysiological recordings in mouse small intestinal smooth muscle. Ascending neuronal contraction (ANC) following electrical field stimulation was studied in rat ileum in a partitioned organ bath separating the aboral stimulation site from the oral recording site. The knockout animals displayed an accelerated upper gastrointestinal transit compared to control animals. The CB1 receptor antagonist AM251 stimulated the force of the ANC in a concentration dependent manner when added in the oral chamber. Anandamide significantly inhibited the ANC when added in the oral chamber. Neither AM251 nor anandamide had an influence on the contraction latency. No effects were observed when drugs were added in the aboral chamber, proving a CB1 mediated action on the neuromuscular junction. Resting membrane potentials and neuronal induced inhibitory junction potentials in CB1(-/-) mice were unchanged as compared to wild type. However, the electrophysiological slow waves were more sensitive to blockade of Ca(2+) channels in CB1(-/-) mice. Our data strongly suggest a physiological involvement of the CB-1 receptor in the regulation of small intestinal motility. Therefore, CB1 receptors are a promising target for the treatment of motility disorders.

17/08/2007 | J Biol Chem
The CB1 cannabinoid receptor mediates excitotoxicity-induced neural progenitor proliferation and neurogenesis
Aguado T, Romero E, Monory K, Palazuelos J, Sendtner M, Marsicano G, Lutz B, Guzman M, Galve-Roperh I

Endocannabinoids are lipid signaling mediators that exert an important neuromodulatory role and confer neuroprotection in several types of brain injury. Excitotoxicity and stroke can induce neural progenitor (NP) proliferation and differentiation as an attempt of neuroregeneration after damage. Here we investigated the mechanism of hippocampal progenitor cell engagement upon excitotoxicity induced by kainic acid administration and the putative involvement of the CB1 cannabinoid receptor in this process. Adult NPs express kainate receptors that mediate proliferation and neurosphere generation in vitro via CB1 cannabinoid receptors. Similarly, in vivo studies showed that excitotoxicity-induced hippocampal NPs proliferation and neurogenesis are abrogated in CB1-deficient mice and in wild-type mice administered with the selective CB1 antagonist rimonabant (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazol ecarboxamide; SR141716). Kainate stimulation increased basic fibroblast growth factor (bFGF) expression in cultured NPs in a CB1-dependent manner as this response was prevented by rimonabant and mimicked by endocannabinoids. Likewise, in vivo analyses showed that increased hippocampal expression of bFGF, as well as of brain-derived neurotrophic factor and epidermal growth factor, occurs upon excitotoxicity and that CB1 receptor ablation prevents this induction. Moreover, excitotoxicity increased the number of CB1+ bFGF+ cells, and this up-regulation preceded NP proliferation. In summary, our results show the involvement of the CB1 cannabinoid receptor in NP proliferation and neurogenesis induced by excitotoxic injury and support a role for bFGF signaling in this process.

07/2007 | Nat Neurosci
Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors
Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin C E, Brenner G J, Rubino T, Michalski C W, Marsicano G, Monory K, Mackie K, Marian C, Batkai S, Parolaro D, Fischer M J, Reeh P, Kunos G, Kress M, Lutz B, Woolf C J, Kuner R

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.

27/06/2007 | J Neurosci
Role of cannabinoid type 1 receptors in locomotor activity and striatal signaling in response to psychostimulants
Corbille A G, Valjent E, Marsicano G, Ledent C, Lutz B, Herve D, Girault J A

A single administration of cocaine or D-amphetamine produces acute hyperlocomotion and long-lasting increased sensitivity to subsequent injections. This locomotor sensitization reveals the powerful ability of psychostimulants to induce brain plasticity and may participate in the alterations that underlie addiction. We investigated the role of cannabinoid receptor type 1 (CB1-R) in the effects of a single injection of psychostimulants. The acute locomotor response to cocaine was normal in mice pretreated with the CB1-R inverse agonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyr azole-3-carboxamide (AM251), whereas no sensitization was observed in response to a second administration a week later. Locomotor responses to cocaine and D-amphetamine were decreased in CB1-R-deficient mice, and sensitization was impaired. To determine how CB1-R controls long-lasting effects of psychostimulants, we studied cocaine-activated signaling pathways. Cocaine-induced cAMP-dependent phosphorylation of glutamate receptor 1 was altered in the striatum of CB1-R mutant mice but not of AM251-treated mice. In contrast, cocaine-induced phosphorylation of extracellular signal-regulated kinase (ERK) was blocked in both CB1-R mutant and antagonist-pretreated mice. Conditional deletion of CB1-R in forebrain principal neurons or GABAergic neurons prevented cocaine-induced ERK activation in dorsal striatum and nucleus accumbens. Our results provide strong evidence for the role of the endocannabinoid system in regulating neuronal circuits critical for long-lasting effects of cocaine, presumably by acting on CB1-R located on terminals of striatal medium spiny neurons.

25/05/2007 | Science
Hardwiring the brain: endocannabinoids shape neuronal connectivity
Berghuis P, Rajnicek A M, Morozov Y M, Ross R A, Mulder J, Urban G M, Monory K, Marsicano G, Matteoli M, Canty A, Irving A J, Katona I, Yanagawa Y, Rakic P, Lutz B, Mackie K, Harkany T

The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB(1) cannabinoid receptors (CB(1)Rs) are enriched in the axonal growth cones of gamma-aminobutyric acid-containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB(1)R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB(1)Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.

25/05/2007 | Neuroscience
Identification of the cannabinoid receptor type 1 in serotonergic cells of raphe nuclei in mice
Haring M, Marsicano G, Lutz B, Monory K

The endocannabinoid system (ECS) possesses neuromodulatory functions by influencing the release of various neurotransmitters, including GABA, noradrenaline, dopamine, glutamate and acetylcholine. Even though there are studies indicating similar interactions between the ECS and the serotonergic system, there are no results showing clear evidence for type 1 cannabinoid receptor (CB1) location on serotonergic neurons. In this study, we show by in situ hybridization that a low but significant fraction of serotonergic neurons in the raphe nuclei of mice contains CB1 mRNA as illustrated by the coexpression with the serotonergic marker gene tryptophane hydroxylase 2, the rate limiting enzyme for the serotonin synthesis. Furthermore, by double immunohistochemistry and confocal microscopy, we were able to detect CB1 protein on serotonergic fibers and synapses expressing the serotonin uptake transporter in the hippocampus and the amygdala. Our findings indicate that the CB1-mediated regulation of serotonin release can depend in part on a direct cross-talk between the two systems at single cell level, which might lead to functional implications in the modulation of emotional states.

04/2007 | Endocrinology
Requirement of cannabinoid receptor type 1 for the basal modulation of hypothalamic-pituitary-adrenal axis function
Cota D, Steiner M A, Marsicano G, Cervino C, Herman J P, Grubler Y, Stalla J, Pasquali R, Lutz B, Stalla G K, Pagotto U

The endocannabinoid system affects the neuroendocrine regulation of hormone secretion, including the activity of the hypothalamus-pituitary-adrenal (HPA) axis. However, the mechanisms by which endocannabinoids regulate HPA axis function have remained unclear. Here we demonstrate that mice lacking cannabinoid receptor type 1 (CB1-/-) display a significant dysregulation of the HPA axis. Although circadian HPA axis responsiveness is preserved, CB1-/- mice are characterized by an enhanced circadian drive on the HPA axis, resulting in elevated plasma corticosterone concentrations at the onset of the dark as compared with wild-type (CB1+/+) littermates. Moreover, CB1-/--derived pituitary cells respond with a significantly higher ACTH secretion to CRH and forskolin challenges as compared with pituitary cells derived from CB1+/+ mice. Both CBL-/- and CB1+/+ mice properly respond to a high-dose dexamethasone test, but response to low-dose dexamethasone is influenced by genotype. In addition, CB1-/- mice show increased CRH mRNA levels in the paraventricular nucleus of the hypothalamus but not in other extrahypothalamic areas, such as the amygdala and piriform cortex, in which CB1 and CRH mRNA have been colocalized. Finally, CB1-/- mice have selective glucocorticoid receptor mRNA down-regulation in the CA1 region of the hippocampus but not in the dentate gyrus or paraventricular nucleus. Conversely, mineralocorticoid receptor mRNA expression levels were found unchanged in these brain areas. In conclusion, our findings indicate that CB1 deficiency enhances the circadian HPA axis activity peak and leads to central impairment of glucocorticoid feedback, thus further outlining the essential role of the endocannabinoid system in the modulation of neuroendocrine functions.

04/2007 | Nat Med
Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells
Maresz K, Pryce G, Ponomarev E D, Marsicano G, Croxford J L, Shriver L P, Ledent C, Cheng X, Carrier E J, Mann M K, Giovannoni G, Pertwee R G, Yamamura T, Buckley N E, Hillard C J, Lutz B, Baker D, Dittel B N

The cannabinoid system is immunomodulatory and has been targeted as a treatment for the central nervous system (CNS) autoimmune disease multiple sclerosis. Using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we investigated the role of the CB(1) and CB(2) cannabinoid receptors in regulating CNS autoimmunity. We found that CB(1) receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB(2) receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB(2)-deficient T cells in the CNS during EAE exhibited reduced levels of apoptosis, a higher rate of proliferation and increased production of inflammatory cytokines, resulting in severe clinical disease. Together, our results demonstrate that the cannabinoid system within the CNS plays a critical role in regulating autoimmune inflammation, with the CNS directly suppressing T-cell effector function via the CB(2) receptor.

10/2006 | Neurobiol Dis
Protective activation of the endocannabinoid system during ischemia in dopamine neurons
Melis M, Pillolla G, Bisogno T, Minassi A, Petrosino S, Perra S, Muntoni A L, Lutz B, Gessa G L, Marsicano G, Di Marzo V, Pistis M

Endocannabinoids act as neuroprotective molecules promptly released in response to pathological stimuli. Hence, they may represent one component of protection and/or repair mechanisms mobilized by dopamine (DA) neurons under ischemia. Here, we show that the endocannabinoid 2-arachidonoyl-glycerol (2-AG) plays a key role in protecting DA neurons from ischemia-induced altered spontaneous activity both in vitro and in vivo. Accordingly, neuroprotection can be elicited through moderate cannabinoid receptor type-1 (CB1) activation. Conversely, blockade of endocannabinoid actions through CB1 receptor antagonism worsens the outcome of transient ischemia on DA neuronal activity. These findings indicate that 2-AG mediates neuroprotective actions by delaying damage and/or restoring function of DA cells through activation of presynaptic CB1 receptors. Lastly, they point to CB1 receptors as valuable targets in protection of DA neurons against ischemic injury and emphasize the need for a better understanding of endocannabinoid actions in the fine control of DA transmission.

17/08/2006 | Neuron
The endocannabinoid system controls key epileptogenic circuits in the hippocampus
Monory K, Massa F, Egertova M, Eder M, Blaudzun H, Westenbroek R, Kelsch W, Jacob W, Marsch R, Ekker M, Long J, Rubenstein J L, Goebbels S, Nave K A, During M, Klugmann M, Wolfel B, Dodt H U, Zieglgansberger W, Wotjak C T, Mackie K, Elphick M R, Marsicano G, Lutz B

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.

21/06/2006 | J Neurosci
Cannabinoid CB1 receptor mediates fear extinction via habituation-like processes
Kamprath K, Marsicano G, Tang J, Monory K, Bisogno T, Di Marzo V, Lutz B, Wotjak C T

The interplay between fear expression and fear extinction provides an important prerequisite for adequate coping with aversive encounters. Current models propose that extinction of conditioned fear is mediated by associative safety learning. Here, we demonstrate that the cannabinoid CB1 receptor, which is crucially involved in fear extinction, is dispensable for associative safety learning. In fact, our results indicate that CB1 mediates fear extinction primarily via habituation-like processes. CB1 null-mutant mice were severely impaired not only in extinction of the fear response to a tone after fear conditioning but also in habituation of the fear response to a tone after sensitization with an inescapable footshock. Surprisingly, long-term habituation was generally affected even in situations with proper short-term adaptation, suggesting the existence of two separated CB1-dependent effector systems for short- and long-term fear adaptation. Our findings underscore the importance of habituation as a determinant of fear extinction in mice and characterize the cannabinoid CB1 receptor as an essential molecular correlate of this process.

06/2006 | J Mol Med
CB1 and TRPV1 receptors mediate protective effects on colonic electrophysiological properties in mice
Sibaev A, Massa F, Yuce B, Marsicano G, Lehr H A, Lutz B, Goke B, Allescher H D, Storr M

CB1 and TRPV1 receptors modulate enteric neurotransmission and colonic inflammation. This study investigates early electrophysiological changes in distal colon of wild-type and receptor deficient mice after an inflammatory insult set by dinitrobenzene sulfonic acid (DNBS). Colitis was induced by DNBS in CB1(-/-) mice, TRPV1(-/-) mice, and their respective wild-type littermates. Electrophysiological properties consisting of membrane potentials and electrically induced inhibitory junction potentials (IJP) of circular smooth muscle cells were evaluated at different time points. Additionally a histological colitis severity score was evaluated in CB1(+/+) and CB1(-/-) mice 24 h after DNBS. Inflammation caused spontaneous atropine insensitive rhythmic action potentials in CB1(-/-) and TRPV1(-/-) mice but not in wild-type animals. This indicates that membrane stability is disturbed, which in turn indicates a lack of protective mechanisms. Focal electrical neuronal stimulation of the myenteric plexus induced IJP in the smooth muscle cells. Twenty-four hours after initiation of inflammation, the duration of the IJP is prolonged in all animals, indicating disturbances within neuromuscular interaction. In CB1(-/-) mice, it is interesting that the duration of IJP was significantly extended, as compared to CB1(+/+) mice pointing toward missing protective mechanisms in the CB1(-/-) mice. Inflammatory insults in the mouse colon induce reproducible changes in the electrophysiological properties and such changes correlate with duration of colitis. In mutants, these electrophysiological changes display different patterns, suggesting the lack of protective properties for neuromuscular interactions and membrane stability.

24/05/2006 | J Neurosci
Cannabinoid receptor type 1 located on presynaptic terminals of principal neurons in the forebrain controls glutamatergic synaptic transmission
Domenici M R, Azad S C, Marsicano G, Schierloh A, Wotjak C T, Dodt H U, Zieglgansberger W, Lutz B, Rammes G

It is widely accepted that cannabinoids regulate GABA release by activation of cannabinoid receptor type 1 (CB1). Results obtained from a variety of brain regions consistently indicate that cannabinoid agonists can also reduce glutamatergic synaptic transmission. However, there are still conflicting data concerning the role of CB1 in cannabinoid-induced inhibition of glutamatergic transmission in cortical areas. Here, we provide direct evidence that activation of CB1 on terminals of principal neurons controls excitatory synaptic responses in the forebrain. In slices of the basolateral amygdala, the CA1 region of the hippocampus, and the primary somatosensory cortex of wild-type mice, application of the CB1 agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4 -benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2; WIN) (5 mum) reduced evoked excitatory postsynaptic responses. In contrast, in slices obtained from conditional mouse mutants lacking CB1 in all principal forebrain neurons but not in GABAergic interneurons (CB1(f/f;CaMKIIalphaCre)), WIN no longer affected glutamatergic synaptic transmission in any of the brain regions tested. Compatible with a presynaptic mechanism, WIN did not change the sensitivity to focally uncaged l-glutamate. WIN reduced glutamatergic responses in slices obtained from mice lacking CB1 exclusively in GABAergic neurons (CB1(f/f;Dlx5/6-Cre)), thus excluding the involvement of CB1 expressed on GABAergic neurons in this effect of the drug. The present data strongly indicate that excitatory synaptic transmission in forebrain areas is directly modulated by CB1 expressed on presynaptic axon terminals originating from glutamatergic neurons.

04/2006 | Int J Obesity (Lond)
How many sites of action for endocannabinoids to control energy metabolism'
Pagotto U, Cervino C, Vicennati V, Marsicano G, Lutz B, Pasquali R

The promising results obtained by clinical trials using Rimonabant to tackle visceral obesity and related disorders recently promoted a remarkable impulse to carry out detailed investigations into the mechanisms of action of endocannabinoids in regulating food intake and energy metabolism. The endocannabinoid system has been known for many years to play an important role in the modulation of the neuronal pathways mediating the rewarding properties of food. However, in the last few years, with the advanced understanding of the crucial role of the hypothalamic neuronal network in the regulation of appetite, several studies have also directed attention to the orexigenic role of the endocannabinoid system, substantiating the well known appetite stimulating properties of derivatives of Cannabis sativa. Furthermore, the last 2 years have seen a number of relevant publications emphasizing the role of endocannabinoids as significant players in various peripheral metabolic processes. To date, the roles of the endocannabinoid system in influencing energy metabolism have proved to be more complex than was formerly believed. However, the diverse ability to modulate both central and peripheral processes highlights the pivotal involvement of the endocannabinoid system in the control of metabolic processes. This review describes the roles of endocannabinoids and the cannabinoid type 1 receptor (CB1) in the control of energy balance.

01/02/2006 | J Neurosci
The endocannabinoid system promotes astroglial differentiation by acting on neural progenitor cells
Aguado T, Palazuelos J, Monory K, Stella N, Cravatt B, Lutz B, Marsicano G, Kokaia Z, Guzman M, Galve-Roperh I

Endocannabinoids exert an important neuromodulatory role via presynaptic cannabinoid CB1 receptors and may also participate in the control of neural cell death and survival. The function of the endocannabinoid system has been extensively studied in differentiated neurons, but its potential role in neural progenitor cells remains to be elucidated. Here we show that the CB1 receptor and the endocannabinoid-inactivating enzyme fatty acid amide hydrolase are expressed, both in vitro and in vivo, in postnatal radial glia (RC2+ cells) and in adult nestin type I (nestin(+)GFAP+) neural progenitor cells. Cell culture experiments show that CB1 receptor activation increases progenitor proliferation and differentiation into astroglial cells in vitro. In vivo analysis evidences that, in postnatal CB1(-/-) mouse brain, progenitor proliferation and astrogliogenesis are impaired. Likewise, in adult CB1-deficient mice, neural progenitor proliferation is decreased but is increased in fatty acid amide hydrolase-deficient mice. In addition, endocannabinoid signaling controls neural progenitor differentiation in the adult brain by promoting astroglial differentiation of newly born cells. These results show a novel physiological role of endocannabinoids, which constitute a new family of signaling cues involved in the regulation of neural progenitor cell function.

In the human colon, vanilloid receptor TRPV1 is overexpressed both in afferent nerve terminals and in epithelial cells during inflammation. In the past years, pharmacological experiments using TRPV1 agonists and antagonists revealed that TRPV1 receptors may play proinflammatory and protective roles in the gastrointestinal tract. Here, we applied a genetic approach to define the role of TRPV1 and analyzed the effects of dinitrobenzene sulfonic acid (DNBS)-induced colitis in TRPV1-deficient (TRPV1-/-) mice. Intrarectal infusion of DNBS induced increased inflammation in TRPV1-/- mice compared to wild-type littermates (TRPV1+/+) as evaluated by macroscopic scoring and myeloperoxidase assays. This finding indicates that TRPV1 receptors are required for the protection within sensory pathways that regulate the response following the initiation of colonic inflammation. Electrophysiological recordings from circular smooth-muscle cells, performed 8 and 24 h after DNBS treatment, revealed strong spontaneous oscillatory action potentials in TRPV1-/- but not in TRPV1+/+ colons, indicating an early TRPV1-mediated control of inflammation-induced irritation of smooth-muscle activities. These unexpected results suggest that TRPV1 receptors mediate endogenous protection against experimentally induced colonic inflammation.

02/2006 | Endocr Rev
The emerging role of the endocannabinoid system in endocrine regulation and energy balance
Pagotto U, Marsicano G, Cota D, Lutz B, Pasquali R

During the last few years, the endocannabinoid system has emerged as a highly relevant topic in the scientific community. Many different regulatory actions have been attributed to endocannabinoids, and their involvement in several pathophysiological conditions is under intense scrutiny. Cannabinoid receptors, named CB1 receptor and CB2 receptor, first discovered as the molecular targets of the psychotropic component of the plant Cannabis sativa, participate in the physiological modulation of many central and peripheral functions. CB2 receptor is mainly expressed in immune cells, whereas CB1 receptor is the most abundant G protein-coupled receptor expressed in the brain. CB1 receptor is expressed in the hypothalamus and the pituitary gland, and its activation is known to modulate all the endocrine hypothalamic-peripheral endocrine axes. An increasing amount of data highlights the role of the system in the stress response by influencing the hypothalamic-pituitary-adrenal axis and in the control of reproduction by modifying gonadotropin release, fertility, and sexual behavior. The ability of the endocannabinoid system to control appetite, food intake, and energy balance has recently received great attention, particularly in the light of the different modes of action underlying these functions. The endocannabinoid system modulates rewarding properties of food by acting at specific mesolimbic areas in the brain. In the hypothalamus, CB1 receptor and endocannabinoids are integrated components of the networks controlling appetite and food intake. Interestingly, the endocannabinoid system was recently shown to control metabolic functions by acting on peripheral tissues, such as adipocytes, hepatocytes, the gastrointestinal tract, and, possibly, skeletal muscle. The relevance of the system is further strenghtened by the notion that drugs interfering with the activity of the endocannabinoid system are considered as promising candidates for the treatment of various diseases, including obesity.

2006 | J Endocrinol Invest
Neuromodulatory functions of the endocannabinoid system
Marsicano G, Lutz B

The endocannabinoid system (ECS) has recently emerged as an important neuromodulatory system in the brain. Several neuronal functions are under the control of the cannabinoid receptor type 1 (CB1 receptor) and of its endogenous lipid ligands (endocannabinoids). CB1 receptors are widely expressed in the brain and their expression pattern reflects the complexity and the variety of functions of the ECS in neuronal activity. In particular, CB1 receptors are present at different levels in several brain regions and in distinct neuronal subpopulations. Endocannabinoids were described to act as retrograde transmitters at synaptic level in many brain regions. Interestingly, the mechanisms governing endocannabinoid-controlled synaptic modulation can vary depending on the region and the neuronal circuit. At physiological and pathophysiological level, the ECS has recently been shown to play important regulatory roles in several brain processes, including the modulation of memory processing and the control of excessive neuronal activity. The discovery of the ECS represents a hallmark in neuroscience research, and the exploitation of its numerous physiological and pathophysiological functions is a promising avenue for therapeutic applications.

10/2005 | Faseb J
The endocannabinoid system drives neural progenitor proliferation.
Aguado T, Monory K, Palazuelos J, Stella N, Cravatt B, Lutz B, Marsicano G, Kokaia Z, Guzman M, Galve-Roperh I

The discovery of multipotent neural progenitor (NP) cells has provided strong support for the existence of neurogenesis in the adult brain. However, the signals controlling NP proliferation remain elusive. Endocannabinoids, the endogenous counterparts of marijuana-derived cannabinoids, act as neuromodulators via presynaptic CB1 receptors and also control neural cell death and survival. Here we show that progenitor cells express a functional endocannabinoid system that actively regulates cell proliferation both in vitro and in vivo. Specifically, NPs produce endocannabinoids and express the CB1 receptor and the endocannabinoid-inactivating enzyme fatty acid amide hydrolase (FAAH). CB1 receptor activation promotes cell proliferation and neurosphere generation, an action that is abrogated in CB1-deficient NPs. Accordingly, proliferation of hippocampal NPs is increased in FAAH-deficient mice. Our results demonstrate that endocannabinoids constitute a new group of signaling cues that regulate NP proliferation and thus open novel therapeutic avenues for manipulation of NP cell fate in the adult brain.

09/2005 | Gastroenterology
Fatty acid amide hydrolase controls mouse intestinal motility in vivo
Capasso R, Matias I, Lutz B, Borrelli F, Capasso F, Marsicano G, Mascolo N, Petrosino S, Monory K, Valenti M, Di Marzo V, Izzo A A

BACKGROUND & AIMS: Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis both of the endocannabinoids (which are known to inhibit intestinal motility) and other bioactive amides (palmitoylethanolamide, oleamide, and oleoylethanolamide), which might affect intestinal motility. The physiologic role of FAAH in the gut is largely unexplored. In the present study, we evaluated the possible role of FAAH in regulating intestinal motility in mice in vivo. METHODS: Motility was measured by evaluating the distribution of a fluorescent marker along the small intestine; FAAH messenger RNA (mRNA) levels were analyzed by reverse-transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by isotope-dilution, liquid chromatography, mass spectrometry. RESULTS: Motility was inhibited by N-arachidonoylserotonin (AA-5-HT) and palmitoylisopropylamide, 2 selective FAAH inhibitors, as well as by the FAAH substrates palmitoylethanolamide, oleamide, and oleoylethanolamide. The effect of AA-5-HT was reduced by the CB1 receptor antagonist rimonabant and by CB1 deficiency in mice but not by the vanilloid receptor antagonist 5'-iodoresiniferatoxin. In FAAH-deficient mice, pharmacologic blockade of FAAH did not affect intestinal motility. FAAH mRNA was detected in different regions of the intestinal tract. CONCLUSIONS: We conclude that FAAH is a physiologic regulator of intestinal motility and a potential target for the development of drugs capable of reducing intestinal motility.

07/03/2005 | Eur J Pharmacol
Cannabinoid CB1 receptor is dispensable for memory extinction in an appetitively-motivated learning task
Holter S M, Kallnik M, Wurst W, Marsicano G, Lutz B, Wotjak C T

The interaction of the cannabinoid CB1 receptor with its endogenous ligands plays an essential role in extinction of aversive memories (Marsicano, G., Wotjak, C.T., Azad, S.C., Bisogno, T., Rammes, G., Cascio, M.G., Hermann, H., Tang, J., Hofmann, C., Zieglgansberger, W., Di, M., V, Lutz, B., 2002. The endogenous cannabinoid system controls extinction of aversive memories. Nature 418, 530-534). The present study tested the generality of this observation in respect to positively-reinforced memories. To this end, male cannabinoid CB1 receptor deficient mice (CB1R-/-) and their wild-type littermate controls (CB1R+/+) were trained in an appetitively-motivated operant conditioning task, in which food-deprived animals received a food reward on nose-poking into an illuminated hole. During training, CB1R-/- turned out to be less motivated to participate in the task. After further restriction of daily food consumption, however, CB1R-/- reached the same level of performance as CB1R+/+ as far as number of correct responses and errors of omission are concerned. The accuracy of performance served as a measure for the memory of the light-reward association and was stable at similarly high levels over a retention period of 9 days without additional training (97.6+/-0.5% vs. 97.0+/-0.9% correct responses). During subsequent extinction training, the positive reinforcement was omitted. As a consequence, both CB1R-/- and CB1R+/+ showed a similar decline in accuracy of performance and total number of correct responses, accompanied by an increase in errors of omission. These data demonstrate that the cannabinoid CB1 receptor is not essential for extinction of the stimulus-response association in an appetitively-motivated learning task.

24/11/2004 | J Neurosci
Prefrontal cortex stimulation induces 2-arachidonoyl-glycerol-mediated suppression of excitation in dopamine neurons.
Melis M, Perra S, Muntoni AL, Pillolla G, Lutz B, Marsicano G, Di Marzo V, Gessa GL, Pistis M

Endocannabinoids form a novel class of retrograde messengers that modulate short- and long-term synaptic plasticity. Depolarization-induced suppression of excitation (DSE) and inhibition (DSI) are the best characterized transient forms of endocannabinoid-mediated synaptic modulation. Stimulation protocols consisting of long-lasting voltage steps to the postsynaptic cell are routinely used to evoke DSE-DSI. Little is known, however, about more physiological conditions under which these molecules are released in vitro. Moreover, the occurrence in vivo of such forms of endocannabinoid-mediated modulation is still controversial. Here we show that physiologically relevant patterns of synaptic activity induce a transient suppression of excitatory transmission onto dopamine neurons in vitro. Accordingly, in vivo endocannabinoids depress the increase in firing and bursting activity evoked in dopamine neurons by prefrontal cortex stimulation. This phenomenon is selectively mediated by the endocannabinoid 2-arachidonoyl-glycerol (2-AG), which activates presynaptic cannabinoid type 1 receptors. 2-AG synthesis involves activation of metabotropic glutamate receptors and Ca2+ mobilization from intracellular stores. These findings indicate that dopamine neurons release 2-AG to shape afferent activity and ultimately their own firing pattern. This novel endocannabinoid-mediated self-regulatory role of dopamine neurons may bear relevance in the pathogenesis of neuropsychiatric disorders such as schizophrenia and addiction.

03/11/2004 | J Neurosci
Circuitry for associative plasticity in the amygdala involves endocannabinoid signaling.
Azad SC, Monory K, Marsicano G, Cravatt BF, Lutz B, Zieglgansberger W, Rammes G

Endocannabinoids are crucial for the extinction of aversive memories, a process that considerably involves the amygdala. Here, we show that low-frequency stimulation of afferents in the lateral amygdala with 100 pulses at 1 Hz releases endocannabinoids postsynaptically from neurons of the basolateral amygdala of mice in vitro and thereby induces a long-term depression of inhibitory GABAergic synaptic transmission (LTDi) via a presynaptic mechanism. Lowering inhibitory synaptic transmission significantly increases the amplitude of excitatory synaptic currents in principal neurons of the central nucleus, which is the main output site of the amygdala. LTDi involves a selective mGluR1 (metabotropic glutamate receptor 1)-mediated calcium-independent mechanism and the activation of the adenylyl cyclase-protein kinase A pathway. LTDi is abolished by the cannabinoid type 1 (CB1) receptor antagonist SR141716A and cannot be evoked in CB1 receptor-deficient animals. LTDi is significantly enhanced in mice lacking the anandamide-degrading enzyme fatty acid amide hydrolase. The present findings show for the first time that mGluR activation induces a retrograde endocannabinoid signaling via activation of the adenylyl cyclase-protein kinase A pathway and the release of anandamide. Furthermore, the results indicate that anandamide decreases the activity of inhibitory interneurons in the amygdala. This disinhibition increases the activity of common output neurons and could provide a prerequisite for extinction by formation of new memory.

11/2004 | Psychopharmacology (Berl)
Reduced sensitivity to reward in CB1 knockout mice.
Sanchis-Segura C, Cline BH, Marsicano G, Lutz B, Spanagel R

RATIONALE: Previous studies have demonstrated that the activation and blockade of the cannabinoid type 1 receptor (CB1) leads to an enhancement and decrease of the consumption of food and other orally ingested reinforcers, respectively. OBJECTIVE: To gain further knowledge about the role of CB1 in sucrose/saccharin reinforcing efficacy and intake, we tested CB1 knockout (CB1-KO) and littermate wild-type (WT) control mice in several self-administration experimental protocols. METHODS: Operant (fixed or progressive ratio schedule) and non-operant conditioning procedures were used. In addition, a choice analysis based on the 'matching law' as well as a microstructural analysis of the intra-session pattern of self-administration was performed. RESULTS: CB1-KO mice consume less sucrose under operant conditions or when using a two-bottle free choice procedure. Moreover, as revealed by additional behavioural analysis, CB1-KO mice exhibit a decreased sensitivity to the rewarding properties of sucrose. In agreement with this finding, the differences between WT and CB1-KO mice faded away when the palatability of sucrose was devaluated by adding quinine, but not when a non-caloric sweetener, saccharin, was available. CONCLUSIONS: These results demonstrate a modulatory role of CB1 in the determination of the rewarding properties of sucrose and probably, as suggested by previous studies, other reinforcers.

09/2004 | Learn Mem
CB1 cannabinoid receptors modulate kinase and phosphatase activity during extinction of conditioned fear in mice.
Cannich A, Wotjak CT, Kamprath K, Hermann H, Lutz B, Marsicano G

Cannabinoid receptors type 1 (CB1) play a central role in both short-term and long-term extinction of auditory-cued fear memory. The molecular mechanisms underlying this function remain to be clarified. Several studies indicated extracellular signal-regulated kinases (ERKs), the phosphatidylinositol 3-kinase with its downstream effector AKT, and the phosphatase calcineurin as potential molecular substrates of extinction behavior. To test the involvement of these kinase and phosphatase activities in CB1-dependent extinction of conditioned fear behavior, conditioned CB1-deficient mice (CB1(-/-)) and wild-type littermates (CB1(+/+)) were sacrificed 30 min after recall of fear memory, and activation of ERKs, AKT, and calcineurin was examined by Western blot analysis in different brain regions. As compared with CB1(+/+), the nonreinforced tone presentation 24 h after auditory-cued fear conditioning led to lower levels of phosphorylated ERKs and/or calcineurin in the basolateral amygdala complex, ventromedial prefrontal cortex, dorsal hippocampus, and ventral hippocampus of CB1(-/-). In contrast, higher levels of phosphorylated p44 ERK and calcineurin were observed in the central nucleus of the amygdala of CB1(-/-). Phosphorylation of AKT was more pronounced in the basolateral amygdala complex and the dorsal hippocampus of CB1(-/-). We propose that the endogenous cannabinoid system modulates extinction of aversive memories, at least in part via regulation of the activity of kinases and phosphatases in a brain structure-dependent manner.

11/04/2004 | J Clin Invest
The endogenous cannabinoid system protects against colonic inflammation.
Massa F*, Marsicano G*, et al.

04/2004 | Eur J Neurosci
Involvement of brain-derived neurotrophic factor in cannabinoid receptor-dependent protection against excitotoxicity.
Khaspekov LG, Brenz Verca MS, Frumkina LE, Hermann H, Marsicano G, Lutz B

Cannabinoid type 1 (CB1) receptors play a central role in the protection against excitotoxicity induced by treatment of mice with kainic acid (KA). As inactivation of CB1 receptor function in mice blocks KA-induced increase of brain-derived neurotrophic factor (BDNF) mRNA levels in hippocampus, the notion was put forward that BDNF might be a mediator, at least in part, of CB1 receptor-dependent neuroprotection [Marsicano et al. (2003) Science, 302, 84-88]. To assess this signalling cascade in more detail, organotypic hippocampal slice cultures were used, as this in vitro system conserves morphological and functional properties of the hippocampus. Here, we show that both genetic ablation of CB1 receptors and pharmacological blockade with the specific CB1 receptor antagonist SR141716A increased the susceptibility of the in vitro cultures to KA-induced excitotoxicity, leading to extensive neuronal death. Next, we found that the application of SR141716A to hippocampal cultures from wild-type mice abolished the KA-induced increase in BDNF protein levels. Therefore, we tried to rescue these organotypic cultures from neuronal death by exogenously applied BDNF. Indeed, BDNF was sufficient to prevent KA-induced neuronal death after blockade of CB1 receptor signalling. In conclusion, our results strongly suggest that BDNF is a key mediator in CB1 receptor-dependent protection against excitotoxicity, and further underline the physiological importance of the endogenous cannabinoid system in neuroprotection.

01/2004 | am j physiol gastrointest liver physiol
Cannabinoid receptor type 1 modulates excitatory and inhibitory neurotransmission in mouse colon.
Storr M, Sibaev A, Marsicano G, Lutz B, Schusdziarra V, Timmermans JP, Allescher HD

The effects of cannabinoid receptor agonists and antagonists on smooth muscle resting membrane potentials and on membrane potentials following electrical neuronal stimulation in a myenteric neuron/smooth muscle preparation of wild-type and cannabinoid receptor type 1 (CB1)-deficient mice were investigated in vitro. Double staining for CB1 and nitric oxide synthase (neuronal) was performed to identify the myenteric CB1-expressing neurons. Focal electrical stimulation of the myenteric plexus induced a fast (f) excitatory junction potential (EJP) followed by a fast and a slow (s) inhibitory junction potential (IJP). Treatment of wild-type mice with the endogenous CB1 receptor agonist anandamide reduced EJP while not affecting fIJP and sIJP. EJP was significantly higher in CB1-deficient mice than in wild-type littermate controls, and anandamide induced no effects in CB1-deficient mice. N-arachidonoyl ethanolamide (anandamide), R-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]- 1,4-benzoxazin-6-yl]-1-naphtalenylmethanone, a synthetic CB1 receptor agonist, nearly abolished EJP and significantly reduced the fIJP in wild-type mice. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxa mide (SR141716A), a CB1-specific receptor antagonist, was able to reverse the agonist effects induced in wild-type mice. SR141716A, when given alone, significantly increased EJP in wild-type mice without affecting IJP in wild-type and EJP in CB1-deficient mice. Interestingly, SR141716A reduced fIJP in CB1-deficient mice. In the mouse colon, nitrergic myenteric neurons do not express CB1, implying that CB1 is expressed in cholinergic neurons, which is in line with the functional data. Finally, excitatory and inhibitory neurotransmission in the mouse colon is modulated by activation of CB1 receptors. The significant increase in EJP in CB1-deficient mice strongly suggests a physiological involvement of CB1 in excitatory cholinergic neurotransmission.

03/10/2003 | Science
CB1 cannabinoid receptors and on-demand defense against excitotoxicity.
Marsicano G, Goodenough S, Monory K, Hermann H, Eder M, Cannich A, Azad SC, Cascio MG, Gutierrez SO, van der Stelt M, Lopez-Rodriguez ML, Casanova E, Schutz G, Zieglgansberger W, Di Marzo V, Behl C, Lutz B

Abnormally high spiking activity can damage neurons. Signaling systems to protect neurons from the consequences of abnormal discharge activity have been postulated. We generated conditional mutant mice that lack expression of the cannabinoid receptor type 1 in principal forebrain neurons but not in adjacent inhibitory interneurons. In mutant mice,the excitotoxin kainic acid (KA) induced excessive seizures in vivo. The threshold to KA-induced neuronal excitation in vitro was severely reduced in hippocampal pyramidal neurons of mutants. KA administration rapidly raised hippocampal levels of anandamide and induced protective mechanisms in wild-type principal hippocampal neurons. These protective mechanisms could not be triggered in mutant mice. The endogenous cannabinoid system thus provides on-demand protection against acute excitotoxicity in central nervous system neurons.

09/2003 | Gastroenterology
An endogenous cannabinoid tone attenuates cholera toxin-induced fluid accumulation in mice.
Izzo AA , Capasso F , Costagliola A , Bisogno T , Marsicano G , Ligresti A , Matias I , Capasso R , Pinto L , Borrelli F , Cecio A , Lutz B , Mascolo N , Di Marzo V

BACKGROUND & AIMS: Cholera toxin (CT) is the most recognizable enterotoxin causing secretory diarrhea, a major cause of infant morbidity and mortality throughout the world. In this study, we investigated the role of the endogenous cannabinoid system (i.e., the cannabinoid receptors and their endogenous ligands) in CT-induced fluid accumulation in the mouse small intestine. METHODS: Fluid accumulation was evaluated by enteropooling; endocannabinoid levels were measured by isotope-dilution gas chromatography mass spectrometry; CB(1) receptors were localized by immunohistochemistry and their messenger RNA (mRNA) levels were quantified by reverse-transcription polymerase chain reaction (PCR). RESULTS: Oral administration of CT to mice resulted in an increase in fluid accumulation in the small intestine and in increased levels of the endogenous cannabinoid, anandamide, and increased expression of the cannabinoid CB(1) receptor mRNA. The cannabinoid receptor agonist CP55,940 and the selective cannabinoid CB(1) receptor agonist arachidonoyl-chloro-ethanolamide inhibited CT-induced fluid accumulation, and this effect was counteracted by the CB(1) receptor antagonist SR141716A, but not by the CB(2) receptor antagonist SR144528. SR141716A, per se, but not the vanilloid VR1 receptor antagonist capsazepine, enhanced fluid accumulation induced by CT, whereas the selective inhibitor of anandamide cellular uptake, VDM11, prevented CT-induced fluid accumulation. CONCLUSIONS: These results indicate that CT, along with enhanced intestinal secretion, causes overstimulation of endocannabinoid signaling with an antisecretory role in the small intestine.

08/2003 | J Clin Invest
The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis.
Cota D, Marsicano G, Tschop M, Grubler Y, Flachskamm C, Schubert M, Auer D, Yassouridis A, Thone-Reineke C, Ortmann S, Tomassoni F, Cervino C, Nisoli E, Linthorst AC, Pasquali R, Lutz B, Stalla GK, Pagotto U

The cannabinoid receptor type 1 (CB1) and its endogenous ligands, the endocannabinoids, are involved in the regulation of food intake. Here we show that the lack of CB1 in mice with a disrupted CB1 gene causes hypophagia and leanness. As compared with WT (CB1+/+) littermates, mice lacking CB1 (CB1-/-) exhibited reduced spontaneous caloric intake and, as a consequence of reduced total fat mass, decreased body weight. In young CB1-/- mice, the lean phenotype is predominantly caused by decreased caloric intake, whereas in adult CB1-/- mice, metabolic factors appear to contribute to the lean phenotype. No significant differences between genotypes were detected regarding locomotor activity, body temperature, or energy expenditure. Hypothalamic CB1 mRNA was found to be coexpressed with neuropeptides known to modulate food intake, such as corticotropin-releasing hormone (CRH), cocaine-amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and preproorexin, indicating a possible role for endocannabinoid receptors within central networks governing appetite. CB1-/- mice showed significantly increased CRH mRNA levels in the paraventricular nucleus and reduced CART mRNA levels in the dorsomedial and lateral hypothalamic areas. CB1 was also detected in epidydimal mouse adipocytes, and CB1-specific activation enhanced lipogenesis in primary adipocyte cultures. Our results indicate that the cannabinoid system is an essential endogenous regulator of energy homeostasis via central orexigenic as well as peripheral lipogenic mechanisms and might therefore represent a promising target to treat diseases characterized by impaired energy balance.

03/2003 | Int J Obes Relat Metab Disord
Endogenous cannabinoid system as a modulator of food intake.
Cota D, Marsicano G, Lutz B, Vicennati V, Stalla GK, Pasquali R, Pagotto U

The ability of Cannabis sativa (marijuana) to increase hunger has been noticed for centuries, although intensive research on its molecular mode of action started only after the characterization of its main psychoactive component Delta(9)-tetrahydrocannabinol in the late 1960s. Despite the public concern related to the abuse of marijuana and its derivatives, scientific studies have pointed to the therapeutic potentials of cannabinoid compounds and have highlighted their ability to stimulate appetite, especially for sweet and palatable food. Later, the discovery of specific receptors and their endogenous ligands (endocannabinoids) suggested the existence of an endogenous cannabinoid system, providing a physiological basis for biological effects induced by marijuana and other cannabinoids. Epidemiological reports describing the appetite-stimulating properties of cannabinoids and the recent insights into the molecular mechanisms underlying cannabinoid action have proposed a central role of the cannabinoid system in obesity. The aim of this review is to provide an extensive overview on the role of this neuromodulatory system in feeding behavior by summarizing the most relevant data obtained from human and animal studies and by elucidating the interactions of the cannabinoid system with the most important neuronal networks and metabolic pathways involved in the control of food intake. Finally, a critical evaluation of future potential therapeutical applications of cannabinoid antagonists in the therapy of obesity and eating disorders will be discussed.

The endogenous cannabinoid system has been shown recently to play a crucial role in the extinction of aversive memories. As the amygdala is presumably involved in this process, we investigated the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN-2) on synaptic transmission in the lateral amygdala (LA) of wild-type and cannabinoid receptor type 1 (CB1)-deficient mice. Extracellular field potential recordings and patch-clamp experiments were performed in an in vitro slice preparation. We found that WIN-2 reduces basal synaptic transmission and pharmacologically isolated AMPA receptor- and GABA(A) receptor-mediated postsynaptic currents in wild-type, but not in CB1-deficient mice. These results indicate that, in the LA, cannabinoids modulate both excitatory and inhibitory synaptic transmission via CB1. WIN-2-induced changes of paired-pulse ratio and of spontaneous and miniature postsynaptic currents suggest a presynaptic site of action. Inhibition of G(i/o) proteins and blockade of voltage-dependent and G protein-gated inwardly rectifying K(+) channels inhibited WIN-2 action on basal synaptic transmission. In contrast, modulation of the adenylyl cyclase-protein kinase A pathway, and blockade of presynaptic N- and P/Q- or of postsynaptic L- and R/T-type voltage-gated Ca(2+) channels did not affect WIN-2 effects. Our results indicate that the mechanisms underlying cannabinoid action in the LA partly resemble those observed in the nucleus accumbens and differ from those described for the hippocampus.

01/08/2002 | Nature
The endogenous cannabinoid system controls extinction of aversive memories.
Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G, Cascio MG, Hermann H, Tang J, Hofmann C, Zieglgansberger W, Di Marzo V, Lutz B

Acquisition and storage of aversive memories is one of the basic principles of central nervous systems throughout the animal kingdom. In the absence of reinforcement, the resulting behavioural response will gradually diminish to be finally extinct. Despite the importance of extinction, its cellular mechanisms are largely unknown. The cannabinoid receptor 1 (CB1) and endocannabinoids are present in memory-related brain areas and modulate memory. Here we show that the endogenous cannabinoid system has a central function in extinction of aversive memories. CB1-deficient mice showed strongly impaired short-term and long-term extinction in auditory fear-conditioning tests, with unaffected memory acquisition and consolidation. Treatment of wild-type mice with the CB1 antagonist SR141716A mimicked the phenotype of CB1-deficient mice, revealing that CB1 is required at the moment of memory extinction. Consistently, tone presentation during extinction trials resulted in elevated levels of endocannabinoids in the basolateral amygdala complex, a region known to control extinction of aversive memories. In the basolateral amygdala, endocannabinoids and CB1 were crucially involved in long-term depression of GABA (gamma-aminobutyric acid)-mediated inhibitory currents. We propose that endocannabinoids facilitate extinction of aversive memories through their selective inhibitory effects on local inhibitory networks in the amygdala.

Neuroprotective effects have been described for many cannabinoids in several neurotoxicity models. However, the exact mechanisms have not been clearly understood yet. In the present study, antioxidant neuroprotective effects of cannabinoids and the involvement of the cannabinoid receptor 1 (CB1) were analysed in detail employing cell-free biochemical assays and cultured cells. As it was reported for oestrogens that the phenolic group is a lead structure for antioxidant neuroprotective effects, eight compounds were classified into three groups. Group A: phenolic compounds that do not bind to CB1. Group B: non-phenolic compounds that bind to CB1. Group C: phenolic compounds that bind to CB1. In the biochemical assays employed, a requirement of the phenolic lead structure for antioxidant activity was shown. The effects paralleled the protective potential of group A and C compounds against oxidative neuronal cell death using the mouse hippocampal HT22 cell line and rat primary cerebellar cell cultures. To elucidate the role of CB1 in neuroprotection, we established stably transfected HT22 cells containing CB1 and compared the protective potential of cannabinoids with that observed in the control transfected HT22 cell line. Furthermore, oxidative stress experiments were performed in cultured cerebellar granule cells, which were derived either from CB1 knock-out mice or from control wild-type littermates. The results strongly suggest that CB1 is not involved in the cellular antioxidant neuroprotective effects of cannabinoids.

The cannabinoid receptor type 1 (CB1) displays unusual properties, including the dual capacity to inhibit or stimulate adenylate cyclase and a brain density considerably higher than the majority of G protein-coupled receptors. Together with overlapping expression patterns of dopamine and serotonin receptors this suggests a potential of CB1 to modulate the function of the dopamine and serotonin system. Indeed, pharmacological studies provide evidence for cross-talks between CB1 and receptors of these neurotransmitter systems. In trying to obtain further insights into possible functional and/or structural interactions between CB1 and the dopamine receptors and the serotonin receptors, we performed double-label in situ hybridization at the cellular level on mouse forebrain sections by combining a digoxigenin-labelled riboprobe for CB1 with 35S-labelled riboprobes for dopamine receptors D1 and D2, and for serotonin receptors 5-HT1B and 5-HT3, respectively. As a general rule, we found that CB1 colocalizes with D1, D2 and 5-HT1B only in low-CB1-expressing cells which are principal projecting neurons, whereas CB1 coexpression with 5-HT3 was also observed in high-CB1-expressing cells which are considered to be mostly GABAergic. In striatum and olfactory tubercle, CB1 is coexpressed to a high extent with D1, D2 and 5-HT1B. Throughout the hippocampal formation, CB1 is coexpressed with D2, 5-HT1B and 5-HT3. In the neocortex, coexpression was detected only with 5-HT1B and 5-HT3. In summary a distinct pattern is emerging for the cannabinoid system with regard to its colocalization with dopamine and serotonin receptors and, therefore, it is likely that different mechanisms underlie its cross-talk with these neurotransmitter systems.

06/2001 | J Clin Endocrinol Metab
Normal human pituitary gland and pituitary adenomas express cannabinoid receptor type 1 and synthesize endogenous cannabinoids: first evidence for a direct role of cannabinoids on hormone modulation at the human pituitary level.
Pagotto U, Marsicano G, Fezza F, Theodoropoulou M, Grubler Y, Stalla J, Arzberger T, Milone A, Losa M, Di Marzo V, Lutz B, Stalla GK

Little is known about the expression and function of cannabinoid receptor type 1 (CB1) in the human pituitary gland. The aim of this study was to investigate CB1 expression in human normal and tumoral pituitaries by in situ hybridization and immunohistochemistry using an antibody against CB1. CB1 was found in corticotrophs, mammotrophs, somatotrophs, and folliculostellate cells in the anterior lobe of normal pituitary. After examination of 42 pituitary adenomas, CB1 was detected in acromegaly-associated pituitary adenomas, Cushing's adenomas, and prolactinomas, whereas faint or no expression was found in nonfunctioning pituitary adenomas. Experiments with cultured pituitary adenoma cells showed that the CB1 agonist WIN 55,212--2 inhibited GH secretion in most of acromegaly-associated pituitary adenomas tested and that the CB1 antagonist SR 141716A was generally able to reverse this effect. Moreover, WIN 55,212--2 was able to suppress GHRH-stimulated GH release, and this effect was not blocked by coincubation with SR 141716A, possibly indicating a non-CB1-mediated effect. In contrast, WIN 55,212--2 was ineffective on GH-releasing peptide-stimulated GH release. In four Cushing's adenomas tested, WIN 55,212--2 was not able to modify basal ACTH secretion. However, simultaneous application of CRF and WIN 55,212--2 resulted in a synergistic effect on ACTH secretion, and this effect could be abolished by SR 141716A, demonstrating a CB1-mediated effect. In the single case of prolactinomas tested, WIN 55,212--2 was able to inhibit basal secretion of PRL. Finally, the presence of endocannabinoids (anandamide and 2-arachidonoylglycerol) was investigated in normal and tumoral pituitaries. All tumoral samples had higher contents of anandamide and 2-arachidonoylglycerol compared with the normal hypophysis. Moreover, endocannabinoid content in the different pituitary adenomas correlated with the presence of CB1, being elevated in the tumoral samples positive for CB1 and lower in the samples in which no or low levels of CB1 were found. The results of this study point to a direct role of cannabinoids in the regulation of human pituitary hormone secretion.

02/2001 | Eur J Neurosci
Differential role of the nitric oxide pathway on delta(9)-THC-induced central nervous system effects in the mouse.
Azad SC, Marsicano G, Eberlein I, Putzke J, Zieglgansberger W, Spanagel R, Lutz B

This study investigated whether the nitric oxide pathway was involved in the central effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive constituent of cannabis sativa. Body temperature, nociception and locomotion were measured in neuronal nitric oxide synthase (nNOS) knock-out (KO) mice and wild-type (WT) controls after intraperitoneal application of Delta(9)-THC. These Delta(9)-THC-induced effects are known to be mediated through the brain-type cannabinoid receptor 1 (CB1). Therefore, in situ hybridization (ISH) experiments were performed in the adult murine brain to determine possible changes in CB1 mRNA levels in nNOS-KO, compared with WT mice, and to reveal brain areas where CB1 and nNOS were coexpressed in the same neurons. We found that an intraperitoneal injection of 10 mg/kg Delta(9)-THC led to the same increase in the hot plate latencies in both genotypes, suggesting that Delta(9)-THC-mediated antinociception does not involve nNOS. In contrast, a significant Delta(9)-THC-induced decrease of body temperature and locomotor activity was only observed in WT, but not in nNOS-KO mice. ISH revealed significantly lower levels of CB1 mRNA in the ventromedial hypothalamus (VMH) and the caudate putamen (Cpu) of the nNOS-KO animals, compared with WT mice. Both areas are known to be among the regions involved in cannabinoid-induced thermoregulation and decrease of locomotion. A numerical evaluation of nNOS/CB1 coexpression showed that approximately half of the nNOS-positive cells in the dorsolateral Cpu also express low levels of CB1. ISH of adjacent serial sections with CB1 and nNOS, revealed expression of both transcripts in VMH, suggesting that numerous nNOS-positive cells of VMH coexpress CB1. Our findings indicate that the nitric oxide pathway is involved in some, but not all of the central effects of Delta(9)-THC.

06/2000 | transgenic res
An in vivo model of hyperacute rejection: characterization and evaluation of the effect of transgenic human complement inhibitors.
Mora M, Lazzer M, Marsicano G, Mulder LC, Carraresi L, Pieri A, Benanchi A, Grifoni D, Nuti S, Bruzzone P, Comporti M, Cortesini R, Rossini M

Hyperacute rejection (HAR) occurring after transplantation within phylogenetically distant species is a severe reaction triggered by preexisting xenoreactive antibodies and complement activation, leading to the destruction of the donor organ. Expression of human complement inhibitors in transgenic pig organs prolongs the survival of xenograft in experimental models. Moreover, the extent of protection from hyperacute rejection is dependent on the level and site of expression of the transgenic molecules and, probably, on the combination of different molecules. In this regard a small animal model to test the efficacy of expression vectors and different human molecules could be very advantageous. A murine model developed in our laboratory was characterized by measurement of several parameters characteristic of HAR in the livers of control and transgenic mice expressing transgenic human DAF (CD55) or MCP (CD46) at the end of 2 h of perfusion with human plasma and after I day. The parameters studied were heamatological values of hepatic functions (GOT and GPT), induction of pro-inflammatory molecules and histopathological evaluation. Cytokines (IL-1alpha, IL-1beta, IL-6) induction and exposure of P-selectin on the endothelial cell surface, was only observed in control animals after 2 h of perfusion, as an early event. GOT and GPT values increase dramatically after 2 h perfusion and 1 day after the treatment according to the histopathological observation of liver damage. On the contrary, the livers of hDAF or hMCP transgenic mice, under the same treatment were significantly protected although the extent of this protection is dependent on the level of expression of transgenic human molecules.

Cannabinoids can modulate motor behaviour, learning and memory, cognition and pain perception. These effects correlate with the expression of the cannabinoid receptor 1 (CB1) and with the presence of endogenous cannabinoids in the brain. In trying to obtain further insights into the mechanisms underlying the modulatory effects of cannabinoids, CB1-positive neurons were determined in the murine forebrain at a single cell resolution. We performed a double in situ hybridization study to detect mRNA of CB1 in combination with mRNA of glutamic acid decarboxylase 65k, neuropeptide cholecystokinin (CCK), parvalbumin, calretinin and calbindin D28k, respectively. Our results revealed that CB1-expressing cells can be divided into distinct neuronal subpopulations. There is a clear distinction between neurons containing CB1 mRNA either at high levels or low levels. The majority of high CB1-expressing cells are GABAergic (gamma-aminobutyric acid) neurons belonging mainly to the cholecystokinin-positive and parvalbumin-negative type of interneurons (basket cells) and, to a lower extent, to the calbindin D28k-positive mid-proximal dendritic inhibitory interneurons. Only a fraction of low CB1-expressing cells is GABAergic. In the hippocampus, amygdala and entorhinal cortex area, CB1 mRNA is present at low but significant levels in many non-GABAergic cells that can be considered as projecting principal neurons. Thus, a complex mechanism appears to underlie the modulatory effects of cannabinoids. They might act on principal glutamatergic circuits as well as modulate local GABAergic inhibitory circuits. CB1 is very highly coexpressed with CCK. It is known that cannabinoids and CCK often have opposite effects on behaviour and physiology. Therefore, we suggest that a putative cross-talk between cannabinoids and CCK might exist and will be relevant to better understanding of physiology and pharmacology of the cannabinoid system.

04/1998 | J Urol
Kidneys derived from mice transgenic for human complement blockers are protected in an in vivo model of hyperacute rejection.
Lazzeri M, Mora M, Mulder LC, Marsicano G, Marinucci G, Boschi M, Bruzzone P, Alfani D, Cortesini R, Rossini M

PURPOSE: The major obstacle to successful discordant kidney xenotransplantation is hyperacute rejection (HAR). Complement plays a key role in the induction of HRA, defined by endothelial cell activation, loss of vascular integrity, hemorrhage and thrombosis. The activation of complement is tightly controlled by a number of species-specific regulatory proteins which inhibit, at different points, the cascade of events leading to the formation of the membrane attack complex (MAC). We have tested the hypothesis that kidneys derived from transgenic mice expressing two human complement inhibitors, Decay Accelerating Factor (hDAF) and Membrane Cofactor Protein (MCP), could be protected from human complement-mediated damage. MATERIALS AND METHODS: Control and transgenic mice were perfused with human plasma by cannulation of the right jugular vein, at a perfusion rate of 10 microL./min. for two hours. Complement C3 deposition was detected on kidney sections by immunohistochemistry using specific FITC antibody. Complement-induced tissue damage was evaluated by histopathological examination. RESULTS: Heavy deposition of complement C3 was observed on kidneys derived from perfused control mice. This was associated with a characteristic HAR pathology of severe interstitial hemorrhage, inflammatory reaction, loss of glomerula and tubuli structure. Kidneys derived from mice transgenic for hDAF or hMCP were partially protected from both complement C3 deposition and tissue damage. The expression of both hDAF and hMCP in double transgenic mice significantly increases the protection from human complement-mediated damage. CONCLUSION: A novel model of in vivo perfusion with human plasma has been adopted to recreate the initial event of HAR. Our data show that this murine model could be very valuable to determine the effect of transgenic human molecules in protecting vascularized organs from human complement attack.

03/1997 | vet immunol immunopathol
Factors controlling haemopoiesis in ovine long-term bone marrow cultures.
Marsicano G, Shehu D, Galli C

We describe an adaptation of the Dexter technique for obtaining ovine long-term bone marrow cultures able to sustain haemopoiesis in vitro for long periods. Two inocula of bone marrow cells collected at three-five weeks interval, in IMDM supplemented with fetal calf serum (10%), horse serum (10%) and hydrocortisone (5 x 10(-7) M), gave rise to the growth of an adherent cell layer which supported, in most cases, active haemopoiesis for up to 15 weeks. The cell layer was composed of macrophages, fibroblasts and adipocytes. Haemopoietic cells formed large foci of 'cobblestone' areas. Haemopoietic progenitors were also released into the supernatant medium and were detectable by clonogenic assay. Granulocytes and monocyte-macrophages differentiated in the cultures in constant proportion until week five, when the monocytic lineage superseded the myelocytic one. These cultures, between weeks five and twelve, produced colony forming cells in a constant pattern, indicating the presence and self renewing of early haemopoietic progenitor cells.

Expression of various developmentally regulated markers was screened throughout the preimplantation stages of in vitro-derived bovine embryos. This was done by investigating the distribution of several nuclear, cytoplasmic and extracellular proteins by means of immunofluorescence microscopy. While lamin B appeared as a constitutive component of nuclei of all preimplantation stages, lamins A/C had a stage-related distribution. The early cleavage stage nuclei contained lamins A/C which generally disappeared in the following stages, with the possible exception of a few positive nuclei in the morula and early blastocyst stage. In the expanded blastocyst stage the nuclei of trophectoderm cells became positive while no positivity was observed in the inner cell mass cells. Starting from day 6, the appearance and/or polarised distribution of various cytoskeletal and cytoskeleton-related components such as F-actin, alpha-catenin and E-cadherin gave an insight into the timing of events related to compaction of bovine embryos. Compaction was correlated with the first differentiation event, i.e. the formation of trophectoderm; this is the first embryonic epithelium, characterised by cytokeratins and desmoplakin. Extracellular fibronectin was first detected in the early blastocyst stage shortly before the morphological differentiation of primitive endoderm, and in the later stages it was localised at the interface between trophectoderm and extraembryonic endoderm. Laminin and collagen IV were expressed by the endoderm cells and contributed to the extracellular matrix underlying the trophectoderm. This study is a first attempt to characterise the cells of in vitro-derived bovine embryos valid for cell line derivation.