Biomedical subjects

Beat Lutz

Publications and source records attributed to Beat Lutz.

35 records · Page 2Linked to original sources

PubMed2004

Circuitry for associative plasticity in the amygdala involves endocannabinoid signaling.

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.

Adenylyl Cyclases↗

PubMed2004

On-demand activation of the endocannabinoid system in the control of neuronal excitability and epileptiform seizures.

Neurons intensively exchange information among each other using both inhibitory and excitatory neurotransmitters. However, if the balance of excitation and inhibition is perturbed, the intensity of excitatory transmission may exceed a certain threshold and epileptic seizures can occur. As the occurrence of epilepsy in the human population is about 1%, the search for therapeutic targets to alleviate seizures is warranted. Extracts of Cannabis sativa have a long history in the treatment of various neurological diseases, including epilepsy. However, cannabinoids have been reported to exert both pro- and anti-convulsive activities. The recent progress in understanding the endogenous cannabinoid system has allowed new insights into these opposing effects of cannabinoids. When excessive neuronal activity occurs, endocannabinoids are generated on demand and activate cannabinoid type 1 (CB1) receptors. Using mice lacking CB1 receptors in principal forebrain neurons in a model of epileptiform seizures, it was shown that CB1 receptors expressed on excitatory glutamatergic neurons mediate the anti-convulsive activity of endocannabinoids. Systemic activation of CB1 receptors by exogenous cannabinoids, however, are anti- or pro-convulsive, depending on the seizure model used. The pro-convulsive activity of exogenous cannabinoids might be explained by the notion that CB1 receptors expressed on inhibitory GABAergic neurons are also activated, leading to a decreased release of GABA, and to a concomitant increase in seizure susceptibility. The concept that the endogenous cannabinoid system is activated on demand suggests that a promising strategy to alleviate seizure frequency is the enhancement of endocannabinoid levels by inhibiting the cellular uptake and the degradation of these endogenous compounds.

Animals↗

PubMed2004

Further evidence for the existence of a specific process for the membrane transport of anandamide.

Indirect evidence for the existence of a specific protein-mediated process for the cellular uptake of endocannabinoids has been reported, but recent results suggested that such a process, at least for AEA [ N -arachidonoylethanolamine (anandamide)], is facilitated uniquely by its intracellular hydrolysis by FAAH (fatty acid amide hydrolase) [Glaser, Abumrad, Fatade, Kaczocha, Studholme and Deutsch (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 4269-4274]. In the present study, we show that FAAH alone cannot account for the facilitated diffusion of AEA across the cell membrane. In particular, (i) using a short incubation time (90 s) to avoid AEA hydrolysis by FAAH, AEA accumulation into rat basophilic leukaemia or C6 cells was saturable at low microM concentrations of substrate and non-saturable at higher concentrations; (ii) time-dependent and, at low microM concentrations of substrate, saturable AEA accumulation was observed also using mouse brain synaptosomes; (iii) using synaptosomes prepared from FAAH-deficient mice, saturable AEA accumulation was still observed, although with a lower efficacy; (iv) when 36 AEA and N -oleoylethanolamine analogues, most of which with phenyl rings in the polar head group region, were tested as inhibitors of AEA cellular uptake, strict structural and stereochemical requirements were needed to observe significant inhibition, and in no case the inhibition of FAAH overlapped with the inhibition of AEA uptake; and (v) AEA biosynthesis by cells and sensory neurons was followed by AEA release, and this latter process, which cannot be facilitated by FAAH, was still blocked by an inhibitor of AEA uptake. We suggest that at least one protein different from FAAH is required to facilitate AEA transport across the plasma membrane in a selective and bi-directional way.

Amidohydrolases↗

PubMed2004

Reduced sensitivity to reward in CB1 knockout mice.

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.

Animals↗

PubMed2004

Generation of a conditional allele of the CBP gene in mouse.

CREB-binding protein (CBP) is an important transcriptional cofactor for various intracellular signaling pathways, including Ca(2+)- and cAMP-mediated gene activation. The loss of one CBP allele causes the human Rubinstein-Taybi syndrome, which is characterized by mental retardation and other severe developmental defects. Deletion of both CBP alleles in the mouse leads to early embryonic lethality. To address the function of CBP in late embryogenesis and in adult physiology, a floxed CBP allele (CBP(fl)) was generated. Using the Cre/loxP recombination system, CBP function was disrupted in principal forebrain neurons by breeding with a transgenic CaMKIIalpha-Cre mouse line to obtain CBP(fl/fl;CaMKIIalphaCre) mice. These mice contain CBP(stop523) alleles specifically in principal forebrain neurons, presumably resulting in the production of a truncated CBP protein unable to interact with a number of transcription factors, including phosphorylated CREB.

Alleles↗

PubMed2004

Involvement of brain-derived neurotrophic factor in cannabinoid receptor-dependent protection against excitotoxicity.

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.

Animals↗

PubMed2004

Dyrk1A potentiates steroid hormone-induced transcription via the chromatin remodeling factor Arip4.

Dyrk1A, a mammalian homolog of the Drosophila minibrain gene, encodes a dual-specificity kinase, involved in neuronal development and in adult brain physiology. In humans, a third copy of DYRK1A is present in Down syndrome (trisomy 21) and has been implicated in the etiology of mental retardation. To further understand this pathology, we searched for Dyrk1A-interacting proteins and identified Arip4 (androgen receptor-interacting protein 4), a SNF2-like steroid hormone receptor cofactor. Mouse hippocampal and cerebellar neurons coexpress Dyrk1A and Arip4. In HEK293 cells and hippocampal neurons, both proteins are colocalized in a speckle-like nuclear subcompartment. The functional interaction of Dyrk1A with Arip4 was analyzed in a series of transactivation assays. Either Dyrk1A or Arip4 alone displays an activating effect on androgen receptor- and glucocorticoid receptor-mediated transactivation, and Dyrk1A and Arip4 together act synergistically. These effects are independent of the kinase activity of Dyrk1A. Inhibition of endogenous Dyrk1A and Arip4 expression by RNA interference showed that both proteins are necessary for the efficient activation of androgen receptor- and glucocorticoid receptor-dependent transcription. As Dyrk1A is an activator of steroid hormone-regulated transcription, the overexpression of DYRK1A in persons with Down syndrome may cause rather broad changes in the homeostasis of steroid hormone-controlled cellular events.

Adenosine Triphosphatases↗

PubMed2004

The endogenous cannabinoid system protects against colonic inflammation.

Excessive inflammatory responses can emerge as a potential danger for organisms' health. Physiological balance between pro- and anti-inflammatory processes constitutes an important feature of responses against harmful events. Here, we show that cannabinoid receptors type 1 (CB1) mediate intrinsic protective signals that counteract proinflammatory responses. Both intrarectal infusion of 2,4-dinitrobenzene sulfonic acid (DNBS) and oral administration of dextrane sulfate sodium induced stronger inflammation in CB1-deficient mice (CB1(-/-)) than in wild-type littermates (CB1(+/+)). Treatment of wild-type mice with the specific CB1 antagonist N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716A) mimicked the phenotype of CB1(-/-) mice, showing an acute requirement of CB1 receptors for protection from inflammation. Consistently, treatment with the cannabinoid receptor agonist R(-)-7-hydroxy-Delta(6)-tetra-hydrocannabinol-dimethylheptyl (HU210) or genetic ablation of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) resulted in protection against DNBS-induced colitis. Electrophysiological recordings from circular smooth muscle cells, performed 8 hours after DNBS treatment, revealed spontaneous oscillatory action potentials in CB1(-/-) but not in CB1(+/+) colons, indicating an early CB1-mediated control of inflammation-induced irritation of smooth muscle cells. DNBS treatment increased the percentage of myenteric neurons expressing CB1 receptors, suggesting an enhancement of cannabinoid signaling during colitis. Our results indicate that the endogenous cannabinoid system represents a promising therapeutic target for the treatment of intestinal disease conditions characterized by excessive inflammatory responses.

Amidohydrolases↗

PubMed2003

CB1 cannabinoid receptors and on-demand defense against excitotoxicity.

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.

Animals↗

PubMed2003

An endogenous cannabinoid tone attenuates cholera toxin-induced fluid accumulation in mice.

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.

Amides↗

PubMed2003

The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis.

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.

Adipocytes↗

PubMed2002

Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein.

CREB-binding protein (CBP) is a multifunctional cofactor implicated in many intracellular signal transduction pathways. We aimed to investigate the involvement of CBP in the cAMP response element-binding protein (CREB)-mediated pathway. The point mutation Tyr658Ala in the CREB-binding domain (CBD) was shown to abolish the binding activity of CBP to phospho-CREB, the activated form of CREB. By using a mutant Cre/loxP recombination system, this point mutation was aimed to be generated in the mouse genome in a tissue- and time-specific manner. A targeting construct in which CBD exon 5 and inverted exon 5* containing the point mutation flanked by two mutant loxP sites (lox66 and lox71) oriented in a head-to-head position was generated. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase, the favorable reaction leads to a product where the mutated exon 5* is placed into the position to be correctly transcribed and spliced. Inversion was observed to be complete in both bacteria and mouse embryonic stem cells. Our results indicate that this Cre- mediated inversion method is a valuable tool to introduce point mutations in the mouse genome in a regulatable manner.

Animals↗

PubMed2002

The endogenous cannabinoid system controls extinction of aversive memories.

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.

Acoustic Stimulation↗

PubMed2002

Neuroprotective properties of cannabinoids against oxidative stress: role of the cannabinoid receptor CB1.

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.

Animals↗

PubMedDate not supplied

Molecular biology of cannabinoid receptors.

During the last decade, research on the molecular biology and genetics of cannabinoid receptors has led to a remarkable progress in understanding of the endogenous cannabinoid system, which functions in a plethora of physiological processes in the animal. At present, two types of cannabinoid receptors have been cloned from many vertebrates, and three endogenous ligands (the endocannabinoids arachidonoyl ethanolamide, 2-arachidonoyl glycerol and 2-arachidonoyl-glycerol ether) have been characterized. Cannabinoid receptor type 1 (CB(1)) is expressed predominantly in the central and peripheral nervous system, while cannabinoid receptor type 2 (CB(2)) is present almost exclusively in immune cells. Cannabinoid receptors have not yet been cloned from invertebrates, but binding proteins for endocannabinoids, endocannabinoids and metabolic enzyme activity have been described in a variety of invertebrates except for molting invertebrates such as Caenorhabditis elegans and Drosophila. In the central nervous system of mammals, there is strong evidence emerging that the CB(1) and its ligands comprise a neuromodulatory system functionally interacting with other neurotransmitter systems. Furthermore, the presynaptic localization of CB(1) together with the results obtained from electrophysiological experiments strengthen the notion that in cerebellum and hippocampus and possibly in other regions of the central nervous system, endocannabinoids may act as retrograde messengers to suppress neurotransmitter release at the presynaptic site. Many recent studies using genetically modified mouse lines which lack CB(1) and/or CB(2) finally could show the importance of cannabinoid receptors in animal physiology and will contribute to unravel the full complexity of the cannabinoid system.

Amino Acid Sequence↗

PubMedDate not supplied

Activation of the cannabinoid receptor type 1 decreases glutamatergic and GABAergic synaptic transmission in the lateral amygdala of the mouse.

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.

Amygdala↗

PubMedDate not supplied

CB1 cannabinoid receptors modulate kinase and phosphatase activity during extinction of conditioned fear in mice.

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.

1-Phosphatidylinositol 4-Kinase↗