Nukleosidoen gehikuntzaren eragina estres erreplikatiboan eta seneszentzia zelularrean: E2F1-/-/E2F2-/- makrofagoen kasua

E2F1 eta E2F2 transkripzio-faktoreek funtzio garrantzitsua dute ziklo zelularraren errgulazioan. Sagu knockout-etan eginiko saioetan deskribatu da E2F1 eta E2F2 geneen absentzian DNAren bikoizketa-tasa emendatuta dagoela makrofagoetan, eta ondorioz seneszentzia goiztiarra aktibatuta dagoela zelula hauetan. Seneszentzia horren mekanismoak ez dira ezagutzen, baina uste da DNAren estres erreplikatiboaren eraginez gertatzen dela. Oraintsu argitaratutako lan batzuren arabera, onkogeneek eragindako desegonkortasun genomikoa nukleosido mailaren urritasunagatik sortzen dela proposatu da. Emaitza hauek kontutan harturik, E2F1-/-/E2F2-/- (DKO) makrofagoen kultiboei nukleosidoak gehitu dizkiet exogenoki, eta zelula hauen proliferazioaren jarraipena egin dut. Nukleosidoekin trataturiko kultiboetan DNAren kalteen jaitsiera eta seneszenziaren saihespena detektatu ditut. Emaitza hauek zera iradokitzen dute, DKO zeluletako seneszentzia goiztiarraren arrazoia DNAren hiper-erreplikazioak sortutako nukleosidoen urritasuna izan daitekeela

Lack of the transient receptor potential vanilloid 1 shifts cannabinoid-dependent excitatory synaptic plasticity in the dentate gyrus of the mouse brain hippocampus

[EN] The transient receptor potential vanilloid 1 (TRPV1) participates in synaptic functions in the brain. In the dentate gyrus, post-synaptic TRPV1 in the granule cell (GC) dendritic spines mediates a type of long-term depression (LTD) of the excitatory medial perforant path (MPP) synapses independent of pre-synaptic cannabinoid CB1 receptors. As CB1 receptors also mediate LTD at these synapses, both CB1 and TRPV1 might be influencing the activity of each other acting from opposite synaptic sites. We tested this hypothesis in the MPP–GC synapses of mice lacking TRPV1 (TRPV1-/-). Unlike wild-type (WT) mice, low-frequency stimulation (10min at 10Hz) of TRPV1-/- MPP fibers elicited a form of long-term potentiation (LTP) that was dependent on (1) CB1 receptors, (2) the endocannabinoid 2-arachidonoylglycerol (2-AG), (3) rearrangement of actin filaments, and (4) nitric oxide signaling. These functional changes were associated with an increase in the maximum binding efficacy of guanosine-5′-O-(3-[35S]thiotriphosphate) ([35S]GTPgS) stimulated by the CB1 receptor agonist CP 55,940, and a significant decrease in receptor basal activation in the TRPV1-/- hippocampus. Finally, TRPV1-/- hippocampal synaptosomes showed an augmented level of the guanine nucleotide-binding (G) Gai1, Gai2, and Gai3 protein alpha subunits. Altogether, the lack of TRPV1 modifies CB1 receptor signaling in the dentate gyrus and causes the shift from CB1 receptor-mediated LTD to LTP at the MPP–GC synapses.This work was supported by the Basque Government (IT1230- 19, to PG); MINECO/FEDER, UE (SAF2015-65034-R, to PG); Ministry of Science and Innovation (PID2019-107548RBI00, to PG); Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III); and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012, to PG); MINECO CTQ2017-85686-R (Spanish Ministry of Economy and Competitiveness, to JS); JE-H is a Postdoctoral Researcher contracted with funds of Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III), and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012), and the Basque Government (IT1230-19). IB-D holds a Postdoctoral Orientation Period contract (BES-2016-076766, BES-C-2016-0051). SA has a Ph.D. contract granted by University of the Basque Country (PIF 16/251). ES-G is funded by Ikerbasque and MINECO (PGC2018- 093990-A-I00; MICIU/AEI/FEDER, UE)

Astrozitoetan kokatutako CB1 hartzaile kannabinoidearen anilisi ultraestrukturala eta fisiologikoa

139 p. (eng.); 145 p. (eusk.)The cannabinoid CB1 receptor-mediated functions in astrocytes are highly dependent on the CB1 receptor distribution in these glial cells relative to neuronal sites, particularly at the nearby synapses under normal or pathological conditions. However, the whole picture of the subcellular CB1 receptor distribution in astroglial compartments remains uncompleted due to the scattered CB1 receptor expression, and therefore difficult to detect, in astrocytes. Our laboratory has in previous studies estimated that about 5-6 % of the total CB1 receptors in the hippocampal CA1 stratum radiatum are localized in astrocytes identified by the marker glial fibrillary acidic protein (GFAP). However, GFAP is a cytoskeleton protein mostly restricted to the astroglial cell bodies and their main branches. This might be distorting the actual proportion and total amount of CB1 receptors in astrocytes. Therefore, the search for alternative astroglial markers to decipher the precise mapping of CB1 receptors in astrocytes is a timely goal in the cannabinoid field. The glutamate aspartate transporter (GLAST) is used as astroglial marker and raises as a good astroglial marker candidate to study in detail the CB1 receptor distribution in astrocytes. To prove this hypothesis, I have used a pre-embedding immunogold method for electron microscopy to compare first the astroglial distribution of GLAST versus GFAP. GLAST labeling was along the plasma membrane of astrocytes, including cell bodies and the smallest astrocytic projections in close contact with neurons, capillaries and other glial cells, covering altogether a much broader labeled area than GFAP. Furthermore, the use of a pre-embedding immunoperoxidase method for electron microscopy served me to assess that almost three times more astroglial area is visualized with GLAST than GFAP, and that GLAST detects four times as much astroglial membranes as GFAP. Finally, a double pre-embedding immunogold/immunoperoxidase method allowed to estimate that about 12 % of the total CB1 receptor particles are localized in GLAST-positive astrocytes, but the value drops to 5-6 % in GFAP-positive astrocytes, as published previously by our laboratory. Once these findings were obtained, I studied in more detail the CB1 receptor localization in astroglial mitochondria. We discovered the presence of functional CB1 receptors in mitochondrial membranes of hippocampal neurons and astrocytes. Accordingly, I used double GLAST-CB1 immunolabeling to analyze in the electron microscope the density of mitochondrial CB1 receptors in neurons and astrocytes in four brain regions: CA1 hippocampus, prefrontal cortex, piriform cortex and nucleus accumbens. The results showed that the CB1 receptor density in astrocytic mitochondria is higher than in neuronal mitochondria. Altogether, despite the lower absolute levels of CB1 receptors in astrocytes than in neurons, the density of mitochondrial CB1 receptors in astrocytes is higher than in neurons in the four brain regions studied. Namely, CB1 receptors are more expressed in astroglial than neuronal mitochondria. Activation of mitochondrial CB1 receptors alters energy production in neurons and can cause memory impairment. Likewise, activation of mitochondrial CB1 receptors in astrocytes interferes with glucose metabolism and lactate production, disrupting neuronal functions and social behavior. In conclusion, the findings that astrocytes of the four brain regions studied contain more CB1 receptors in their mitochondria than the neuronal mitochondria, and that the cannabinoid-induced reduction of oxygen consumption is absent in mitochondria isolated from the forebrain of GFAP-CB1-KO mice, suggest that mitochondrial CB1 receptors in astrocytes play a crucial role in the global effects of cannabinoids on brain mitochondrial respiratio

CB1 Receptors in the anterior piriform cortex control odor preference memory

The retrieval of odor-related memories shapes animal behavior. The anterior piriform cortex (aPC) is the largest part of the olfactory cortex, and it plays important roles in olfactory processing and memory. However, it is still unclear whether specific cellular mechanisms in the aPC control olfactory memory, depending on the appetitive or aversive nature of the stimuli involved. Cannabinoid-type 1 (CB1) receptors are present in the aPC (aPC-CB1), but their potential impact on olfactory memory was never explored. Here, we used a combination of behavioral, genetic, anatomical, and electrophysiological approaches to characterize the functions of aPC-CB1 receptors in the regulation of appetitive and aversive olfactory memory. Pharmacological blockade or genetic deletion of aPC-CB1 receptors specifically impaired the retrieval of conditioned odor preference (COP). Interestingly, expression of conditioned odor aversion (COA) was unaffected by local CB1 receptor blockade, indicating that the role of aPC endocannabinoid signaling is selective for retrieval of appetitive memory. Anatomical investigations revealed that CB1 receptors are highly expressed on aPC GABAergic interneurons, and ex vivo electrophysiological recordings showed that their pharmacological activation reduces miniature inhibitory post-synaptic currents (mIPSCs) onto aPC semilunar (SL), but not pyramidal principal neurons. COP retrieval, but not COA, was associated with a specific CB1-receptor-dependent decrease of mIPSCs in SL cells. Altogether, these data indicate that aPC-CB1 receptor-dependent mechanisms physiologically control the retrieval of olfactory memory, depending on odor valence and engaging modulation of local inhibitory transmission.Dissection des mécanismes hypothalamiques impliqués dans la détection du statut nutritionnel et régulation de la prise alimentaire via les interactions entre mTORC1, les mélanocortines et les endocannabinoïdes.Représentation sensorielle lors d'états psychotiquesRecepteurs aux cannabinoides dans le codage visuel corticalRecepteurs aux cannabinoides dans le codage visuel corticalNeurocircuitry of endocannabinoid regulation of food intakeDevelopment of pregnenolone derivatives as allosteric inhibitors of CB1 cannabinoid receptors for thetreatment of schizophrenia and psychotic syndrome

Cannabidiol Administration Prevents Hypoxia-Ischemia-Induced Hypomyelination in Newborn Rats

Neonatal hypoxia-ischemia (HI) is a risk factor for myelination disturbances, a key factor for cerebral palsy. Cannabidiol (CBD) protects neurons and glial cells after HI insult in newborn animals. We hereby aimed to study CBD's effects on long-lasting HI-induced myelination deficits in newborn rats. Thus, P7 Wistar rats received s.c. vehicle (HV) or cannabidiol (HC) after HI brain damage (left carotid artery electrocoagulation plus 10% O-2 for 112 min). Controls were non-HI pups. At P37, neurobehavioral tests were performed and immunohistochemistry [quantifying mature oligodendrocyte (mOL) populations and myelin basic protein (MBP) density] and electron microscopy (determining axon number, size, and myelin thickness) studies were conducted in cortex (CX) and white matter (WM). Expression of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) were analyzed by western blot at P14. HI reduced mOL or MBP in CX but not in WM. In both CX and WM, axon density and myelin thickness were reduced. MBP impairment correlated with functional deficits. CBD administration resulted in normal function associated with normal mOL and MBP, as well as normal axon density and myelin thickness in all areas. CBD's effects were not associated with increased BDNF or GDNF expression. In conclusion, HI injury in newborn rats resulted in long-lasting myelination disturbance, associated with functional impairment. CBD treatment preserved function and myelination, likely as a part of a general neuroprotective effect.This work was supported by grants from the Carlos III Research Institute (ISCiii) according to the Spanish Plan for R+D+I 2008-2011 and the State Plan for Scientific and Technical Research and Innovation 2017-2019, with co-funding from the European Regional Development Funds (FEDER) (FIS-PS1600689), from the Biomedicine Program, Community of Madrid (S2010/BMD-2308) and from GW Research Ltd (GWCRI09119)

Endocannabinoid long-term depression revealed at medial perforant path excitatory synapses in the dentate gyrus

The endocannabinoid system modulates synaptic plasticity in the hippocampus, but a link between long-term synaptic plasticity and the type 1 cannabinoid (CB1) receptor at medial perforant path (MPP) synapses remains elusive. Here, immuno-electron microscopy in adult mice showed that similar to 26% of the excitatory synaptic terminals in the middle 1/3 of the dentate molecular layer (DML) contained CB1 receptors, and field excitatory postsynaptic potentials evoked by MPP stimulation were inhibited by CB1 receptor activation. In addition, MPP stimulation at 10 Hz for 10 min triggered CB, receptor-dependent excitatory long-term depression (eCB-eLTD) at MPP synapses of wild-type mice but not on CB1-knockout mice. This eCB-eLTD was group I mGluR-dependent, required intracellular calcium influx and 2-arachydonoyl-glycerol (2-AG) synthesis but did not depend on N-methyl-d-aspartate (NMDA) receptors. Overall, these results point to a functional role for CB1 receptors with eCB-eLTD at DML MPP synapses and further involve these receptors in memory processing within the adult brain.We thank all members of P. Grandes laboratory for their helpful comments, suggestions, and discussions during the performance of this study. The authors thank Giovanni Marsicano (INSERM, U1215 Neurocentre Magendie, Endocannabinoids and Neuroadaptation, Bordeaux, France. University de Bordeaux, France), Beat Lutz (Institute of Physiological Chemistry and German Resilience Center, University Medical Center of the Johannes Gutenberg University Mainz, Germany) and Susana Mato (Achucarro Basque Center for Neuroscience, Science Park of the UPV/EHU, Leioa, Vizcaya, Spain) for providing the CB1 receptor knock-out mice. This work was supported by MINECO/FEDER, UE (grant number SAF2015-65034-R to PG); The Basque Government (grant number BCG IT764-13 to PG); Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III) and European Regional Development Funds-European Union (ERDF-EU; grant RD16/0017/0012 to PG); PhD contract from MINECO (BES-2013-065057 to SP); Vanier Canada Graduate Scholarship (NSERC to CJF)

Endokannabinoide sistemaren parte-hartzea arratoien garuntxoaren jaio osteko garapenean

Endokannabinoide-sistema (EKS) zelulen arteko komunikazio-sistema fisiologiko neuromodulatzaile garrantzitsuenetako bat da, eta helduaroan beraren funtzio ezagunena neurotransmisoreen doikuntza da. Azken urteotako ikerketek, aldiz, funtzio garrantzitsu horrez gain, garapen-prozesuetan ere parte-hartze handia duela erakutsi dute. Izan ere, zenbait artikuluk burmuinaren arauzko enbrioi-garapenerako EKSren beharra agerian utzi dute. Enbrioi-aroko ikerketek muga metodologiko handiak dituzte, eta orain arteko ikerketa gehienak hazkuntza zelularretan egin dira. Halere, burmuineko garuntxoaren kortexean, EKSren adierazpena handia da, eta jaio osteko garapen-prozesu guztiak biltzen ditu: pikor-zelulen morfogenesia, hain zuzen ere. Lan honetan, ultraegitura mailan, EKSren zenbait osagairen kokapena aztertu da; pikor-neuronen garapenaren faseetan baita helduaroan ere. CB1 kannabinoideen hartzailea, eta 2-arakidonil-glizerol (2-AG) endokannabinoidea sintetizatzen eta degradatzen dituzten entzimak —diazilglizerol lipasa (DAGL-α) eta monoazilglizerol lipasa (MAGL), hurrenez hurren— batera ageri dira hazkuntza- eta migrazio-prozesuetan dauden zuntz paraleloen axoietan jaio osteko garapen goiztiarrean. Konkretuki, CB1 eta DAGL-α zuntz paraleloen mintzean kokatzen dira, eta MAGL, mintzean ez ezik, zitoplasman ere adierazten da. Jaio osteko 12. egunean, zuntz paraleloek Purkinje neuronen arantza dendritikoekin kontaktu sinaptikoak ezartzen dituztenean, CB1 eta MAGL zuntz horien bukaera sinaptikoan mantenduko dira. CB1 bukaera sinaptiko horien mintzean kokatzen da, eta MAGL, aldiz, mintzaren hurrentasunean ez ezik, zitoplasman ere adierazten da. DAGL-α, zuntz paraleloen bukaera sinaptikotik desagertuko da, eta alde postsinaptikoan adieraziko da —hots, Purkinje neuronen arantza den- dritikoetan—. EKSn aztertutako 3 osagai horiek batera ageri dira zuntz paraleloetan garapenean zehar, kontaktu sinaptikoak ezarri arte. Horrela, zuntz horiek helduaren ezaugarriak lortzen dituztenean, hartzailea eta degradazio-entzima zuntz paraleloen bukaera sinaptikoan mantenduko dira. Sintesi-entzima, aldiz, axoi-profil horietatik desagertuko da, eta elementu postsinaptikoan adierazten hasiko da —hots, Purkinje neuronen arantza dendritikoetan—. Modu horretan, aztertutako aldi bakoitzean, EKSko osagaien kokapen espezifikoa bistaratu zen. Lortutako emaitzek EKS moldakorra dela eta funtzio desberdinetara egoki daitekeela iradokitzen dute, bere jarduna modu desberdinetan betez. Testuinguru horretan, garuntxoaren garapenean EKSren osagaien kokapen zehatza eta izaera identifikatzea alderdi kritikoa da, haurdun dauden emakumeen eta baita nerabeen kalamu-kontsumoak garunean egitura, funtzio eta portaera mailan eragiten dituen aldaketak ulertzeko. Horrez gain, jakintza hori terapeutikoki erabil daiteke, endokannabinoideen manipulazioak aplikazio klinikoak izan baititzake jaio osteko garapenean eta haurtzaroan gertatzen diren nerbio-sistemako gaixotasunen tratamenduan.; The endocannabinoid system (ECS) is widely distributed throughout the or-ganism. This system influences brain development and suppresses neurotransmitter re-lease in the mature brain, thus contributing to brain circuit formation and neural com-munication. Because of methodological limitations, most investigations focused on the study of the embryo development have been carried out in cell culture. In the central nervous system, the ECS is highly expressed in the cerebellar cortex where it plays a crucial role in the postnatal development of the granule cells. However, little is known about how the precise localization of the components of the ECS takes place during the early postnatal development, which is needed for the correct wiring of the cerebellar circuits. In this study, we used high resolution immunoelectron microscopy to investi-gate the subcellular localization of the cannabinoid CB1 receptor and the main enzymes for the synthesis and degradation of the endocannabinoid 2-arachidonoyl-glycerol (2-AG), diacylglycerol lipase (DAGL-α) and monoacylglycerol lipase (MAGL), re-spectively, in the granule cells during the early postnatal development of the cerebel-lum. The CB1 receptor, DAGL-α and MAGL were localized at the granule cell axons, the parallel fibers, during granule cell migration and axon elongation occurring at early postnatal age. Moreover, CB1 receptor and DAGL-α immunoparticles were placed in the parallel fiber membranes, while MAGL labeling was at the parallel fiber mem-branes and cytoplasm. At postnatal day 12, when parallel fibers make already synapses with the Purkinje cell dendritic spines, the CB1 receptor exhibited the typical presynap-tic localization in parallel fiber terminal membranes, and MAGL was close to the membrane and in the cytoplasm of the parallel fiber terminals. However, DAGL-α was excluded from the parallel fiber terminals but it was expressed at postsynaptic sites. Al-together, the expression of CB1 receptors, DAGL-α, and MAGL in the granule cell parallel fibers starts at early stages of the postnatal development. Later on, the CB receptors and MAGL remain in the mature parallel fiber synapses, while DAGL-α disap-pears from them and switches to the postsynaptic dendritic spines of the Purkinje cells. These structural changes correlate with the functions in which the ECS is involved dur-ing postnatal development. Hence, the identification of the specific localization of the components of the ECS in the developing cerebellum is crucial for understanding the structural, functional and behavioral changes taking place in the brain by cannabis con-sume. Furthermore, this knowledge could be exploited therapeutically as the ECS ma-nipulation might have potential clinical applications in the treatment of brain diseases caused by abnormal development of the brain