© 2012 Nature America, Inc. All rights reserved. receptor signaling in the brain. Thus, Thus, mtCB mechanisms contributed to endocannabinoid-dependent ronal energetics and function is still a matter of on neuronal mitochondria and their potential ability to modulate neu- protein families controlling neuronal activity. The p Edgar Soria-Gómez Edgar receptors, receptors, exogenous and teins mitochondria that evidence consistent now is there such as soluble adenylyl cyclase adenylyl such as soluble of of effectors downstream G potential localization production respi mitochondrial regulating proteins, eventually mitoc of phosphorylation and PKA of activation to leading kinase A (PKA) A kinase complex complex I enzymatic activity and respiration in neu physiology, remain poorly understood poorly remain physiology, activity mitochondrial coupling mechanisms the and i cascade signaling cAMP–PKA intramitochondrial the its its functions. Here we show that the type-1 cannabi The mammalian brain is one of the organs with the h Giovanni Bénard metabolism mitochondria mitochondria (mtCB activity to brain functions are still poorly documented poorly still are functions brain to activity Etienne Hebert-Chatelain Etienne Germany. key elements of eukaryotic cell functions cell eukaryotic of elements key mi supplies, energy cellular regulating and Ensuring Received Received 11 November 2011; accepted 20 January 2012 NATURE NEUROSCIENCE NATURE 1 but it up consumes to 20% of the body’s ene resting weight body total the of 2% only represents brain The Luz López-Rodríguez María Mitochondrial CB Matthias Klugmann Matthias Foundation, Foundation, Rome, Italy. getics in brain physiology and pathology is the foc the research is pathology and physiology brain in getics regulation of brain functions brain of regulation Chemistry, Complutense University, Madrid, Spain. Bordeaux, Bordeaux, Maladies Rares: Génétique et Métabolisme la la plasticité neuronale, U862, Bordeaux, France. Australia. Australia. Leioa, Leioa, Spain. INSERM, INSERM, Neurocentre de Magendie, Physiopathologie l G protein–coupled receptors (GPCRs) represent one of the represent (GPCRs) receptors G protein–coupled 7,8 . . Moreover, several reports have shown the intramitocho 8 1,4,5 9 Translational Neuroscience Facility, Department of Institute Institute of Biochemistry and Molecular Medicine, B 10,11,13,14 5 , but the molecular mechanisms linking mitochondria linking mechanisms molecular the but , Laboratoire Laboratoire Physiologie Cellulaire de la Synapse, C 1 receptors directly modulate neuronal energy metabo 12 . Thus, cAMP can be produced in mitochondria, mitochondria, in produced be can cAMP Thus, . . However, the upstream mechanisms regulating regulating mechanisms . However,upstream the 11 1–3,11 1 ), ), where it directly controls cellular respiration These These authors contributed equally to this work. Cor

1,3 7,8 ADVANCE ONLINE PUBLICATION PUBLICATION ONLINE ADVANCE , , Isabel Matias , , Federico Massa , Stephan Guggenhuber , Stephan 1,4 9 . The involvement of neuronal ener- neuronal of involvement The . , , phosphodiesterase 1,3 , Christophe Mulle , Christophe 6 , , Pedro Grandes 1,4 . 4 1,3 Department Department of Faculty Neurosciences, of Medicine an in in situ 7 Institute Institute of Chemistry,Physiological University Me 1 that are crucial for the the for crucial are that 1,3 (MRGM), (MRGM), EA 4576, Bordeaux, France. speculation receptors regulate neuronal energy a a plasticité and neuronale, Endocannabinoids Neuroa , , Anna Delamarre 1,3,11 ; ; published online 4 March 2012; endocannabinoids decreased endocannabinoids cyclic AMP concentratio rgy production rgy ration ration and energy resence resence of GPCRs protein signaling, protein signaling, Physiology, School of Medical Sciences, University 10,11 contain G pro- G contain us of intensive intensive of us ern, ern, Switzerland. tochondria are are tochondria , , Nagore Puente . in mammals, mammals, in and neuronal neuronal and ronal mitochondria. ronal In mitochondria. addition, CB intracellular entre entre National de la Recherche Scientifique UMR 509 noid noid receptor (CB and protein 4 ighest ighest energy demands, and mitochondria are key det 6 n neurons, neurons, n 7 , Rodrigue Rossignol , Rodrigue . However, , Beat Lutz , Beat 3,5 hondrial hondrial depolarization-induced suppression of depolarization-induced inhibition i , Silvia Ortega-Gutiérrez , Silvia largest largest ndrial ndrial respondence respondence should be addressed to G.M. (giovanni.m 1,2 l . 10 Present Present address: European Brain Research Institute, 1,3 lism, lism, revealing a new mechanism of action of G prot affect mitochondrial functions mitochondrial affect juana)-derivative juana)-derivative functions m activity, neuronal control tightly they where branes, showed that CB that showed evidence evidence points also to the ability of CB of cannabinoid receptors as typical plasma membrane plasma typical as receptors cannabinoid of lipophilic lipophilic nature of most cannabinoids to the regulation of endocannabinoid metabolism compart intracellular different Indeed, cells. inside membran plasma at only not occur might interactions ascribed to nonspecific alterations of alterations membrane prop to nonspecific ascribed on cannabinoids lipophilic of effects mitochondrial mutant mice constitutively lacking the CB the lacking constitutively mice mutant this of specificity the investigate to us prompted tion Th labeling. background nonspecific considered generally experiments, microscopy electron in mitochondria nal mitochondrial biogenesis in peripheral non-neural t non-neural peripheral in biogenesis mitochondrial somal intracellular membranes intracellular somal lyso in signal functionally to shown been have tors that that CB 7 , , Mathilde Metna-Laurent , , Jürg Gertsch and and energy production. Through activation of mtCB 4 CB The The present study stemmed from the neuroanatomical ob , , Joana Lourenço 1 ) ) is present at the membranes of mouse neuronal 1 doi:10.1038/nn.3053 receptors are GPCRs highly enriched in neuronal pl 3 1 Université Université de Bordeaux, Neurocentre Magendie, Physi receptor immunogold particles are on receptor often particles immunogold detected 15–17 2,3 . . Early studies suggested that the dical dical Center of the Johannes Gutenberg University, & Giovanni Marsicano & Giovanni d d Dentistry, University of the Basque Country UPV/E 1 receptors are present on neuronal mitochondrial mitochondrial neuronal on present are receptors 9 , Francis Chaouloff , Francis daptation, daptation, U862, Bordeaux, France. 6 , , Mar Martín-Fontecha of of New South Wales, Sydney, New South Wales, 1,3,5,10 1, 1, Bordeaux, France. ∆ , , Luigi Bellocchio 25 18 9 - (THC) could . . However, with the identification identification the with However, . 1,3 1 receptors and mitochondrial , , Astrid Cannich n, n, protein kinase A activity, 15 1 receptor signaling to regulate [email protected]). implies that receptor– “Rita Levi-Montalcini” erminants erminants of n n the hippocampus. 1 1,3 1,3 receptor sativa , 6 Department Department of Organic 23,24 S E L C I T R A 6 ments contribute contribute ments 2 , Université Université de , , and CB somal or endo- or somal opathologie opathologie de 1,3 erties issues signal by using using by signal neurons were were neurons 26 etabolism and etabolism ein–coupled ein–coupled GPCRs . The results results The . , but they are are they but es, but also also but es, asma mem- 1,3 1 is is observa- Mainz, Mainz,

20 21,22 servation servation HU, HU, , . . Recent 1 neuro- (mari- recep- . The The . 15,19 1 , © 2012 Nature America, Inc. All rights reserved. approximately 95% of mtCB of 95% approximately without digitonin presolubilization, suggesting that suggesting tr presolubilization, digitonin with without treated mitochondria purified of noblotting ( mitochondria of S E L C I T R A purified wild-type brain mitochondria ( mitochondria brain wild-type purified i. 3 Fig. region of wild-type ( wild-type of region hippocampal CA1 the in neurons of membranes of CB of mitochondria. ( mitochondria. detection of CB of detection (WT, total mitochondrial area, 35.7 35.7 area, (WT,mitochondrial total mitochondria of subset immunopositive the specific CB specific Arrows, mitochondria. single of details Insets, 2 2 1 Figure C intracellular of stimulation Furthermore, getics. membranes, and their direct activation regulates mito 200 nm. ( nm. 200 (WT, mitochondria total of a proportion as (mito) n CB blotting confirmed the specific presence of CB presence specific the confirmed blotting CB total CB total CB RESULTS functions. neuronal specific and metabolism ne providing the a hippocampus, link between possible cannabinoid-dependent regulation of short-term synaptic plasticity participate activity mitochondrial of presynaptic inhibition Further quantifications revealed that 15.5% 15.5% that revealed quantifications Further and dropped to 0.4 0.4 to dropped and 0.7% of CA1 mitochondria in in mitochondria of CA1 0.7% mitochondria from WT and and WT from mitochondria ( wild-type (WT) and and (WT) wild-type from (TTL) lysate tissue total brain whole in pal neurons of wild-type mice (mtCB mice wild-type of neurons pal h CA1 of membranes mitochondrial on microscopy tron labeling labeling was 18.3 Fig. 1a Fig. ling ling ( mitochondria displayed CB displayed mitochondria Figure 3c Figure in shown are blots Full-length mice ( in mice compartments somato-dendritic and terminals axon membrane. Scale bar, 200 nm. ( bar,nm. 200 Scale membrane. mitochondrial outer the on particles and and digitonin assay of CB of assay digitonin mitochondria. Resistance of mtCB of Resistance mitochondria. shown in in shown are blots Full-length space. intermembrane mitochondrial the facing protein the of portion C-terminal the with membrane outer the on localized is receptor the that indicates (digit) digitonin by permeabilization membrane mild to not but digestion trypsin to domain mean mean outer of Tom20, translocase matrix; mat., membrane; e = 458 mitochondria from 3 mice). ( 3 mice). from mitochondria = 458 ) Immunoblotting of CB of ) Immunoblotting 1 1 1 −/− receptor immunoreactivity was detected by immunogo by detected was immunoreactivity receptor receptor protein is localized on neuronal mitochon Supplementary Fig. 1b Fig. Supplementary n 1 Fig. Fig. 1c ± = 463 mitochondria from 3 mice; 3 mice; from mitochondria = 463 , total mitochondrial area, 29.4 29.4 area, mitochondrial , total receptor immunoparticles per area of area per immunoparticles receptor . eiuniiain f muood mgs revealed images immunogold of Semiquantification ). Supplementary Fig. 2a Fig. Supplementary s.e.m. *** s.e.m. ), but not not but ), CB 1 . . ( Supplementary Figure 4 Figure Supplementary protein was localized to mitochondria. Western immun Western mitochondria. to localized was protein c ) ) CB 1 f ) Detail of CB of ) Detail 1 receptor localization. Scale bar, Scale localization. receptor ). ). MtCB receptors in neuronal neuronal in receptors a 1 1 , receptors on mitochondrial mitochondrial on receptors -immunopositive mitochondria mitochondria -immunopositive b P ) Electron immunogold immunogold ) Electron ± CB Cnr1 < 0.001 as compared to WT.to compared as < 0.001 a Fig. 1f Fig. 2.0 per particles 1 ) and ) and ± 1 receptor expression in expression receptor 1 receptors were between comparably distributed 0.1 particles per per particles 0.1 −/− −/− 1 1 mice and purified purified and mice receptor protein protein receptor CB receptor gold gold receptor CB ). This was confirmed by Western immu- Western by confirmed was This ). (here called called (here 1 1 Supplementary Supplementary 1 ). Approximately 30% of wild-type CA1 CA1 wild-type of 30% Approximately ). −/− −/− immunolabeling, whereas only 2.9% 2.9% only whereas immunolabeling, 1 was localized to the outer membrane membrane outer the to localized was g CB ). The density of mtCB of density The ). mice ( mice . Den, dendrites; ter, terminals; m, mitochondria; mitochondria; m, ter, terminals; dendrites; . Den, mice. mice. ) ) Trypsin and 1 C-terminal C-terminal µ 1 m −/− d µ µ ) Density ) Density 2 m m ; b mice showed nonspecific labe- nonspecific showed mice CB 1 ). ). µ 2 2 ; ; ). ). of in mitochondria wild type CB m Fig. 1a Fig. Fig. 1e Fig. 1 −/− 2 in in 1 −/− , ± 4.2% of CA1 neuronal neuronal of CA1 4.2% CB 1 ) littermates ( littermates ) and and immunoreactivity in immunoreactivity and and mitochondrial membranes 20 kDa; CIII, core 2 prote core CIII, kDa; 20 membranes mitochondrial 1 f e d c a −/− CB1-labeled B Supplementary Supplementary Supplementary Supplementary mito/total mito (%) N-terminal the mice ( mice 1 chondrial ener-chondrial 1 20 30 40 10 receptors and and receptors ypsin with or or with ypsin uronal energy uronal energy immunogold immunogold 0

WT wild-type wild-type dria ippocam- TTL WT in endo-

CB 1d Fig. ld elec- ld –/– 1b Fig. 1 *** den that that CB TTL in 1 o- –/– ter ). ). ±

Density (CB1 particles 2 Mito WT

per µm positive mito) m Supplementary Fig. 4 Fig. Supplementary anatomical distribution of mtCB of distribution anatomical behavior and functions brain affects tially likely likely located in the intermembrane mitochondrial sp portion of portion mtCB protein is present at the surface of neuronal mitochondri neuronal of surface at the present is protein glutamatergic neurons glutamatergic excit and GABA containing neurons inhibitory include These where they negatively regulate excitability and neuro conditional mutant mice lacking lacking mice mutant conditional mtCB tive to that in wild-type mice ( mice wild-type in that to tive mitoc labeled of percentage the in reductions similar tively toneally, 5 mg per kilogram body weight) reduced mi reduced weight) body (i kilogram per mice mg 5 C57BL6/N toneally, to THC of administration acute The MtCB glutamatergic in than neurons GABAergic hippocampal ( both mutant strains ( strains mutant both mtCB observed observed concerning the proportion of mtCB ( mutants gic (Glu- gic dria dria of presumably inhibitory symmetric synapses in ern aymti snpe (rsmby excitatory) CB (presumably synapses asymmetric bearing sion ( sion 10 15 20 25 Supplementary Fig. 2b Fig. Supplementary 0 5 CB 1 −/− CB WT Mito OMM, outer mitochondrial membrane; IMM, inner mitoc inner IMM, membrane; mitochondrial outer OMM, 27,28 1 1 Fig. 2e Fig. 1 m 1 1 eetr ae xrse i dfeet ernl popu neuronal different in expressed are receptors was reduced by 50.4% in GABA- mice, whereas it was almost exclusively present on present exclusively it was almost whereas mice,

CB expres densely more however, are, which receptors, –/– directly regulates brain mitochondrial activity mitochondrial brain regulates directly m –/– 1 *** CB . MtCB . Fig. 2d Fig. ADVANCE ONLINE PUBLICATION PUBLICATION ONLINE ADVANCE 1 ). As expected, most mtCB most expected, As ). −/− ) or in GABAergic (GABA- GABAergic in or ) mat. 1 1 ). No difference between these two genotypes was was genotypes two these between difference No ). is exposed to is the whereas the exposed C cytosol, terminus i was still present in CA1 hippocampal neurons of neurons hippocampal CA1 in present still was Cytosol IMM Fig. 2a Fig. 24 ). Thus, a substantial portion of CB of portion substantial a Thus, ). b , where activation of CB activation , where ). Thus, both neuronal populations contain contain populations neuronal both Thus, ). in of mitochondrial complex III. Values, Values, III. complex mitochondrial of in OMM , b and and den Fig. 2c Fig. 1 CB receptors in the hippocampus of hippocampus the in receptors Supplementary Fig. 1c Fig. Supplementary 1 Tom20 g either in cortical glutamater- cortical in either m CB ). In contrast, the density of of density the contrast, In ). CB ter CIII 1 was detected in terminals terminals in detected was 1 1 16,17,27,28 −/− m CB NATURE NEUROSCIENCE NATURE 1 versus total CB , , but not in Glu- 01 15 15 10 0 1 −/− 1 m receptors differen- receptors ) neurons, respec- neurons, ) transmission . We analyzed the the We . analyzed Trypsin (min) Glu- ace ace ( hondria rela- hondria ones. tochondrial tochondrial CB hondrial hondrial n GABA- in a. mitochon- Fig. Fig. 1g 1 ntraperi- receptor receptor 1 , 1 d −/− expres- lations, lations, CB + digit ), with with ), sed in in sed mice atory atory 15–17 1 and −/− s . , © 2012 Nature America, Inc. All rights reserved. number of mitochondria in the CA1 hippocampal regio hippocampal CA1 the in mitochondria of number mitochondria from 3 mice). ( 3 mice). from mitochondria ( ( GABA- the CA1 hippocampal region of Glu- of region hippocampal CA1 the cannabinoids, we measured the effects of THC the on effects we measured res cannabinoids, total CB total wild-type values. wild-type CB hippocampal neurons. ( neurons. hippocampal NATURE NEUROSCIENCE NATURE 3 Figure bioenergetics) respiratory chain complex I activity (particularly 2 Figure P complex I activity and mitochondrial respiration mitochondrial and activity I complex suggest been have (PKA) A kinase protein and cyclase cAMP of reduction Values, mean mean Values, GABA- and fields) due to a direct activation of mtCB injected with AAV-CB with injected WIN on complex I activity in purified mitochondria mitochondria purified in I activity complex on WIN was fully rescued by rescued was fully m hippocampal purified of activity I complex on WIN (vehicle, 0.60 0.60 (vehicle, THC treatment decreased mitochondrial cAMP by appro 6.2; THC, 65.9 THC, 65.9 6.2; protein; protein; WIN on respiration of purified mitochondria from wi from mitochondria purified of respiration on WIN ## # n cM cnet ( content cAMP and treatment of purified mitochondria with 100 nM WIN WIN nM 100 with mitochondria purified of treatment pal CB pal purified from wild-type and receptors on located the plasma membrane, brain mito fluorescent protein, AAV-GFP) or CB or AAV-GFP) protein, fluorescent control a either (AAV)expressing viruses associated WIN WIN specifically decreased mitochondrial complex I activity of complex I in the presence of 100 nM WIN nM 100 of presence the I in complex of activity we injected the hippocampi of of hippocampi the injected we in in brain butfrom mitochondria wild-type, not c oxygen decreased dose-dependently drug The (WIN). mtCB ra otie te -iae nhr rti 11 (AKA 121 protein organelles these anchor 1e Fig. A-kinase Supplementary the contained dria and treated treated and c n P P < < 0.05). To test whether the effects of CB effects the To < 0.05). whether test ) Proportion of CB of ) Proportion e d c b a = 473 mitochondria from 3 mice) and GABA- and 3 mice) from mitochondria = 473 P

WIN effect on mitochondrial To investigate whether mtCB 1 < 0.01 as compared to Glu- to compared as < 0.01 −/− 0.05, 0.05, respiration (% control) 100 1 CB 50 1 -dependent -dependent manner ( hippocampal mitochondria ( mitochondria hippocampal 0 1 expression is sufficient for the mitochondrial effec for is the expression mitochondrial sufficient Mt-CB Direct regulation of mitochondrial activity by mtC by activity mitochondrial of regulation Direct signal in the CA1 hippocampal region of Glu- of region hippocampal CA1 the in signal 1 n −/− = 6, 6, = ## 02 50 20 10 0 mice. ( mice. P in vitro in ± < 0.01 as compared to WT. Dashed horizontal lines, WT.to horizontal Dashed compared as < 0.01 s.e.m. ** s.e.m. 4 ± CB WT * # 1 in purified hippocampal mitochondria (vehicle, 94. ± is present in both GABAergic and glutamatergic CA1 CA1 glutamatergic and GABAergic both in present is 0.06; THC, 0.41 0.41 THC, 0.06; P 12 Dose WIN(nM) 8.1 nmol oxidized NADH per minute per milligram NADH milligram minute per 8.1 per nmol oxidized CB 1 < 0.05). CB 0.05). < –/– . WIN decreased mitochondrial PKA activity in an PKA activity mitochondrial . decreased WIN 1 e 1 -immunopositive mitochondria normalized to the tota the to normalized mitochondria -immunopositive 1 # * −/− ) Proportion of mtCB of ) Proportion ( with the synthetic CB synthetic the with in in vivo 15,19,29 a n i. 3c Fig. ( , = 6 per group). Values, mean mean Values, group). = 6 per P b n < 0.01; *** < 0.01; ) Immunogold localization of mtCB of localization ) Immunogold ), confirming that PKA activity occurs in in occurs activity PKA that confirming ),

= 27 fields) mice. Scale bar, 500 nm. bar,nm. 500 Scale mice. fields) = 27 d ADVANCE ONLINE PUBLICATION PUBLICATION ONLINE ADVANCE CB viral re-expression of CB the re-expression viral , and mitochondrial forms of adenylyl adenylyl of forms mitochondrial and , 100 ) Density of mtCB of ) Density Fig. Fig. 3d ## * CB . ipcma nuoa mitochon- neuronal Hippocampal ). 1 1 receptor signaling typically involves involves typically signaling receptor −/− 1 CB is responsive to natural plant-derived 1 1 −/− CB ± littermates ( or to an indirect stimulation of CB 1 0.06 nmol per mg protein; protein; mg per nmol 0.06 ). ). To establish whether hippocam- −/− mice. Dashed horizontal lines, lines, horizontal Dashed mice. 1 WIN effect on complex I P −/− Fig. 3e Fig. < 0.001 as compared to WT;to compared as < 0.001

activity (% control) adeno- neurotropic with mice ( 1 1 100 (AAV-CB receptor normalized to the the to normalized receptor 50 a 0 ) and GABA- ) and 1 ). receptor activation were were activation receptor 1 CB 1 0 in Glu- in Supplementary Fig. 3a WIN55,212-2 WIN55,212-2 agonist CB 1 from WT and and WT from ld-type (WT) and and (WT) ld-type involved in neuronal −/− CB WT in purified mitochondria from WT and and WT from mitochondria purified in 5 1 ( 7,8,11,13,14,30 n of Glu- n of mice ( mice −/− n CB 1 1 ± –/– = 4). ( = 4). ) CB s.e.m. * s.e.m. Time (min) activity activity ( 31 CB 10 piration, cAMP piration, 1 , , mice ( * # protein (green (green protein −/− B . The effect of of effect The . chondria were chondria 1 ed to regulate regulate to ed 1 −/− 1 ximately 30% 1 onsumption onsumption ( −/− n receptors in the brain. ( brain. the in receptors itochondria itochondria 1 receptor in receptor (arrows) in (arrows) = 320 = 320 n and and d CB ts ts of WIN, mice ( mice = 31 ) Effect of 100 nM WIN on mitochondrial PKA activit PKA mitochondrial on WIN nM 100 of ) Effect CB P Fig. Fig. 3a 1 Fig. 3b < 0.05, ** < 0.05, . Acute Acute . 20 ## −/− * values for vehicle-treated controls. vehicle-treated for values 1 n P121; P121; −/− CB = 6, 6, =

b hippocampi locally injected with AAV-GFP, with injected from and locally hippocampi 7 7 1 ). ). ). ). −/− ± 1 ) ) l

brains ( brains WIN effect on cAMP P degrading activity ( activity degrading on the type of ligand. of type the on cannabinoids regulate mitochondrial activity activity mitochondrial regulate cannabinoids ( nrae mtcodil -rcioollcrl ( 2-arachidonoylglycerol mitochondrial increased dose- JZL However, shown). not (data quantification i but its mitochondria, neuronal were levels below the detected was endocannabinoid The tion. a levels endocannabinoid mitochondrial measured and and and fr mitochondria neuronal in activity PKA and levels (MAGL), the two main endocannabinoid degrading enzy degrading endocannabinoid main two the (MAGL), du monoacylgl and (FAAH) a hydrolase amide (JZL), acid fatty of JZL195 with mitochondria brain purified P CB wild from mitochondria brain in respiration reduced chondrial respiration in wild-type brain mitochondr brain wild-type in respiration chondrial le 2-arachidonoylglycerol between observed was tion content (% control) mtCB activate to able endocannabinoids and in arerespiration the brain, and (ii) mitochondria PKA activity, compl accumulation, cAMP mitochondrial Fig. Fig. 4a 100 150 < 0.01; *** < 0.01; < 0.0001; 0.0001; < a c 50 Brain mitochondria have substantial levels of endoc of levels substantial have mitochondria Brain Together, these data show that (i) mtCB (i) that show data Together, these CB -labeled 0

1 1 −/− CB mito/total mito (%) n 10 20 30 40 0 = 4 independent experiments). ( experiments). = 4 independent *** WT , mice ( , mice a 1

– CB Glu- ) Dose–response curve for the effect of the CB the of effect the for curve ) Dose–response −/− c

1 –/– *** ), indicating ), that indicating of the functions mtCB littermates. THC induced effects similar to those of to W those similar effects THC induced littermates. CB

Fig. 4f Fig. GABA- CB P ## CB 1 < 0.001 as compared to control (vehicle or time 0) time or (vehicle control to compared as < 0.001 –/– Fig. 4e Fig. –/– *** 1 1 −/− brains ( brains ).

WIN effect on PKA Table 1 Supplementary activity (% control) correla- inverse significant Notably, a highly ). 100 150 Glu- e d 50 Density (CB particles

0 1 n 2 CB = 3). ( = 3). per µm positive mito) 25 10 15 20 0 5 WT 1 **

CB Glu- –/–

1 –/– c CB GABA- ) cAMP content after 5 min 5 min after content ) cAMP b ## CB y y ( 1 b –/– ## 1 –/– ** ) Time course of enzymatic enzymatic ) Timeof course n = 3). ( = 3). 1

Genotype

in situ in 1 CB

WIN effect on complex I affects directly signaling ). To test whether endo- To). whether test

1 activity (% control) AAV e −/− 100 150 ) Effect of 100 nM 100 of ) Effect 50 0 . hippocampi locally locally hippocampi

n situ in % CB immunogold 1 1 are not dependent S E L C I T R A 1 particles in mito/total receptor agonist agonist receptor F F CB GFP GFP WT limits limits of reliable

* gold particles equipped with equipped 10 15 20 vels and mito- and vels i. 4d Fig. GABA- -type, but not not but -type, om wild-type wild-type om 0 5 dependently dependently ycerol lipase lipase ycerol ia ( ia , we treated treated we , CB Glu- CB al inhibitor inhibitor al annabinoid annabinoid ex I activity ex I activity nd respira- nd

n purified purified n 1 –/– 1 r –/– CB mes GABA- = 0.82, 0.82, =

; ; CB 1 ) and and ) –/– CB 1 –/– 15,32 * 1 IN IN –/– 1 3 , © 2012 Nature America, Inc. All rights reserved. S E L C I T R A important endocannabinoid-dependenttransmi formGABAergic of synaptihippocampal of control Retrograde neuronal endocannabinoid-dependent in involved dria noanbnis wih ergaey ciae presy activate retrogradely which endocannabinoids, neuron pyramidal postsynaptic CA1 of Depolarization of inhibition (DSI) inhibition of depolarization-induced as known process a in mission inhibitory GABAergic decreasing transiently receptors, 4 4 mtCB If CB Intracellular 4 Figure CB and WT in content cAMP mitochondrial ( endocannabinoids. active biologically contain mitochondria and THC, PKA activity in WT and and WT in activity PKA and and (WT) wild-type from mitochondria purified of respiration on THC of effect the for curve time from 10–15 mice). ( mice). 10–15 from time ( mitochondria brain purified of respiration 2-AG and respiration in purified brain brain purified in respiration and 2-AG ( mice). 10–15 from time ( scale a logarithmic on presented are Data (JZL). JZL195 FAAH and inhibitor MAGL double the with treatment after mitochondria brain purified in (2-AG) arachidonoylglycerol ( and is occluded by CB is and occluded and the action of endogenously mobilized endocannab mobilized endogenously of action the and scaffold and the biotin moiety should preserve the the preserve should moiety biotin the and between scaffold spacer a of presence the and compound, this cell the impede should bulk biotin hydrophilic the pr The HU-biot). hereafter (HU210-biotin, HU210 agonist # Fig. 5d Fig. we synthesized a biotinylated version of the lipoph the of version biotinylated a synthesized we be able to fully block and occlude DSI, respectively. DSI, occlude and block to fully able be membrane and intracellular CB intracellular and membrane influe relative the discriminate to and DSI on cell-impermeant CB cell-impermeant intracellular mtCB intracellular receptor antagonist. receptor mitochondria from WT mice (values are from from are (values mice WT from mitochondria receptor antagonist pressin and the lipophilic CB lipophilic the and pressin incubat slices hippocampal from extracts of analysis plasma membranes plasma hypothesis. As a , hemopressin should not be of hemopressin ( hemopressin of of fluorescence cells treated with intracellular a biot displayed a comparable affinity for recombinant for affinity comparable a displayed biot reveal experiments competition Radioligand receptors. to that of HU210 (HU210, (HU210, HU210 of that to etrate plasma membranes and stimulate mtCB stimulate and membranes plasma etrate H and HU210 of ability the To characterize nM). 0.4 fected primary mouse fibroblast cultures derived from from derived cultures fibroblast mouse primary fected Neither HU210 nor HU-biot had any effect on cellular res on cellular any had nor effect HU-biot HU210 Neither with a plasmid expressing the CB the expressing plasmid a with n n d P = 3–5 pools of mitochondria purified each each purified mitochondria of pools = 3–5 = 3–5 pools of mitochondria purified each each purified mitochondria of pools = 3–5 2- endocannabinoid the of ) Levels < 0.05, < 0.05, To investigate the occluding effects of cell-imperm 1 −/− CB littermates. ( littermates. 1 – −/− MtCB f 1 ). Thus, hemopressin can be used as can a be used ). cell-impermean Thus, hemopressin can control brain mitochondrial activity, are mito are activity, mitochondrial brain control can brains. ( brains. ## P < 0.01, < 0.01, 1 receptors are activated by by activated are receptors 1 Supplementary Fig. 5a Fig. Supplementary b c receptors participate in DSI in participate receptors 15–17 1 ) Effect of THC on THC of ) Effect ) Effect of THC on THC of ) Effect 25 receptors participate in the expression of DSI, DSI, of expression the in participate receptors 1 . This was confirmed both by measurements of by measurements both confirmed was . This CB receptor antagonists and agonists should not not should agonists and antagonists receptor ### 1 receptor agonists, which prevent the binding binding the prevent which agonists, receptor f e . DSI is blocked by CB by blocked is DSI . ) Correlation between between ) Correlation 1 ) Effect of JZL on JZL of ) Effect a P −/− ) Dose–response ) Dose–response < 0.001 as compared to WT. Dashed lines, vehicle v vehicle WT.to lines, Dashed compared as < 0.001 littermates. littermates. K i = 0.2 0.2 = 1 antagonist AM251 ( AM251 antagonist 25,33,34 1 1 receptors on cellular respiration, respiration, cellular on receptors receptor (creating MF-CB (creating receptor ± 0.07 nM; HU-biot, HU-biot, nM; 0.07 provided a first tool to test this d – , c e ) and by spectrometric spectrometric by and ) ). Values, mean mean Values, ). 1 1 fluorescent derivative fluorescent a d receptor antagonists antagonists receptor receptors, we trans- we receptors, JZL ( JZL 2-AG THC effect on eant CB The peptide CB peptide The (pmol per mg protein) ilic CB ilic µ able able to penetrate 100 Supplementary Supplementary affinity for CB for affinity

M) mitochondrial 10 c plasticity ed with hemo- with ed U-biot to pen- to U-biot penetration of of penetration CB 1

nce of plasma plasma of nce respiration (% control) suppression suppression 100 ed that HU- that ed the HU210 HU210 the CB neurotrans- atc CB naptic 50 s mobilizes mobilizes s functions? functions? inoids 1 .51 0.25 0 ssion is an an is ssion 0 piration of piration K receptors receptors 1 1 1 esence of esence receptor receptor receptor ± i −/− = 1.5 1.5 = 1 s.e.m. * s.e.m. 400 0 cells). cells). chon- pups pups t t CB 15–17 16 CB WT . If . ** 1 ± Dose THC(nM) –/– 1 1 1 1 .

P e # * < 0.05, ** 0.05, < JZL effect on the the fluorescent protein mCherry (mock) or the CB obtained obtained from f Mouse cells. intact in respiration cellular alter ( and Hemo became able to fully block the depression depression the block fully to able became Hemo and Figure 5 Figure transfected transfected ( independent experiments). Values, mean mean Values, experiments). independent HU210 in intact MF-CB intact in HU210 antagonize to able not was Hemo consumption. oxygen signifi HU210 agonist lipophilic the whereas cells, to vehicle control. control. vehicle to and hemopressin (Hemo) did not alter cellular respi cellular alter not did (Hemo) hemopressin and mock-transfected mock-transfected HU210, but not HU-biot, decreased cellular respiratio cellular decreased HU-biot, not but HU210, cells ( cells depressing cell respiration in permeabilized MF-CB permeabilized in respiration cell depressing efficie as became HU-biot Accordingly, effect. this indicating that both drugs activate mtCB activate drugs both that indicating and compared their effects to those of the cell-impermean AM251 and agonist HU210 on inhibitory synaptic tran able to alter the effect of HU210 on intact MF-CB intact on HU210 of effect the alter to able hemop Notably, permeance. cell their on relies ence mitochondrial respiration of living cells. living of respiration mitochondrial notably, (iii) only the activation of intracellular CB of intracellular activation the notably,only (iii) cellula alter not do and cells intact penetrate not h and HU-biot (ii) respiration, cellular alter and cells data indicate that (i) the lipophilic CB that (i) lipophilic the indicate data but it fully blocked this effect in permeabilized cell permeabilized in effect this blocked fully it but c a ) In permeabilized MF-CB permeabilized ) In ) Neither HU210 nor HU-biot altered cellular respir cellular altered HU-biot nor HU210 ) Neither mitochondrial c b a Cellular respiration CB cell-permeant the of impact the Weanalyzed next alues. respiration (% control) (% vehicle) 100 150 50 Fig. 5b Fig. –60 –40 –20 0 800 Hemopressin and HU-biot do not penetrate cells and cells penetrate not do HU-biot and Hemopressin 0 * # P 0.25 0 Mock-transfected ADVANCE ONLINE PUBLICATION PUBLICATION ONLINE ADVANCE < 0.01, *** 0.01, < CB HU210 CB CB WT ), suggesting that cell penetration is a necessary s is a necessary penetration cell that ), suggesting CB ** 1 Dose JZL ( Dose JZL 1 −/− –/–

HU-biot 1 b 1 –/–

−/− THC effect on cAMP MFs MFs ( MFs ## CB content (% control) P pups and transfected with a vector expressing eith 100 150 200 < 0.01. 50 1 0 −/− 1 µ P n cells ( cells M) < 0.001 as compared to vehicle control; control; vehicle to compared as 0.001 < = 6 independent experiments). ( experiments). = 6 independent –60 –40 –20 mouse fibroblasts ( ### *** WT 1 1 * 0 cells, HU-biot reduced cellular respiration respiration cellular reduced HU-biot cells,

HU-biot CB n f = 4–7 independent experiments). experiments). independent = 4–7

Respiration # 1 –/–

Hemo MF-CB (nanogram-atoms HU210 + Hemo oxygen per min HU210 Intact per mg protein) 1 100 150 ± 1 agonist HU210 can penetrate can penetrate HU210 agonist cells 50 * s.e.m. * s.e.m. 0 c

1 THC effect on PKA * receptors, but the differ- the but receptors, 20 0 Fig. 5a ibroblasts (MFs) were were (MFs) ibroblasts

NATURE NEUROSCIENCE NATURE activity (% control) 2-AG (pmolpermgprotein) JZL 1 JZL 0.25 Vehicle 1 100 receptor (MF-CB 50 0 cantly decreased decreased cantly –60 –40 –20 P ration in MF-CB in ration < 0.05 as compared compared as < 0.05 by HU210 ( HU210 by µ 0 ation in mock- in ation M WT r respiration, and, and, respiration, r this effect of effect this ). ). Conversely, only ** 1 µ s ( s 06 0100 80 60 40 smission and DSI, receptors affects affects receptors 1 HU-biot M nt as HU210 in in HU210 as nt 1 cells ( cells cells ( cells Fig. 5c Fig. emopressin do do emopressin ressin was not not was ressin CB b Permeabilized n in MF-CB in n * MF-CB ) HU-biot ) HU-biot ## 1 1 t t antagonist cannot cannot HU210 –/– HU210 + Hemo antagonist Fig. 5b Fig. n * Fig. 5c Fig. ). These These ). 1 1 = 3–5 = 3–5 cells ). ). ## tep for tep 1

er er ), ), ), ), 1

© 2012 Nature America, Inc. All rights reserved. electrophysiological traces (1 and 2, traces before traces 2, and (1 traces electrophysiological CB membrane–impermeant cell the of and cells) of results. ( results. of summary bottom, respectively); depolarization, after and before traces 2, and (1 traces electrophysiological Top, representative cells). CB 10 10 ** mean mean pyramidal neurons with postsynaptic intracellular a intracellular postsynaptic with neurons pyramidal 5 5 course. ( course. electrophysio Top, representative HU-biot. or HU210 postsynaptic pyramidal neurons (Control, (Control, neurons pyramidal postsynaptic CA1 of 0 mV to −70 from step depolarization a 5-s by induced DSI strong of amplitude the hemopressin at a high dose (10 (10 dose high a at hemopressin weak DSI induced by a 1-s depolarization step of CA of step depolarization a 1-s by induced DSI weak (500 nM) displayed a very similar ability to reduce eI reduce to ability similar a very displayed nM) (500 Supplementary Fig. 6c representative electrophysiological traces. Right, Right, traces. electrophysiological representative hemopressin (Hemo, 10 10 (Hemo, hemopressin (4 (4 impermeant CB impermeant the lipophilic CB lipophilic the neednl o te rqec o siuain f af of stimulation of ( frequency the of independently to depolarization-induced DSI. ( DSI. depolarization-induced to NATURE NEUROSCIENCE NATURE 7 Figure b a ( AM251 by CA blocked of fully was neurons depolarization e 5-s a As by induced respectively. DSI ‘strong’ a HU-biot, agonist and hemopressin 6 Figure slices ( slices to applied when (eIPSCs) currents postsynaptic tory CA1 postsynaptic pyramidal neurons before and after and before neurons pyramidal postsynaptic CA1 after 25–30 min of incubation ( of min incubation 25–30 after was HU-biot and HU210 both of effect eIPSCs-depressing The transmission. GABAergic hippocampal on HU210 of and effect that cannabinoid the biotin n moiety does only partially and reversibly reduced DSI amplitude DSI reduced reversibly and partially only induction in o hippocampal slices ( fully HU210 with incubation 30-min a expected, As and HU-biot ( HU-biot and b by eIPSCs of depression the blocked fully hemopressin lular CB lular In contrast, HU-biot only partially occluded DSI at DSI occluded partially only HU-biot contrast, In ∆eIPSC 6a Fig. Supplementary µ P µ 1 1 M M ( µ < 0.01, *** < 0.01, receptor agonist HU-biot (500 nM; nM; (500 HU-biot agonist receptor amplitude (%) nM; (500 HU210 agonist receptor M, M, M; M; –80 –70 –60 –50 –40 –30 –20 –10 ± 10 n s.e.m. ** s.e.m. n 0 Supplementary Fig. 6a Fig. Supplementary = 5 cells) on the amplitude of strong DSI induced induced DSI strong of amplitude the on = 5 cells) n Inhibition of mitochondrial activity potentiates D potentiates activity mitochondrial of Inhibition CB Intracellular c = 5 cells) and of the cell membrane– cell the of and = 5 cells) 1 = 7 cells) on the amplitude of DSI induced by a 1- by induced DSI of amplitude the on = 7 cells) ) Occlusion effect of 30-min incubations of slices slices of incubations 30-min of effect ) Occlusion receptor signaling is not required for this pharmaco for this required not is signaling receptor

Vehicle b ) Effects of the lipophilic synthetic synthetic lipophilic the of ) Effects 1 DSI 5 s DSI 5 P Supplementary Fig. 6a Fig. Supplementary receptor antagonist peptide peptide antagonist receptor 1 P

Rotenone DSI. control to compared as < 0.0001 receptor antagonist AM251 AM251 antagonist receptor < 0.01 as compared to control. NS, not significant not NS, control. to compared as < 0.01 NS , µ d 1 M; M; receptors contribute contribute receptors ). High concentrations of HU210 and HU-biot

, b ADVANCE ONLINE PUBLICATION PUBLICATION ONLINE ADVANCE n ). Notably, the extracellular antagonist antagonist extracellular the Notably, ). = 8 cells) on = 8 cells) Rotenone a at 0mV at 0mV Vehicle ) Effects of ) Effects , 1 s 1 1 s 1 Fig. 6b Fig. b Fig. 6c ). However, the peptide antagonist antagonist peptide the However, ). µ Pre-DSI M) did not alter evoked inhibi- evoked alter not did M) n n = 7 = 6 cells) on normalized eIPSCs in CA1 pyramidal h pyramidal CA1 in eIPSCs normalized on = 6 cells) n and and , = 9 b Fig. 6a Fig. ), suggesting that intracel- that suggesting ), Supplementary Fig. 6a Fig. Supplementary Supplementary Supplementary Fig. 6d S Post-DSI DSI summary data plot. ( plot. data summary and after depolarization, respectively); bottom, s bottom, respectively); depolarization, after and pplication of vehicle ( vehicle of pplication ). The application of of application The ). 1 postsynaptic pyramidal neurons before and after b after and before neurons pyramidal 1 postsynaptic logical traces (1 and 2, traces before and after dr after and before traces 2, and (1 traces logical bath application of the mitochondrial complex I in complex mitochondrial the of application bath

∆elPSC a ot ot alter the effect ## concentrations concentrations PSCs ( PSCs SI. ( SI. amplitude (%) 50 ms by a 5-s depolarization step of CA1 postsynaptic py postsynaptic CA1 of step depolarization a 5-s by P hippocampal hippocampal ( –50 –40 –30 –20 –10 50 pA with HU210 ( HU210 with s depolarization step of CA1 postsynaptic pyramidal postsynaptic CA1 of step s depolarization ferent fibers fibers ferent < 0.01; < 0.01; 1 pyramidal pyramidal 1 ccluded DSI DSI ccluded 10 Fig. 6a Fig. 0 oth HU210 HU210 oth a xpected ) Amplitudes of strong DSI induced by a 5-s depolar a 5-s by induced DSI strong of ) Amplitudes observed observed maximal maximal Control Fig. 6b Fig. 50 ms

logical logical AM251 2 1 50 pA DSI and and ### . c *** ) Amplitude of DSI induced by a 1-s depolarization depolarization a 1-s by induced DSI of ) Amplitude

16 Hemo ## P n ), ), ). ). ). n , = 6 cells) or rotenone (2.5 (2.5 rotenone or = 6 cells) < 0.001. ** 2 1 ∆eIPSC 50 of concentrations at HU-biot with or = 8 cells) ( CB endocannabinoids ( endocannabinoids i. 7b Fig. DSI is mediated by presynaptic CB presynaptic by mediated is DSI endocan local in increase to due not was it that and inhibitory neurotransmission, that it was dependent on CB of that the effect rotenone on DSI was not due to a depolarization ( depolarization ( enhanced reliably depol rotenone step, long depolarization a 1-s by induced s; DSI (5 period strong on effect no had Thus, cell penetration of CB of penetration cell Thus, inhibitor. The application of rotenone to hippocampal Rotenone is a well known specific and potent mitochondri to contributes activity mitochondrial of Inhibition neurons. pyramidal postsynaptic of depolarization in DSI of occlusion and blockade full respective the for amplitude (%) 5 and nM 500 both of Supplementary Fig. 7c Fig. Supplementary Fig. 7b Fig. 1 –80 –70 –60 –50 –40 –30 –20 –10 2 10 1 0 −/−

Vehicle mice in the presence of the depolarization ( depolarization the of presence the in mice b

, o le te niioy fet o H-it n eI on HU-biot of effects inhibitory the alter to ), eIPSCs amplitude ). Rotenone was unable to affect eIPSCs in the abse the in eIPSCs affect to unable was Rotenone ). (% of baseline) DSI 1 s 100 150

Rotenone 50 Fig. 7a Fig. 0 ippocampal neurons. Arrow, time of application of application of time Arrow, neurons. ippocampal Supplementary Fig. 7a Supplementary ** 1 5 01 02 03 40 35 30 25 20 15 10 ummary of results. Values, mean mean Values, results. of ummary Supplementary Fig. 7d Fig. Supplementary ). However, if a weaker DSI was induced by a by induced was DSI weaker a if However, ). U1 HU-biot HU210 µ ug application, respectively); bottom, time time bottom, respectively); application, ug Time (min) M; M; 2 1 hibitor rotenone (2.5 (2.5 rotenone hibitor µ ath application of rotenone (2.5 (2.5 rotenone of application ath d c –60 –50 –40 –30 –20 –10 ), or to modify the levels of mitochondrial mitochondrial of levels the modify to or ), 10 M ( M n 0 = 6 cells). ( = 6 cells). 1 ramidal neurons. Top, representative Top, representative neurons. ramidal

Vehicle 6c Fig. antagonists and agonists is necessary necessary is agonists and antagonists neurons (gray bar, (gray neurons 1 2 DSI 1 s

ization step from −70 to 0 mV of 0 mV to −70 from step ization Rotenone 1 and and receptors 2 d NS ), ), to induce DSI in slices from ) Effect of hemopressin (Hemo, (Hemo, hemopressin of ) Effect step of CA1 postsynaptic postsynaptic CA1 of step c Supplementary Fig. 6d Fig. Supplementary

∆elPSC 50 ms 0 nM ( 0 nM µ ). These results indicate indicate results These ). 50 pA

M). ( M). amplitude (%) –50 –40 –30 –20 –10 10 16 –60 –50 –40 –30 –20 –10 0 10 . Rotenone did not not did Rotenone . 0 n b

n Control direct alteration of alteration direct S E L C I T R A ± = 7 cells). Values, Values, = 7 cells). ) Amplitudes of ) Amplitudes = 7 cells) or = 7 cells) s.e.m. * s.e.m.

Vehicle Supplementary HU-biot 0.5 nabinoid levels. levels. nabinoid 1 2 DSI amplitude amplitude DSI HU210 slices slices (2.5 DSI µ M). Left, Left, M). duced by 5-s 5-s by duced * * * *** 1 2 al al complex I DSI 1 s

HU-biot 5 ### 1

receptors, µ P arization arization

M ### Hemo < 0.05, < 0.05, 2 1 ** nce of of nce µ

PSCs PSCs M µ 1 M) M) 2 ). ). 5 © 2012 Nature America, Inc. All rights reserved. experimental data demonstrate that the metabotropic metabotropic the that demonstrate data experimental of DSI. of t contributes and likely metabolism energy regulates it where of neurons, membranes on mitochondrial is present membrane (approximately 15% of total cell CB cell total of 15% (approximately membrane dniid iohnra poen ( proteins mitochondrial identified known several of values the to comparable is and GPCRs Notably, approximately 30% of neuronal mitochondria brain purified in respiration of reduction dependent seem low.seem However, this expression accounts for up MitoProt II (ref. 35) predicted the CB the predicted 35) (ref. II MitoProt S E L C I T R A 93 poaiiy f iohnra lclzto ( 2 Table localization mitochondrial of probability 39.3% CB tions. Although a minimal amount (less than 10%) of subset of subset organelles expressing mtCB a receptors, suggesting strong impact of cannabinoi mitochondrial alterations are thought to be of etio 6 6 ( postsynaptically infused when any effect produce activity has been recently proposed recently been has activity synaptic plasticity. synaptic bioenergetics, which might be involved in endocannabi by a DSI a weak induced abolish 1-s depolarization hemopressin data, these with strong DSI. Consistent ing the involvement of in the mitochondria presynaptic key roles in both physiological and pathological br a new mechanism of modulation of neuronal activity, receptor proteins are expressed in glutamatergic neurons glutamatergic in expressed are proteins receptor confirming that intracellular CB intracellular that confirming regulating synaptic plasticity and possibly behavio However, mitochondria also influence the normal fun tions o nele ay ucin o edcnaiod signaling brain endocannabinoid of functions many underlie to tionand functional characterization of brain mtCB naling naling are mainly exerted by glutamatergic-neuron C of en or properties the orexigenic totoxic seizures of limited percentage receptors. For instance, prot e are system endocannabinoid the of functions the of functional point of view.of point functional a negligible a considered be cannot membranes drial Intracellular CB Intracellular product energy and and respiration organelle’s mitochondria the in late occur to proposed recently been activ PKA and levels cAMP regulating by activity, I modulation the through exerted be may function This brai mitochondrial regulates of directly it where mitochondria, hippocampus) the in 30% (approximately tion Therefore, the 15% of hippocampal CB ence of mtCB of ence establ data these Thus, activity. I complex of inhibition mimic is function this and hippocampus, the in sion form dependent of of plasticity short-term inhibitory The present results show that the CB the that show results present The DISCUSSION DSI. of forms weak, not h pooto o CB of proportion The The hypothesis that GPCRs may directly regulate mit regulate directly may GPCRs that hypothesis The CB Neuronal bioenergetics is emerging as a key player key a as emerging isbioenergetics Neuronal 1 receptors can bear crucial functional significance 1,2,4,5 15–17 1 -dependent forms of synaptic plasticity such as DSI DSI as such plasticity synaptic of forms -dependent ). This theoretical value is much higher than that that than higher much is value theoretical This ). . This holds true for neurodegenerativefordisorders, i true holds This . Nuornmte rlae s vr energy-demandin very a is release Neurotransmitter . 1 receptors and their role in the regulation of neuronal neuronal of regulation the in role their and receptors 1 receptors are involved in DSI, an endocannabinoid- 1 receptors located on the mitochondrial mitochondrial the on located receptors 1 receptors contribute to strong, but strong, to contribute receptors upeetr Tbe 2 Table Supplementary 1 6,30 . . Moreover, a low abundance of 1 protein located on mitochon- 1 1 . The open source program program source open The . GPCR is present in a por- a in present is GPCR receptor protein to bear a bear to protein receptor pathological relevance r ain processes. 1,2,4,5 docannabinoid sig- docannabinoid ection against ection exci- 1 ctioning of the brain, 1 receptors provides receptors) might might receptors) d signaling d on signaling the which might have to to 30% of mtCB Fig. 7c Fig. ( total cortical CB B Fig. 7d Fig. was able to fully Supplementary Supplementary o the expression o expression the neurotransmis- in specific loca- noid-dependent ion ity, which have have which ity, do contain CB . The identifica- 1 mitochondria. mitochondria. mount from a from mount receptors in brain func- brain in xerted by this this by xerted expression of ked by direct direct by ked CB ish the exist- the ish of complex complex of 7,8,11,13,14,30 respiration. respiration. n neuronal neuronal n or recently recently or ochondrial ochondrial o modu- to 24,27 ), suggest- ), 1 ), further ), further are likely likely are receptor receptor of many many of n which n in the the in acutely acutely , many many , ). Our Our ). 27,28 1 4 g 1 1 - . . .

functioning in health and disease. and health in functioning unde better to frameworks theoretical new of opment and cellular regulation, energy with unforeseeable signalin GPCR between relationship the detail deeper pave will the way ronal for mitochondria new studies demonstration that CB demonstration sys endocannabinoid of the exploitation therapeutic for the pharmacol new add possibly will and functions brain signaling endocannabinoid which through mechanisms are ex involved in regulation DSI further chondrial forms of synaptic regulation and plasticity by differen plasticity and regulation synaptic of forms spatial aspects spatial on rely might they but unknown, are difference cal lized endocannabinoids. As yet, the mechanistic bas induced by exogenously applied cannabinoids or endo results suggest a mechanistic difference between the dec mitter (for example, glutamate) example, (for mitter o stimulation by signaling endocannabinoid potentiate rote of effects indirect to due be also could effect D in activity mitochondrial of involvement the gesting synaptic of form this potentiated rotenone by activity DSI, respectively. In addition, inhibition of presy occlud partially only hemopressin antagonist the and mtCB Conversely, our data clearly show that intracellula Thus, Thus, mtCB due to efficient endogenous degradation of endocanna of degradation endogenous efficient to due participate in DSI and suggest that mtCB that suggest and DSI in participate raising the possibility that mtCB one were stimulating endocannabinoid mobil indirectly synaptic depolarization did not alter inhibitory tr the application of rotenone or postsynaptically in t mobilization endocannabinoid acting at presynaptic mtCB presynaptic at acting En signaling. endocannabinoid intracellular this of neurotransmitters ong the for ATPneeded of form the in supply energy an respiration mitochondrial decreasing temporarily activity activity and neurotransmitter release calcium levels and redox potential, which can also m transmission in the hippocampus one and much are longer applicationneeded periods previous studies have shown that much higher concen its application to slices should result into alterations of eIPSCs. application of cannabinoids does not seem to involve means example, tolerance) induce might and drug, the of properties cokinetics duration their are depending long-lasting, generally lipophi of (especially applications Conversely,drug dria has also been recently proposed to has dria recently proposed been sy also modulate nabinoid signaling, which would otherwise remain si would otherwise which nabinoid signaling, so far events cellular unknown to favor additional able to endogenously mobilize endocannabinoids might trig thereby different triggering mechanisms. Finally, dep oth for preference display might cannabinoids enous C subdomains. cellular specific to limited be might synapti and/or depolarization by endocannabinoids of process The discovery that intracellular CB intracellular that discovery The Taken together, our results show that intracellular that show results our together, Taken 1 4 priiae n tog S. hs aprnl contr apparently These DSI. strong in participate ) . The membrane-impermeant CB membrane-impermeant The . 5 , and mitochondria can modulate its efficiency by d by efficiency its modulate can mitochondria and , ADVANCE ONLINE PUBLICATION PUBLICATION ONLINE ADVANCE 1 receptors might receptors to contribute DSI to and possibly o 17 . DSI is short-lasting (a few tens of seconds), likel seconds), of tens few (a short-lasting is DSI . 4,5 29 . Moreover, mitochondria regulate intracellular intracellular regulate Moreover,mitochondria . . . From the spatial point of view, the mobilization 1 receptors are functionally expressed on expressed neu- are receptors functionally 1 receptors might contribute to DSI by DSI to contribute might receptors 16 36 1 37 might interfere with these processes. . This is, however, unlikely because because unlikely however, is, This . release, which can in turn increase increase turn in can which release, . Reduction of eIPSCs by exogenous 4 . . Rapid trafficking of mitochon- 1 receptor signaling and mito- and signaling receptor 1 receptor agonist HU-biot HU-biot agonist receptor 1 NATURE NEUROSCIENCE NATURE might be the mediator mediator the be might r CB naptic mitochondrial ansmission. If roten- none, which might might which none, means means of endocan- he he absence of post- effect oneffect the devel- es of this paradoxi- tends the range tends of naptic naptic plasticity lic cannabinoids) cannabinoids) lic only on pharma- extra effects (for effects extra intracellular CB odulate odulate neuronal 1 temporal and/or and/or temporal trations of roten- onversely, exog- onversely, docannabinoids docannabinoids olarization olarization steps investigating in investigating receptors (likely to to alter synaptic lent. er subdomains, subdomains, er genously mobi- ed and blocked blocked and ed plasticity,sug- CB rease of eIPSCs oing release of of release oing t means. t d altering the the altering d g in the brain brain the in g ogical targets targets ogical c stimulation stimulation c ization ization f neurotrans- f SI. The latter latter The SI. rstand brain brain rstand binoids 1 influences influences receptors receptors ger ger other, tem. The tem. adictory adictory Indeed, ifferent ifferent per per se 15,17 ther ther 1 1 y . , , . © 2012 Nature America, Inc. All rights reserved. Post-Doc ANR-09RDPOC-006-01 (G.B.), European Union AVENIR/INSERM (G.M.), INSERM–Agence Nationale de la P.V. Piazza and C. Wotjak for reading critically th G.M.’s laboratory for discussions; and A. Bacci, D. the NeuroCentre Magendie for mouse care and genotyp We thank D. Gonzales, N. Aubailly and all the perso NATURE NEUROSCIENCE NATURE Note: Supplementary information is available on the at http://www.nature.com/nature paper of the version available are references associated any and Methods METHODS E.S.-G. E.S.-G. performed the electrophysiological studies. N.P. performed th study. G.B. performed the biochemical experiments. different parts supervised of this work. G.B., P.G. N.P., J.L. and L.B. equally contributed to experime (FOR926, B.L.) and CONACyT (E.S.-G.). Ramon y Cajal program (S.O.-G.), Deutsche Forschung MICINN P.G.;(SAF2009-07065, SAF2010-22198-C02-01, P.G.),(UFI11/41, Comunidad de Madrid (S2010/BMD-23 (GIC07/70-IT-432-07, P.G.), University of the Basqu Instituto de Salud Carlos III P.G.(RD07/0001/2001, Foundation University(E.S.-G.), of Bordeaux (J.L.) Recherche Medicale (G.M. and M.M.-L.), Region Aquit Research Council (ENDOFOOD, ERC-2010-StG-260515, G. Program (REPROBESITY, HEALTH-F2-2008-223713, G.M. a . ato, .. Gecmn, . Ceg A Mtcodi i nuolsiiy and neuroplasticity in Mitochondria A. Cheng, & M. Gleichmann, M.P., Mattson, 4. . agln SB, e utr a Seeic, .. Adro, .. h metabolic The J.C. Anderson, & R.R. Steveninck, van Ruyter de S.B., Laughlin, 5. hemopressin and performed and measured intracellular hemopressin and AM251. J M.M.-F. and synthesized and M.L.L.-R. biochemically endocannabinoids and endocannabinoid-degrading enzy M.M.-L., E.H.-C. and A.C. participated in biochemic provided forthe viruses re-expressionlocal of CB . eos A.E. Belous, 6. 1. MacAskill, A.F. & Kittler, J.T. Control of mitochondrial transport and localization in localization and transport mitochondrial of A.F.J.T.Kittler,Control MacAskill, & 1. reprints/index.html. o available is information permissions and Reprints Published online at http://www.nature.com/natureneu The authors declare no competing interestfinancial manuscript. All authors edited the manuscript. experiments. G.M. conceived the andwhol supervised of the work. P.G. the anatomical supervised studies 2. Attwell, D. & Laughlin, S.B. An energy budget for signaling in the grey matter of matter grey the in signaling for budget energy An S.B. Laughlin, & D. Attwell, 2. . ihls DG & egsn S.J. Ferguson, & D.G. Nicholls, 3. 7. Lyssand, J.S. & Bajjalieh, S.M. The heterotrimeric G protein subunit G alpha i is i alpha G subunit protein G heterotrimeric The S.M. Bajjalieh, & J.S. Lyssand, 7. 8. Andreeva, A.V., Kutuzov, M.A. & Voyno-Yasenetskaya, T.A. G alpha12 is targeted to targeted Voyno-Yasenetskaya,A.V.,is & T.A. Andreeva, alpha12 Kutuzov,M.A. G 8. ACKNOWLEDGMENTS AUTHOR CONTRIBUTIONS COMPETING FINANCIAL INTERESTS COMPETING neurological disorders. neurological 2002). cost of neural information. neural of cost n PY-ie iohnra receptors. mitochondrial P2Y2-like and neurons. the brain. the present on mitochondria. on present (2006). 2821–2831 (2008). 2821–2831 the mitochondria and affects mitochondrial morphology and motility.and morphology mitochondrial affects and mitochondria the Trends Cell Biol. TrendsCell J. Cereb. Blood Flow Metab. Flow Blood Cereb. J. t al. et in vivo iohnra clim rnpr i rgltd y P2Y1- by regulated is transport calcium Mitochondrial studies and stereotactic injections of A. viruses. Neuron

FEBS Lett. FEBS in vitro 20

Nat. Neurosci. Nat. , 102–112 (2010). 102–112 , ADVANCE ONLINE PUBLICATION PUBLICATION ONLINE ADVANCE

60 studies on cell penetration. M.K., S.G. and B.L. , 748–766 (2008). 748–766 , ieegtc 3 Bioenergetics

581

21 , 5765–5768 (2007). 5765–5768 ,

, 1133–1145 (2001). 1133–1145 , 1 , 36–41 (1998). 36–41 , . el Biochem. Cell J. , R.R. and, R.R. G.M. the designed e e manuscript. Supported by 1 Cota, M. Cota, Guzman, U. Pagotto, nts. P.G. and equally R.R. receptors. F.C. part supervised e e anatomical studies. L.B. and . R.R. supervised biochemical supervised . R.R.

, , Red de Trastornos Adictivos, Nature Neuroscience s. s. ), Basque Country ), GovernmentBasque Country nline at http://www.nature.com/ at nline nnel nnel of the Animal Facility of e Country e UPV/EHU Country F.M., J.L. and C.M. performed al al experiments. I.M. measured ocec/ roscience/. characterized HU-biotcharacterized e e study and wrote the .G. .G. generated fluorescent Aaei Pes London, Press, (Academic sgemeinschaft aine (G.M.), Fyssen ing; all the members of matic S.O.-G.,activities. 53, M.L.L.-R.), 53, M.L.L.-R.), M.L.L.-R.), M.L.L.-R.), 7 M.), Fondation pour la th Recherche (Retour nd B.L.), European Framework

neuroscience/. in the online online the in 99 1165–1174 , FASEBJ. website. D.,

22 , 3 hn R, erly IM, ekCe, .. Wle, .. h phosphorylation The J.E. Walker, & S.Y. Peak-Chew, I.M., Fearnley, R., 13. Chen, 2 y, . Le J, me, . Rtn RR & erne RJ Atoiat modulate Antioxidants R.J. Ferrante, & R.R. Ratan, S., Impey, J., Lee, H., 12. Ryu, 2 qia S. 22. Aquila, 6 ascn, G. 26. Marsicano, 21. Tedesco, L. 16. Kano, M., Ohno-Shosaku, T., Hashimotodani, Y., Uchigashima, M. & Watanabe, M. Watanabe, & Y.,T.,M. Hashimotodani, Ohno-Shosaku, 16. Uchigashima, M., Kano, 14. S. Helling, 15. Piomelli,D.The molecular logic ofendocannabi 27. Bellocchio, L. T.I.Bonner,Structure & A.C. Young, M.J., Brownstein, S.J., Lolait, 19. L.A., Matsuda, 28. K. Monory, A.I. 23. Gulyas, 25. Rozenfeld, R. & Devi, L.A. Regulation of CB1 trafficking by trafficking receptor cannabinoid CB1 of Regulation L.A. Devi, & R. 25. Rozenfeld, and ligands receptors, of distribution Anatomical R. Kuner, & G. 24. Marsicano, 11. R. Acin-Perez, 0 cnPrz R. 10. Acin-Perez, 17. Marsicano, G. & Lutz, B. Neuromodulatory functions of the . 8 atv, . Briga, .. fet f et9ttayrcnaio on delta9-tetrahydrocannabinol of Effect M.K. Birmingham, & A. 18. Bartova, 29. Pertwee, R.G. Pharmacology of cannabinoid receptor ligands. receptor cannabinoid of Pharmacology 29. R.G. Pertwee, 0 aa S De cM pa a at n h rglto o te iohnra electron mitochondrial the of regulation the in part a play cAMP Does S. 30. Papa, 0 atn BR Clua efcs f cannabinoids. of effects Cellular B.R. 20. Martin, 9. Zippin, J.H. Zippin, 9. 1 ugnue, . Mnr, . Lt, . Kumn, . A vector-mediated AAV M. Klugmann, & B. Lutz, K., Monory, S., 31. Guggenhuber, 32. J.Z. Long, 3 emn, A.S. 33. Heimann, 34. Gomes, I. 34. Gomes, 5 lrs MG & icn, . opttoa mto t peit mitochondrially predict to method Computational P. Vincens, & M.G. 35. Claros, 37. C. Costa, 36. Kilbride,S.M.,Telford, J.E.,Tipton, K.F. D & f uuis f ope I rm oie er mitochondria. heart bovine from I complex of subunits of genome in neurons. in genome mitochondrial the of DNA D-loop to binding CREB increase and PKA mitochondrial n os wie dps tsu, uce ad ie: h rl o eO, 3 MAPK, p38 eNOS, pathways. of AMPK and role the liver: and muscle, tissue, adipose white mouse in anbni1 eetr n a oeta rl o aadmd i sem uvvl and survival reaction. sperm acrosome in anandamide of role potential a and receptor cannabinoid1 aversive memories. aversive (1986). noanbni-eitd oto o snpi transmission. synaptic of control Endocannabinoid-mediated (2003). 873–884 Mol. Cell Proteomics Cell Mol. 26036–26045 (2004). 26036–26045 phosphorylation. system. of a cannabinoid receptor and functional expression in the hippocampus. the in and cerebellum hippocampus, rat the in amygdala. compartments postsynaptic and pre- the adaptor protein AP-3. protein adaptor the in neurochemistry. and circuitries 2008). Brain cord: the spinal the and and Cannabinoids brain the in enzymes regulates respiration. regulates J. Endocrinol. Invest. Endocrinol. J. (2009). 309–380 mitochondrial NADH-oxidase activity. NADH-oxidase mitochondrial transport chain in mammalian cells? mammalian in chain transport (1999). 635–664 561–564 (1990). 561–564 in distinct signaling microdomains. signaling distinct in protects against seizure-induced excitoxicity. seizure-induced against protects overexpression of CB1 cannabinoid receptor in pyramidal neurons of the hippocampus regulated by endocannabinoid crosstalk in vivo. in crosstalk endocannabinoid by regulated receptors. 20270–20275 (2009). 20270–20275 ciiy nrae C-needn guaae ees rts rm depolarized from rates release glutamate synaptosomes. Ca-independent increases activity molecules. imported proteins and their targeting sequences. targeting their and proteins imported nue b mtcodil ope I inhibition. I complex mitochondrial by induced (1996). (2008). Nat. Neurosci. Nat. et al. et Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. et al. et et al. et Eur. J. Neurosci. J. Eur. et al. et AAPS J. AAPS et al. et al. et t al. et et al. et t al. et et al. Electrophysiology and pharmacology of striatal neuronal dysfunction neuronal striatal of pharmacology and Electrophysiology et al. et J. Neurochem. J. t al. et t al. et -derived as novel type of bioactive signaling bioactive of type novel as peptides Hemoglobin-derived Dual blockade of FAAH and MAGL identifies behavioral processes behavioral identifies MAGL FAAHand of blockade Dual Phosphorylation and kinetics of mammalian cytochrome c oxidase. c cytochrome mammalian of kinetics and Phosphorylation Cannabinoid receptor stimulation impairs mitochondrial biogenesis t al. et The endocannabinoid system controls key epileptogenic circuits epileptogenic key controls system endocannabinoid The Cell Metab. Cell Compartmentalization of bicarbonate-sensitive adenylyl cyclase adenylyl bicarbonate-sensitive of Compartmentalization ergto o to noanbni-yrlzn ezms into enzymes endocannabinoid-hydrolyzing two of Segregation ua sem ntm: lrsrcua lclzto o the of localization ultrastructural anatomy: sperm Human Bimodal control of stimulated food intake by the endocannabinoid

Anat. Rec. (Hoboken) Rec. Anat. Nature Cyclic AMP produced inside mitochondria regulates oxidative regulates mitochondria inside produced AMP Cyclic 12 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. h edgnu cnaiod ytm otos xicin of extinction controls system cannabinoid endogenous The

popoiseae A sfr lclzd o mitochondria to localized isoform 2A phosphodiesterase A

Neuron 7 J. Biol. Chem. Biol. J. 29 Diabetes eorsi i a ivre gns o C1 cannabinoid CB1 of agonist inverse an is Hemopressin

, 1714–1724 (2008). 1714–1724 , , 658–669 (2010). 658–669 , 13 , 27–46 (2006). 27–46 ,

, 281–283 (2010). 281–283 , FASEB J. FASEB 418

20

9 51

e. oav, . 1122 Srne, e York, New (Springer, 161–202 A.) Kofalvi, (ed. 106

, 265–276 (2009). 265–276 , , 441–458 (2004). 441–458 , , 530–534 (2002). 530–534 , 59 , 455–466 (2006). 455–466 , , 2826–2836 (2010). 2826–2836 , , 826–834 (2008). 826–834 ,

FASEB J. FASEB

22 286 IUBMB Life IUBMB

J. Biol. Chem. Biol. J. 104 , 2311–2322 (2008). 2311–2322 , , 30423–30432 (2011). 30423–30432 ,

293 , 20588–20593 (2007). 20588–20593 , avey,G.P. Partialinhibition complexofI

PLoS ONE PLoS noidsignalling. , 298–309 (2010). 298–309 , 17

102 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. , 82–84 (2003). 82–84 ,

Eur. J. Biochem. J. Eur. of the cloned cDNA. 58 . Neurosci. J. hrao. Rev. Pharmacol.

, 13915–13920 (2005). 13915–13920 , , 173–175 (2006). 173–175 , 251

5 , 5002–5006 (1976). 5002–5006 , , e15707 (2010). e15707 , S E L C I T R A Nat.Rev. Neurosci. . il Chem. Biol. J. Curr. Med. Chem. Med. Curr.

hso. Rev. Physiol. 28

241 8040–8052 ,

Nature 38 , 779–786 , 45–74 ,

279 106 346 89

6 4 7 , , , , , , © 2012 Nature America, Inc. All rights reserved. Synthesis of and CB and of Synthesis same amounts of solvents (0.1–0.2% ethanol; 0.1% DM Vehicles the containedPBS. inhemopressins DMSO; in below) (see HU-biot WIN55,252 ethanol; in dissolved was THC Scientific. respectively) were as described condensation between HU210 (ref. 40) and 40) (ref. HU210 between condensation GABA- Drugs. 22 male mice were used. Experimenters were blind to genotypes and treatment acid in the presence of dicyclohexylcarbodiimide, l o bt sxs ee sd o electrophysiology. for used were sexes both of old mice (2–4 months old) were used for anatomy and bio Female conditions.standard under maintained were Mice 3306369). number of INSERM and the French Ministry of Agriculture an and doi:10.1038/nn.3053 Mice. ONLINE METHODS roomtemperature) roomtemperath,incubated and (2 (2-carboxyethyl)phosphinewe hydrochloride(Pierce) ml mg 5 Co.). Bio (CS standard Fmoc solid-phase synthesis chemistry on a CS Synthesis of fluorescent hemopressin. previously described Purity (elemental analysis) > 95%. The CB The 95%. > analysis) (elemental Purity column chromatography. Spectroscopic data were consi The peptide was incubated with 1 × 10 hemopressin was determined using CHO-K1 cells expre CB DMSO and purified and analyzed with preparative HPLC rescein-5-maleimide(Pierce) to the cysteine. The p one-stea bydone was peptide the tofluorescein of SDS-PAGE following Coomassie brilliant blue G-250 sta peptide–protein carrier conjugate was purified, conc imidomethyl)-cyclohexane-1-carboxylate (SMCC) cross solutionof maleimide-activated ans mcKLH carrying (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl total CB (one-site binding). The apparent no byanalyzed were Concentration curves cells.response determined after washing and subtracting of signals (TecanFarcyte, 485-nm excitation and 535-nm emissi bindingbuffer, fluorescence intensities were measu plastic tubes (30 min, 30 °C). After centrifugation muoyohmsr fr lcrn microscopy. electron for Immunocytochemistry scope pre-embedding immunogold labeling as recently NormalizedmtCB mitochondria, in density of mtCB differenNoelements. dendritic with synapses dria, used in 50- with mtCB1 versus total asymmetric or symmetric synapti littermate type controls. Normalizedasymmetric CA1 for each parameter, one-way ANOVA), allowing the poo where Semiquantification of mtCB1. AKAP 149/121, Santa Cruz Biotechnology). GABA- Go-Af450-1, FrontierGo-Af450-1,Ltd); Co.12goatAKAP Science dria. MtCB determined the proportion of hippocampal CA1 CB observed between the wild-type littermates of the d 1 binding assay with fluorescent hemopressin. N Experiments were approved by the Committee on Anim 42 -hydroxybenzotriazolecouplingas reagents (yield: Drugs were from Sigma, Tocris, Abbott Laboratories Abbott Tocris, Sigma, from were Drugs CB CB . The number of labeled mitochondria normalized to 1 . Axon terminals identification: abundant synaptic ves 1 1 µ 1 −/− −/− labeling density was calculated on the area of labeled m-thick hippocampal sections: goat CB mice (elsewhere called and 1 CB labeling versus total CB 41 1 1 . binding assay for HU-biot. for assay binding −/− −1 hemopressin in PBS (pH 7.2) containing 5 mM tris tris mM 5 containing 7.2) (pH PBS in hemopressin mice ( 26–28,38,39 The detailed procedure has been described else- K n 1 = 3 each) were processed for electron micro- d or proportion of mtCB value for the CB CB 5 cells suspended in 200 Rat hemopressin was synthesized using . C57BL/6N were from Janvier (France). 1 -KO, Glu- 1 1 showed mtCB binding assay was performed as performed was assay binding N 2 -(+)-biotinyl-6-aminohexanoic , 0.5% BSA, pH 7.4) in silanized and three washes with ice-cold The N CB 1 ifferent mutant mice ( 1 red96-wellain plate reader -immunopositive mitochon- from untransfected control roductwas reconstituted in 1 antiserum (2 p procedurecouplingfluo-p , ce in percentage of labeled percentageof in ce HU-biot was prepared by prepared was HU-biot entrated and analyzed with N receptor was 119 s. A total of 312 female and idtp, Glu- Wild-type, 1 K on).Specific binding was d Forestry (authorization -dimethylaminopyridine -KO and GABA- CB ssing human CB chemistry. Mice 20–30 d 336S Peptide Synthesizer or symmetric terminalssymmetricor uccinimidyl-4-( re incubated (20 min at min (20 incubated re SO). 1 antiserum(2 1 linker (Pierce). The final described stent with the structure. d -2 (WIN), HU210 and HU210 (WIN), -2 ining. The introduction ure) with a 10 mg ml mg 10 a withure) ling of respective wild- value of the fluorescent 1 and ESI-MS. 1 32%) and32%)bypurified c boutons containing −/− versus total CB nlinear curve fitting curve nlinear 1 total mitochondria proportion versus al Health and Care Glu- , µ icles, mitochon- Inc. or Ascent or Inc. l binding buffer mitochondria. 42 µ g ml . Antibodies CB ± 1 CB µ 1 N −1 (hCB −/− 11 nM. CB P g ml g -male- ; CB1- > 0.6, 1 1 and -KO, 1 was −/− −1 1 −1 ). ; ,

Glu- mitochondria revealed the percentages of axon termin ated from P0–P1 transfection. and culture Cell death, the brains or hippocampioforbrains the death, in a 2-mm-gap cuvette using 30 using cuvette2-mm-gap a in Cell Cell were electroporated in Optimem medium (Invitrog (Harvard apparatus, 175V, 1-ms pulse, five pulses, Transfectionsflasks. wereoutcarriedB by usinga sected sected and mitochondria were purified as previously d Trypsin and digitonin assays. Mitochondrial purification from mouse hippocampi or hippocampi mouse from purification Mitochondrial resuspended in culture medium and experiments were penicillin/streptomycin solution (Invitrogen). Cells were seeded onto 25-cm onto seeded were Cells (Invitrogen).solutionpenicillin/streptomycin 2% and l-glutamine 1% FBS, 10% with DMEM in resuspended and ugation solution. Cell trypsin 0.25% incubated in thenwith All samples were centrifuged (12,000 ature). The reaction was stopped by adding protease Trypsin digestion (0.1% w/v) wasroo w/v,performed (0.25%, fordigitonin 0, with 5 not or treated were cadherin (Santa Cruz, overnight, 4 °C). The presenc 1 h, room temperature), synaptophysin (Millipore, 2 4 °C), calreticulin (Santa Cruz, overnight, 4 °C), K GAPDH againstrespectively antibodies using tigated reticulum (ER), synaptosomes and plasma membrane co temperature)and Tom-20 (Santa overnight,Cruz, ° 4 immunodetected Mitochond using antibodies (0.05%). against complex PBS-Tween20 III in milk 5% in soaked 4–16% acrylamide gels and transferred to PVDF membra THC were performed at 30 °C for 1 h. to the manufacturers’ instructions. Reactions in pre (Assay Designs Inc) and an ELISA kit (Enzo Life Sci were assayed on isolated mitochondria using the Direct CyclicandPKAAMP activityassays. by the Bio-Rad Quantity One system. HRP signal was revealed using the ECL plus reagent bated with secondary HRP-coupled antibodies (1 h, r receptormembranesThen(Cayman, overnight,°C). 4 fractions was analyzed using an antiserum directed to the chamber. A coupled respiratory state was obt chamber. Respiratory substrates (10 mM pyruvate nitol,and 25 mM sucrose, 10 mM KCl, 10 mM Tris-HCl,mitochondria pH (1 mg) were suspended in oxygenchamberClarkglasselectrequippeda1 awith ml of the r Oxygenconsumption assays. in sample buffer and analyzed by western blot. experiments ( w omitted) DTT (beta-mercaptoethanolconditionsand ~ for boiled and DTT mM 50 beta-mercaptoethanoland were Samples buffer. sample in diluted and (Roche) Immunoblotting. reaction of NADH oxidation into NAD permeabilized with digitonin (20 respiratorywith substratespyruvate mM (10 andmal was recorded. For experiments with permeabilized ce t in directly transferredwere cells Intact medium. NADH-ubiquinone oxidoreductase (complex I) activity by adding the drugs directly to the chamber. The experiments using MFs were performed on 2 × 10 The effects of hemopressin, WIN, JZL195, HU210 and H CB 1 −/− and GABA- Supplementary Fig. 3b CB1 All samples were supplemented with proteases inhib proteases with supplemented were samples All −/− CB mice. Dorsal skin was excised and minced in PBS an 1 −/− Oxygen consumption monitoredwas in °Cat30 Mitochondria diluted in isolation buffer (100 mice. Mouse primary fibroblasts (MFs) were gener- were (MFs) fibroblasts primary Mouse µ µ g of DNA. After electroporation, cells were cellselectroporation, AfterDNA. of g g ml CB g ). Proteins were separated on Tris-glycine , 4 °C, 10 min). Each pellet was suspended + Cyclic activityAMPand PKA activities 1 −1 by complex I as previously described −/− ). and wild-type littermatesdis-were wild-type and 0.5-s interval between pulses). espiration buffer (75 mM man- ained by adding 2 mM ADP. DEL (ER; Enzo Life Sciences, e of the CB ence), respectively, according TX electroporator TX 830 ECM , 10 or 15 min (room temper- against the C terminus of the NATURE NEUROSCIENCE NATURE he chamber and respirationand chamber he sence or absence of WIN or s werebycentrif- collecteds lls, MFs were supplemented malate) were added directly inhibitor cocktail (Roche). oom temperature). Finally, (Amersham) and detected h, room temperature) and ode (Hansatech).ode Purified m temperature, 15 min). min). 15 temperature, m C). Cytosol,C). endoplasmic (Mitosciences, 2 h, room ate) and 2 mM ADP andADPate)mM and2 7.4, 50 mM EDTA) in the escribed (Santa Cruz, overnight, Cruz, (Santa Correlate-EIA cAMP kit als containing mtCB1 in ntaminations were inves- performed 3 d later. supplemented with 2% 2% with supplemented were washed and incu- andwerewashed en) at 2 × 10 assay. ere also used in some in used also ere nes. Membranes were 6 U-biot were analyzed 5 min. Nonreducingmin. 5 cells ml whole brain. whole il rtis were proteins rial 1 in mitochondrial 43 We followed the . −1 7 in growth cells ml After itors µ 43 g) −1 d 2 .

© 2012 Nature America, Inc. All rights reserved. NATURE NEUROSCIENCE NATURE Purification and quantification of endocannabinoids of quantification and Purification mides. previously described as performed were mitochondria purified and fraction membrane total brain the from and (2-AG), Fatty acid amide hydrolase and lipase monoacylglycerol and hydrolase amide acid Fatty reliable quantification. Therefore, only data relating t tively. Anandamide was detected in the samples, but calibration curve and were expressed as nmol or pmol p . mi mmol mCi 5.0 (50 mitochondria tota purified or brain using by detected were activities MAGL or (20 min, 37 °C) for FAAH and MAGL, respectively. Th 50 mM Tris-HCl, pH 9 (30 min, 37 °C) for FAAH and i 1.0 mCi mmol 2 volumesCHClof2 t ofextractionaqueousafter countingphase the of FAAH)[ or was dried under a stream of argonstreamofreconstitutedand a under dried was −2.0 mm DV; −3.5 mm AP, Tris-HCl, pH 7.4, 2.5 mM EDTA and 5 mM MgCl mM 5 EDTAand mM 2.5 7.4, Tris-HCl,pH tively). Animals were used for experiments five wee (HA)-tagged rat CB (HA)-taggedrat Viral rescue of CB1 expression in the hippocampus. pmol of hydrolyzed anandamide or 2-AG per min per mg CHO-K1 cells wereincubated10CHO-K1cells with and determination of genomic titers were as describ as were titers genomic of determination and intoAAVanGFP) expression cassette,packaging of Cellular localization of fluorescent hemopressin. fluorescent of localization Cellular different times ( 150 nl min to corresponding standards. Fractions were quantified by measuring the area unde B (95:5 methanol:water) with 0.1% ammonium hydroxid ESI source in SIM mode. The mobile phases were A (9 1200LC-MSDinstrumentVL anEclipsewithXDB-C18 co 100 argon of stream a under dried was phase aqueous the and the organic and aqueous layers separated by cen centrifuged, Dounce-homogenized in chloroform:metha (sameconditions electrophysiologyinas experiment and 50 50 and spectrography. chro liquid by AM251 hemopressinandMeasurement of D software. Nikon TS100 fluorescence microscope and video camera beforeincubation.NP-40PBSIma 0.5% in with lized was measured by flow cytometry (FACScan, Cytek). Som detached with trypsin, and fixed with 0.01% formald AAV-GFP or AAV-CB1 (6 × 10 µ L H Purification and quantification ofanandamide, 2-a µ 2 L were analyzed by LC/MS. LC/MS was carried out in in out carried was LC/MS LC/MS. by analyzed were L O, and 60 −1 44 3 into the dorsal and ventral hippocampus (−2.0 mm A . The amountsThe anandamide. of 2-AG andweredetermine H]glycerol (for MAGL) produced were measured by sci by measuredwere produced MAGL) (forH]glycerol −1 Slices previously incubated with 10 −1 Supplementary Fig. 4a–c , 25 , 1.8 1.8 , 3 µ 1 /CH µ receptor(AAV-CB L were analyzed by LC/MS. For AM251, the organic ph M, ARC Inc.) as substrates. Reactions were carried ou µ , R Ic) r 2-oleoyl-[ or Inc.) ARC M, 3 OH (1:1 v/v). Enzymatic activities wereexpressedEnzymatica activitiesv/v). (1:1 OH ± 3.4 mm ML, −3.0 mm DV from bregma, respec- µ 11 g protein), and [ and protein), g viral genomes ml µ M fluorescentM hemopressinmM 50 in 1 ) or green fluorescentprotein(AAV-green or ) ) in incubation buffer plus 0.5% BSA, 14 trifugation. For hemopressin, ehyde (10 min). Fluorescence −1 he incubationhemixture with µ C]anandamide (for FAAH,(for C]anandamide ks after viral injection 2 1 × 10 × 1 ) was injected bilaterally at M hemopressin or AM251 o 2-AG are presented. at levels below the limit of Cloning of hemagglutinin (30 min, 30 °C), washed °C), 30 min, (30 s) weres) withwater,rinsed in 100 in AAV1/2vectorschimeric 3 r the peak and compared ges were acquired with a acquiredwerewithges e [ ed 5:5 water:methanol) and ]lcrl fr MAGL, (for H]glycerol er mg of protein, respec- n Tris-HCl buffer, pH 7 e and 0.1% formic acid. using the NIS Elements 14 and nol:water (2:1:1; 2 ml), 31,45 e cells were permeabi- and reconstituted in in reconstituted and l membrane fraction fraction membrane l protein. C]ethanolamine (for rachidonoylglycerol lumncoupled anto matography/mass 6 . One . µ hCB N L of acetonitrile,of L P, -acylethanola- assays. ± 1 2.0 mm ML, µ -expressing n Agilent an l of either of l ntillation d using a usingd FAAH 31 . t in ase s 8 ascn, G. 38. Marsicano, t ferent drugs on DSI. In some experiments ( experiments some In DSI. on drugs ferent 9 ooy K. 39. Monory, Electrophysiology. 44. Lourenço, J., Matias, I., Marsicano, G. & Mulle, C. Pharmacological activation of activation Pharmacological C. Mulle, & G. Marsicano, I., Matias, J., 44. Lourenço, calculated as described voltage step from −70 to 0 mV applied to the postsy was then applied every 10 min after drug application applied and patched cells were left undisturbed. Th Th 0). (time recorded was baseline stable a and s 3 afferent stimulation as during DSI protocols (see b rotenone on basal inhibitory synaptic transmission usin (2.5 ent data points GraphPad software (version 5.0). Results were express analyses. Statistical intracellular solution in the patch pipette. 40. Lander,R., Mechoulam, University,N., individual, the of Synthesis J. Zahalka, & A. 1 atnCue L, atnFneh, . Cplcho S, óe-orge, .. & M.L. López-Rodríguez, S., Capolicchio, M., Martín-Fontecha, L., 41. Martín-Couce, 2 une N. 42. Puente, no effect on following recorded DSI (not shown). wereaspreviously described depolarization protocol e starting before checked was expressionlayer.DSI 46. J. Lourenço, 43. G. Benard, tion positioned in the stratumpositionedtionthe in radiatum, 100 were evoked using a patch pipette filled with a HEPE reachingwhole-cell configuration for infusion of i 10–15 s. To determine the effects of HU210, HU-biot, In other experiments ( experiments other In obtained, was baseline stable a after and,patched CA1 A min. 30 forvehicle) (or drugs with incubated 45. Klugmann,M. ANOVA (followed by Bonferroni’s of postsynaptic rotenone on DSI was studied by addin and a new DSI protocol was applied after 15 min of b obtained a stable baseline, a DSI protocol was appl pairwise schedule was used: CA1 pyramidal neurons were -test, one-way ANOVA (followed by Newman-Keuls Twodifferent treatmentevalua to schemeswere used For DSI, IPSCs were evoked 3 s,every and DSI was i To determine the effects of HU210 (500nM), HU-biot ( excitotoxicity. Delta(9)-tetrahydrocannabinol in mice. in Delta(9)-tetrahydrocannabinol ant rcpos rvs noanbni mobilization. endocannabinoid drives receptors kainate hraooial dsic, nnimr o a erhdoanbnl derivative. tetrahydrocannabinol a of TetrahedronAsymmetry enantiomers distinct, pharmacologically the study of cannabinoid receptors. cannabinoid of study for the probes chemical endocannabinoid-based of Development S. Ortega-Gutiérrez, extended amygdala. extended Am. J. Physiol. Cell Physiol. Cell Physiol. J. Am. Neurosci. rats. adult in activity seizure and cognition affect differentially proteins signaling at GABAergic synapses. GABAergic at signaling 3243–3248 (2011). 3243–3248 µ M) on basal inhibitory synaptic transmission, IPSCstransmission,synapticinhibitory basal on M)

28 et al. et t al. et t al. et ± et al. et , 347–360 (2005). 347–360 , Science s.e.m. Data were analyzed using a paired or unpair etal. t al. et Physiological diversity of mitochondrial oxidative phosphorylation. oxidative mitochondrial of diversity Physiological Preparation of hippocampal slices and recording of Synaptic activation of kainate receptors gates presynaptic CB(1) presynaptic gates receptors kainate of activation Synaptic oyoa atvto o te noanbni sse i the in system endocannabinoid the of activation Polymodal eei dseto o bhvorl n atnmc fet of effects autonomic and behavioural of dissection Genetic All graphs and statistical analyses were performed using performed were analyses statistical and graphs All AAV-mediated hippocampal expression ofshort and l B cnaiod eetr ad ndmn dfne against defense on-demand and receptors cannabinoid CB1 Nat. Neurosci. Nat. 46 46 Fig. 7a Fig.

302 .

. Control experiments showed that this ‘initial’ DS 1 , 315–318 (1990). 315–318 , , 84–88 (2003). 84–88 , 28,46

291 – c . After. reaching stable baseline (~10minafter and , C1172–C1182 (2006). C1172–C1182 , Nat. Neurosci. Nat. post hoc

14 J. Med. Chem. Med. J. Supplementary Figs. 6a Figs. Supplementary , 1542–1547 (2011). 1542–1547 , PLoS Biol. PLoS test), as appropriate. Fig. 6a Fig. µ m from the CA1 pyramidalCA1from cellthe m

elow), IPSCs were evoked every 13

ied. Then, drugs were applied 5 naptic pyramidal neuron and ntracellularsolution), IPSCs

a DSI protocol was applied.wasprotocol DSI a e same stimulation protocol 54 aseline recording. The effect , 197–204 (2010). 197–204 , , e269 (2007). e269 , en, drugs (or vehicle) werevehicle) (or drugs en, , ach experiment with a 5-s a with experiment ach c s up to 30 min. S-based extracellular solu- hemopressin (10 g the drug (2.5 ed as means of independ- post hoc , 5265–5269 (2011). 5265–5269 , pyramidal cell was then was cell pyramidal and g the same conditions of nduced by a 1-s or a 5-s te the effect of the dif- theof effect the te 500nM) and rotenone patched and, after we 7d doi:10.1038/nn.3053 were evoked every every evoked were ), slices were first were slices ), . Neurosci. J. test), or two-way – c and ed Student’s ongHomer 1 µ M) to the µ 7b Mol. Cell Mol. M) and eIPSCs

I had , c ), a ), 31 ,