Mitochondrial CB1 Receptors Regulate Neuronal Energy Metabolism

© 2012 Nature America, Inc. All rights reserved. Foundation, Foundation, Rome, Italy. Australia. Germany. Chemistry, Complutense University, Madrid, Spain. Leioa, Spain. la plasticité neuronale, U862, Bordeaux, France. Bordeaux, Maladies Rares: Génétique et Métabolisme (MRGM), EA 4576, Bordeaux, France. 1 understood poorly remain physiology, neuronal and activity mitochondrial coupling mechanisms the and neurons, in cascade signaling cAMP–PKA intramitochondrial the production and respiration energy mitochondrial regulating proteins, eventually mitochondrial of phosphorylation and PKA of activation to leading (PKA) A kinase cyclase adenylyl such as soluble of of effectors downstream G potential protein localization signaling, teins pro G contain mitochondria that evidence consistent now is there ronal energetics and function is still a matter of speculation on neuronal mitochondria and their potential ability to modulate neu protein families controlling neuronal activity. The presence of GPCRs documented. poorly still are functions brain to activity intensive of focus the research is pathology and physiology brain in getics functions brain of regulation functions cell eukaryotic of elements key are mitochondria supplies, energy cellular regulating and Ensuring but it up consumes to 20% of the body’s production energy resting mammals, in weight body total the of 2% only represents brain The receptor signaling in the brain. Thus, mtCB suppression of mechanisms depolarization-induced contributed to inhibition in endocannabinoid-dependent the hippocampus. complex I enzymatic activity and respiration in neuronal In mitochondria. addition, CB intracellular receptors, exogenous cannabinoids and mitochondria (mtCB its functions. Here we show that the type-1 cannabinoid receptor (CB The mammalian brain is one of the organs with the highest energy demands, and mitochondria are key determinants of Luz López-Rodríguez María Klugmann Matthias Hebert-Chatelain Etienne Soria-Gómez Edgar Giovanni Bénard metabolism Mitochondrial CB t r a 558 Received 11 November 2011; accepted 20 January 2012; published online 4 March 2012; corrected online 27 March 2012 (details online); INSERM, INSERM, Neurocentre Magendie, de Physiopathologie la plasticité and neuronale, Endocannabinoids U862, Neuroadaptation, Bordeaux, France. G protein–coupled receptors (GPCRs) represent one of the largest one of largest the represent (GPCRs) receptors G protein–coupled

7 , 8 . . Moreover, several reports have shown the intramitochondrial C I 8 1 9 Translational Neuroscience Facility, Department of Physiology, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, , Institute Institute of Biochemistry and Molecular Medicine, Bern, Switzerland. 4 , 5 10 5 , but the molecular mechanisms linking mitochondrial mitochondrial linking mechanisms molecular the but , e l Laboratoire Laboratoire Physiologie Cellulaire de la Synapse, Centre National de la Recherche Scientifique UMR 5091, Bordeaux, France. 1 , 11 receptors directly modulate neuronal energy metabolism, revealing a new mechanism of action of G protein–coupled , 13 1 s 2 , . Thus, cAMP can be produced in mitochondria, mitochondria, in produced be can cAMP Thus, . 1 4 . However, the upstream mechanisms regulating regulating mechanisms . However,upstream the 11 1 1 –3 ), ), where it directly controls cellular respiration and energy production. Through activation of mtCB These These authors contributed equally to this work. should Correspondence be addressed to G.M. ([email protected]). 1 , 7 11 , 3 , 8 , , 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 Department Department of Faculty Neurosciences, of Medicine and Dentistry, University of the Basque Country UPV/EHU, in in situ , 7 3 Institute Institute of Chemistry,Physiological University Medical Center of the Johannes Gutenberg University, Mainz, 1 that are crucial for the the for crucial are that 1 , 3 receptors regulate neuronal energy , , Anna Delamarre 1 , 3 endocannabinoids decreased endocannabinoids cyclic AMP protein concentration, kinase A activity, , 11 10 , , Nagore Puente , 1 1 and protein 4 6 7 , Rodrigue Rossignol , Rodrigue . However, , Beat Lutz , Beat 3 , 5 , Silvia Ortega-Gutiérrez , Silvia 1 , 2 - - - . 10 Present Present address: European Brain Research Institute, “Rita Levi-Montalcini” 1 , evidence evidence points also to the ability of CB were of alterations membrane properties to nonspecific neurons ascribed on cannabinoids lipophilic of effects mitochondrial GPCRs membrane plasma typical as receptors cannabinoid of functions mitochondrial affect juana)-derivative cannabinoid functions and metabolism activity, neuronal control tightly they where branes, showed that CB that showed CB the lacking constitutively mice mutant using by signal this of specificity the investigate to us prompted tion This observa labeling. background nonspecific considered generally are they but experiments, microscopy electron in mitochondria nal that CB membranes endo intracellular or somal lysosomal in signal functionally to shown been have tors to the regulation of endocannabinoid metabolism contribute compartments also intracellular different but Indeed, cells. inside membranes, plasma at only not occur might interactions lipophilic nature of most cannabinoids tissues non-neural peripheral in biogenesis mitochondrial 3 7 , , Mathilde Metna-Laurent , , Jürg Gertsch 4 CB The The present study stemmed from the neuroanatomical observation , , Joana Lourenço 1 ) ) is present at the membranes of mouse neuronal 1 VOLUME 15 | NUMBER 4 | APRIL 2012 APRIL | 4 NUMBER | 15 VOLUME receptors are GPCRs highly enriched in neuronal plasma mem 3 1 Université Université de Bordeaux, Neurocentre Magendie, de Physiopathologie receptor immunogold particles are on receptor particles often immunogold detected neuro 15– 1 2 7 , . . Early studies suggested that the 3 & Giovanni Marsicano & Giovanni 1 receptors are present on neuronal mitochondrial mitochondrial neuronal on present are receptors 9 , Francis Chaouloff , Francis 6 , , Mar Martín-Fontecha 1 , 3 , 5 , 10 ∆ , , Luigi Bellocchio 2 1 9 5 8 -tetrahydrocannabinol (THC) could . . However, with the identification identification the with However, . 1 , 1 3 receptors and mitochondrial , , Astrid Cannich 1 1 5 receptor signaling to regulate implies that receptor

1 nature nature 1 1 receptor , , 3 Cannabis sativa 3 , 6 doi:10.1038/nn.305 Department Department of Organic

23 , 2 6 2 4 , NEUR Université Université de , , and CB 1

2 , 3 6 . The results results The . , 1 1

OSCI 3 2 21 0 , . . Recent 1 – , 2 (mari recep ligand ligand 2 . The The . EN 15 , 3 C 1 9 E ------, © 2012 Nature America, Inc. All rights reserved. membrane; mat., matrix; Tom20, translocase of outer mitochondrial membranes 20 kDa; CIII, core 2 protein of mitochondrial complex III. Values, Values, III. complex mitochondrial of 2 protein core CIII, kDa; 20 membranes mitochondrial outer of Tom20, translocase matrix; mat., membrane; 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 mtCB of Resistance mitochondria. CB of assay digitonin ( bar,nm. 200 Scale membrane. mitochondrial outer the on particles 3c Figure in shown are blots Full-length and WT from mitochondria and (WT) wild-type from (TTL) lysate tissue total brain whole in ( CB 35.7 area, (WT,mitochondrial total mitochondria of subset immunopositive the CB of n (WT, mitochondria total of a proportion as (mito) ( nm. 200 CB specific Arrows, mitochondria. single of details Insets, ( wild-type of region hippocampal CA1 the in neurons of membranes CB of detection ( mitochondria. 1 Figure or N-terminal the that with suggesting trypsin presolubilization, digitonin with without treated mitochondria purified of noblotting ( mitochondria of mtCB of 95% approximately 3 Fig. ( mitochondria brain wild-type purified of CB presence the specific confirmed blotting CB total 15.5% that revealed quantifications Further 0.4 to dropped and labeling was 18.3 wild-type ( in mice compartments somato-dendritic and terminals axon ling ( in mitochondria of CA1 0.7% CB displayed mitochondria and 1a Fig. (mtCB mice wild-type of neurons pal hippocam CA1 of membranes mitochondrial on microscopy tron CB CB RESULTS functions. neuronal specific and metabolism neuronal energy providing the a hippocampus, link between possible cannabinoid-dependent regulation of short-term synaptic plasticity in in endo participate activity mitochondrial of presynaptic inhibition CB intracellular of stimulation Furthermore, getics. membranes, and their direct activation ener regulates mitochondrial nature nature mean e = 458 mitochondria from 3 mice). ( 3 mice). from mitochondria = 458 ) Immunoblotting of CB of ) Immunoblotting 1 1 1 −/− receptor immunoreactivity was detected by immunogold elec immunogold by detected was immunoreactivity receptor receptor protein is localized on neuronal mitochondria Supplementary Fig. 1b Fig. Supplementary n 1 Fig. Fig. 1 ± = 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 ). Semiquantification of immunogold images revealed that that revealed images immunogold of Semiquantification ). Supplementary Fig. 2a Fig. Supplementary s.e.m. *** s.e.m. ), but not not but ),

1 NEUR CB . . ( Supplementary Figure 4 Figure Supplementary protein was localized to mitochondria. Western immuno Western mitochondria. to localized was protein c ) ) CB 1 f c ) Detail of CB of ) Detail 1 receptor localization. Scale bar, Scale localization. receptor ). ). MtCB receptors in neuronal neuronal in receptors a OSCI 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. 1 Fig. 2.0 per particles 1 ) and ) and EN ± 1 receptor expression in expression receptor 1 receptors were between comparably distributed 0.1 particles per per particles 0.1 −/− C −/− 1 E 1 mice and purified purified and mice receptor protein protein receptor f

CB receptor gold gold receptor CB ). This was confirmed by Western immu Western by confirmed was This ). (here called called (here VOLUME 15 | NUMBER 4 | APRIL 2012 APRIL | 4 NUMBER | 15 VOLUME 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; OMM, outer mitochondrial membrane; IMM, inner mitochondrial mitochondrial inner IMM, membrane; mitochondrial outer OMM, 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. 1 Fig.

Fig. 1 Fig. 1 −/− 2

in in 1 −/− , ± 4.2% of CA1 neuronal neuronal of CA1 4.2% CB a e 1 ) littermates ( littermates ) and and immunoreactivity in immunoreactivity and and 1 a e c −/− CB1-labeled Supplementary Supplementary Supplementary Supplementary

mito/total mito (%) ( mice 1 1 10 20 30 40 receptors and and receptors immunogold immunogold 0

WT TTL WT

CB 1 Fig. –/– 1 Fig. 1 *** den

CB TTL d 1 –/ ter ). ). ± b ------–

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per m positive mito) m dria dria of presumably inhibitory symmetric synapses in Glu- GABA- in excitatory) CB (presumably synapses asymmetric bearing ( sion observed concerning the proportion of mtCB ( mutants mtCB ( mice wild-type in that to rela tive mitochondria labeled of percentage the in reductions similar ( strains mutant both tively (Glu- gic lacking mice mutant conditional mtCB of distribution anatomical behavior and functions brain affects tially neurons glutamatergic excitatory and GABA containing neurons inhibitory include These where they negatively regulate excitability and neurotransmission mitochondria. neuronal of surface at the present is protein 4 Fig. Supplementary likely located in the intermembrane mitochondrial space ( of portion mtCB µ mitochondrial reduced weight) body (intraperi kilogram per mice mg 5 C57BL6/N toneally, to THC of administration acute The MtCB ones. glutamatergic in than neurons GABAergic hippocampal mtCB ( 10 15 20 25 Supplementary Fig. 2b Fig. Supplementary 0 5 CB 1 −/− CB WT Mito 27 1 1 Fig. 2 Fig. 1 m 1 1 receptors are expressed in different neuronal populations, populations, neuronal different in expressed are receptors was reduced by 50.4% in GABA- mice, whereas it was almost exclusively present on present mitochon exclusively it was almost whereas mice, receptors, which are, however, more densely expressed in in expressed densely more however, are, which receptors,

–/ CB , directly regulates brain mitochondrial activity mitochondrial brain regulates directly m 2 –

–/– 8 ***

1 CB . MtCB . Fig. 2 Fig. e 1 f ). As expected, most mtCB most expected, As ). −/− d ) 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 is was still present in CA1 hippocampal neurons of neurons hippocampal CA1 in present still was Cytosol IMM Fig. 2a Fig. 2 ). Thus, a substantial portion of CB of portion substantial a Thus, ). 4 b , where activation of CB activation , where ). Thus, both neuronal populations contain contain populations neuronal both Thus, ). OM , b M and and den Fig. 2 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 c 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 −/− m CB , 1 17 versus total CB , , but not in Glu- , 1 0 1 27 −/− 1 , m 2 receptors differen receptors 8 t r a ) neurons, respec neurons, ) . We analyzed the the We . analyzed Trypsin (min) 1 0 CB Fig. Fig. 1 C I 1 1 5 receptor receptor 1 , 1 d −/− expres CB + digit ), with with ), e l g mice

15– 5 1 and 559 −/− 1 s 7 ------, .

© 2012 Nature America, Inc. All rights reserved. purified from wild-type and receptors on located the plasma membrane, brain were mitochondria due to a direct activation of mtCB P respiration mitochondrial and activity I complex regulate to suggested been have (PKA) A kinase protein and cyclase cAMP of reduction protein; THC, 65.9 6.2; bioenergetics) respiratory chain complex I activity (particularly involved in neuronal values. wild-type ## mean Values, GABA- and fields) CB total GABA- ( 3 mice). from mitochondria ( Glu- of region hippocampal CA1 the in mitochondria of number ( Glu- of region hippocampal CA1 the ( neurons. hippocampal 2 Figure t r a 560 (WIN). The drug dose-dependently decreased oxygen consumption consumption oxygen decreased dose-dependently drug The (WIN). treated and 0.60 (vehicle, THC treatment decreased mitochondrial cAMP by approximately 30% # AAV-CB with injected and WT from mitochondria purified in I activity complex on WIN ( WIN nM 100 with mitochondria purified of treatment and WT from mitochondria purified in WIN nM 100 of presence the I in complex of activity and (WT) wild-type from mitochondria purified of respiration on WIN 3 Figure of THC the on cAMP effects we measured respiration, cannabinoids, CB by rescued was fully mitochondria hippocampal purified of activity I complex on WIN CB or AAV-GFP) protein, fluorescent (green protein control a either (AAV)expressing viruses associated of hippocampi the injected we pal CB mtCB (AKAP121; 121 protein organelles these anchor 1e Fig. A-kinase Supplementary the contained dria ( content cAMP and WIN specifically decreased mitochondrial complex I activity ( in brain butfrom mitochondria wild-type, not n c P P < < 0.05). To test whether the effects of CB effects the To < 0.05). whether test ) Proportion of CB of ) Proportion 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 effects of WIN, effects for is the expression mitochondrial sufficient

C I signal in the CA1 hippocampal region of Glu- of region hippocampal CA1 the in signal Mt-CB Direct regulation of mitochondrial activity by mtCB by activity mitochondrial of regulation Direct 1 n −/− = 6, 6, = ## 1 0 mice. ( mice. e l P n vitro in ± 2 0 < 0.01 as compared to WT. Dashed horizontal lines, values for vehicle-treated controls. vehicle-treated for values lines, WT.to horizontal Dashed compared as < 0.01 s.e.m. ** s.e.m. 4 ± CB WT * # 1 in purified hippocampal mitochondria (vehicle, 94.7 ± 5 0 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 1 Dose WIN(nM 8.1 nmol oxidized NADH per minute per milligram NADH milligram minute 8.1 per per nmol oxidized CB s 1 2 < 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 total total the to normalized mitochondria -immunopositive 1 # * −/− ) Proportion of mtCB of ) Proportion 0 ( with the synthetic CB synthetic the with in in vivo 15 a n i. 3 Fig. ( , , = 6 per group). Values, mean mean Values, group). = 6 per P 19 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,nm. 500 Scale mice. fields) = 27 2 9 d CB ) viral re-expression of the CB re-expression viral , and mitochondrial forms of adenylyl adenylyl of forms mitochondrial and , 100 ) Density of mtCB of ) Density Fig. Fig. 3 ## * c 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 ( d 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. b 1 WIN effect on complex I P −/− Fig. 3 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 e 1 ). receptor activation were were activation receptor 1 CB 1 0 in Glu- in Supplementary Fig. 3a agonist WIN55,212-2 WIN55,212-2 agonist CB 1 −/− CB WT 5 1 7 mice ( mice −/− n CB 1 1 ± , –/ 8 = 4). ( = 4). ) CB s.e.m. * s.e.m. – Time (min) , 3 11 CB 10 1 , , mice ( 1 * # −/− . The effect of of effect The . 1 , 13 1 −/− 1 1 ( −/− n receptors in the brain. ( brain. the in receptors 1 , receptor in receptor 14 (arrows) in (arrows) = 320 = 320 n and and d CB mice ( mice = 31 ) Effect of 100 nM WIN on mitochondrial PKA activity ( activity PKA mitochondrial on WIN nM 100 of ) Effect , CB 3 P Fig. Fig. 3 0 1 Fig. 3

< 0.05, ** < 0.05, . Acute Acute . 20 ## −/− * 1

n −/− CB = 6, 6, =

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

c brains ( brains WIN effect on cAMP P endocannabinoids able to activate mtCB activate to able endocannabinoids and in are respiration with the brain, and equipped (ii) mitochondria PKA activity, I complex activity accumulation, cAMP mitochondrial ( mitochondria P brain wild-type in respiration chondrial mito and levels 2-arachidonoylglycerol between observed was tion CB not but wild-type, from mitochondria brain in respiration reduced ( wild-type and from mitochondria neuronal in activity PKA and levels content (% control) ( 2-arachidonoylglycerol mitochondrial increased dose JZL However, shown). not (data quantification purified in but its neuronal mitochondria, were levels below the limits detected of reliable was anandamide endocannabinoid The tion. respira and levels endocannabinoid mitochondrial measured and enzymes degrading endocannabinoid main two the (MAGL), lipase inhibitor dual monoacylglycerol and (FAAH) a hydrolase amide (JZL), acid fatty of JZL195 with mitochondria brain purified activity mitochondrial regulate cannabinoids ( activity degrading ligand. of type the on Fig. Fig. 4a 100 150 < 0.01; *** < 0.01; < 0.0001; 0.0001; < c a 50 Together, these data show that (i) mtCB (i) that show data Together, these Brain mitochondria have substantial levels of endocannabinoid endocannabinoid of levels substantial have mitochondria Brain CB -labeled 0

1 1 −/− CB mito/total mito (%) n VOLUME 15 | NUMBER 4 | APRIL 2012 APRIL | 4 NUMBER | 15 VOLUME 10 20 30 40 0 = 4 independent experiments). ( experiments). = 4 independent *** WT , mice ( , mice a 1

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

–/– ***

), indicating ), that indicating of the functions mtCB 1 littermates. THC induced effects similar to those of to WIN those similar effects THC induced littermates. CB

Fig. 4 Fig. GABA- CB P ## CB 1 < 0.001 as compared to control (vehicle or time 0); 0); time or (vehicle control to compared as < 0.001 –/– Fig. 4 Fig. –/– *** 1 1 −/− f brains ( brains ). Supplementary Table 1 Supplementary d WIN effect on PKA e activity (% control) correla inverse significant Notably, a highly ). 100 150 Glu- d 50 Density (CB particles 0 1 n 2 CB = 3). ( = 3). per µm positive mito) 10 15 20 25 0 5 WT 1 ** CB Glu- –/ – –/– 1 c CB GABA- ) cAMP content after 5 min 5 min after content ) cAMP b ## CB 1 b –/ ## –/– ** –

) Time course of enzymatic enzymatic ) Timeof course 1 n = 3). ( = 3). 1

Genotype in situ in 1 CB e

WIN effect on complex I affects directly signaling nature nature ). 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

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–/ CB –/– EN – CB

15 1 * 1 , –/ 1 C 3 – 2 E - - - - ,

© 2012 Nature America, Inc. All rights reserved. with a plasmid expressing the CB the expressing plasmid a with from derived cultures fibroblast mouse primary fected mtCB stimulate and membranes plasma etrate pen to HU-biot and HU210 of ability the To characterize nM). 0.4 (HU210, HU210 of that to CB recombinant for affinity comparable a displayed biot HU- that revealed experiments competition Radioligand CB receptors. for affinity the preserve should moiety HU210 biotin the the and between scaffold spacer a of presence the of and penetration compound, this cell the impede should bulk biotin hydrophilic the of presence The HU-biot). hereafter (HU210-biotin, HU210 agonist CB lipophilic the of version biotinylated a synthesized we CB intracellular and membrane plasma of influence relative the discriminate to and DSI on agonists antagonist. receptor 5d Fig. CB lipophilic the and pressin hemo with incubated slices hippocampal from extracts of analysis ( hemopressin of of fluorescence cells treated intracellular with a derivative fluorescent membranes plasma hypothesis. As a peptide, hemopressin should not be able to penetrate receptor antagonist hemopressin CB peptide The respectively. DSI, and occlude block to fully able be CB cell-impermeant mtCB intracellular endocannabinoids mobilized endogenously of action the and by CB is and occluded (DSI) inhibition of suppression depolarization-induced as known process a in mission CB presynaptic neurotrans inhibitory GABAergic decreasing activate transiently receptors, retrogradely which mobilizes endocannabinoids, neurons pyramidal postsynaptic CA1 of Depolarization important an is endocannabinoid-dependent transmission formGABAergic of synaptichippocampal of plasticity control Retrograde functions? neuronal endocannabinoid-dependent in involved dria mtCB If CB Intracellular # from are (values mice WT from mitochondria brain purified in respiration and 2-AG ( mice). 10–15 from time ( mitochondria brain purified of respiration ( 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 WT in activity PKA CB and WT in content cAMP mitochondrial and (WT) wild-type from mitochondria purified of respiration on THC of effect the for curve ( endocannabinoids. active biologically contain mitochondria and THC, 4 Figure nature nature of respiration on cellular any had nor effect HU-biot HU210 Neither n n d P = 3–5 pools of mitochondria purified each each purified mitochondria of pools = 3–5 each purified mitochondria of pools = 3–5 2- endocannabinoid the of ) Levels < 0.05, < 0.05, To investigate the occluding effects of cell-impermeant CB 1 −/− CB littermates. ( littermates. 1 – −/−

f 1 NEUR MtCB ). Thus, hemopressin can be used as a cell-impermeant CB as can a be used ). cell-impermeant Thus, hemopressin can control brain mitochondrial activity, are mitochon are activity, mitochondrial brain control can brains. ( brains. ## P < 0.01, < 0.01, 1 OSCI receptors are activated by activated are receptors 1 Supplementary Fig. 5a Fig. Supplementary b c receptors participate in DSI in participate receptors 15– 1 EN ) Effect of THC on THC of ) Effect ) Effect of THC on THC of ) Effect 2 receptors participate in the expression of DSI, DSI, of expression the in participate receptors 5 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 ### C 1 7 receptor agonists, which prevent the binding binding the prevent which agonists, receptor f e 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 values. vehicle WT.to lines, Dashed compared as < 0.001 VOLUME 15 | NUMBER 4 | APRIL 2012 APRIL | 4 NUMBER | 15 VOLUME littermates. littermates. K i = 0.2 0.2 = 1 antagonist AM251 ( AM251 antagonist 25 1 1 receptors on cellular respiration, respiration, cellular on receptors , 33 receptor (creating MF-CB (creating receptor , 3 ±

4 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 d a receptor antagonists antagonists receptor receptors, we trans we receptors, JZL 2-AG THC effect on

( (pmol per mg protein) µ 100 Supplementary Supplementary

M) mitochondrial 10 1 respiration (% control) 100 CB 50 1 0 0 K receptors receptors 1 1 1 receptor receptor receptor ± i −/− 0.25 = 1.5 1.5 = 1 s.e.m. * s.e.m. 0 cells). cells). pups pups

15– 1 CB WT 6 . If . ** 1 1 1 7 ± Dose THC(nM –/ 1 1 1 1 - - - - - . –

P 400 e # * < 0.05, ** 0.05, < JZL effect on HU210, but not HU-biot, decreased cellular respiration in MF-CB in respiration cellular decreased HU-biot, not but HU210, mock-transfected to vehicle control. control. vehicle to mean Values, experiments). independent ( HU210 by depression the block fully to able became Hemo and ( MF-CB intact in HU210 of effect this antagonize to able not was Hemo consumption. oxygen decreased significantly HU210 agonist lipophilic the whereas cells, MF-CB in respiration cellular alter not did (Hemo) hemopressin and transfected ( the fluorescent protein mCherry (mock) or the CB obtained from were (MFs) fibroblasts Mouse cells. intact in respiration cellular alter 5 Figure and compared their effects to those of the cell AM251 and agonist HU210 on inhibitory synaptic transmission and DSI, cells. living of respiration mitochondrial CB of intracellular activation the notably, only and, (iii) respiration, cellular alter not do and cells intact penetrate not do hemopressin and HU-biot (ii) respiration, cellular alter and cells CB that (i) lipophilic the data indicate ( cells permeabilized in effect this blocked fully it but MF-CB intact on HU210 of effect the alter to not able was hemopressin Notably, permeance. cell their on relies ence mtCB activate drugs both that indicating MF-CB permeabilized in in respiration cell HU210 depressing as efficient as became HU-biot Accordingly, effect. this ( cells c a

) In permeabilized MF-CB permeabilized ) In mock- in respiration cellular altered HU-biot nor HU210 ) Neither mitochondrial a Cellular respiration CB cell-permeant the of impact the Weanalyzed next respiration (% control) (% vehicle) 100 150 50 Fig. 5 Fig. –60 –40 –20 0 ) 800

0 * # Hemopressin and HU-biot do not penetrate cells and cannot cannot and cells penetrate not do HU-biot and Hemopressin P 0 Mock-transfected < 0.01, *** 0.01, < CB HU210 b CB 0.25 CB WT ), suggesting that cell penetration is a necessary step for step is a necessary penetration cell that ), suggesting CB ** 1 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 either 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 ( ### *** b 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 –/ MF-CB

Hemo (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, 2 0 Fig. 5 activity (% control) 2-AG (pmolpermgprotein) JZL 1 JZL 0.25 Vehicle 1 - 100 receptor (MF-CB impermeant impermeant antagonist 50 0 –60 –40 –20 P 0 < 0.05 as compared compared as < 0.05 µ 0 a M c WT t r a ). ). Conversely, only ** 1 µ 40 receptors affects affects receptors 1 HU-biot M 1 cells ( cells cells ( cells Fig. 5 Fig. CB b Permeabilized * 60 MF-CB ) HU-biot ) HU-biot ## 1 1

HU210 –/ HU210 + Hemo antagonist C I – c 80 Fig. 5 Fig. n * Fig. 5 Fig. ). These These ). 1 1 = 3–5 = 3–5 cells ). ). ## 1 e l

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© 2012 Nature America, Inc. All rights reserved. 10 10 ( vehicle of application intracellular postsynaptic with neurons pyramidal ( plot. data summary Right, traces. electrophysiological representative (2.5 rotenone of application bath after and before neurons pyramidal postsynaptic CA1 of step depolarization a 1-s by induced DSI weak (2.5 rotenone I inhibitor complex mitochondrial the of application bath after and before neurons pyramidal postsynaptic CA1 7 Figure a concentrations at DSI occluded partially only HU-biot contrast, In DSI induction in occluded hippocampal slices ( fully HU210 with incubation 30-min a expected, As ( of min incubation 25–30 after observed was HU-biot and HU210 both of effect eIPSCs-depressing maximal The transmission. GABAergic hippocampal on HU210 of and effect that cannabinoid the biotin not moiety does alter the effect CB lular ( HU-biot and HU210 both by eIPSCs of depression the blocked fully hemopressin fibers afferent of stimulation of ( frequency the of independently ( eIPSCs reduce to ability similar a very displayed nM) (500 ( amplitude Supplementary DSI Fig. 6c reduced reversibly and partially only ( slices hippocampal to applied when (eIPSCs) currents postsynaptic tory (10 dose high a at hemopressin ( AM251 pyramidal by CA1 blocked of fully was neurons depolarization expected 5-s a As by induced respectively. DSI ‘strong’ a HU-biot, agonist and hemopressin ** mean Values, results. of summary bottom, respectively); depolarization, after and before traces 2, and (1 traces electrophysiological 5 ( course. time bottom, respectively); application, drug after and before traces 2, and (1 traces electrophysiological Top, representative HU-biot. or HU210 CB membrane–impermeant cell the of and cells) CB ( results. of summary bottom, respectively); depolarization, after and before traces 2, and (1 traces electrophysiological Top, representative cells). (Control, neurons pyramidal postsynaptic CA1 of 0 mV to −70 from step depolarization a 5-s by induced DSI strong of amplitude the 10 (Hemo, hemopressin CB impermeant (4 CB lipophilic the ( DSI. depolarization-induced to 6 Figure t r a 562 mean 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 nM; (500 HU210 agonist receptor

amplitude (%) 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 by a 5-s depolarization step of CA1 postsynaptic pyramidal neurons. Top, representative Top, representative neurons. pyramidal postsynaptic CA1 of step depolarization a 5-s by induced DSI strong of amplitude the on = 5 cells) n

C I 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-s depolarization step of CA1 postsynaptic pyramidal neurons (gray bar, (gray neurons pyramidal postsynaptic CA1 of step depolarization a 1-s by induced DSI of amplitude the on = 7 cells) Intracellular CB Intracellular Inhibition of mitochondrial activity potentiates DSI. ( DSI. potentiates activity mitochondrial of Inhibition ) Occlusion effect of 30-min incubations of slices with HU210 ( HU210 with slices of incubations 30-min of effect ) Occlusion receptor signaling is not required for this pharmacological pharmacological for this required not is signaling receptor

Vehicle b ) Effects of the lipophilic synthetic synthetic lipophilic the of ) Effects e l 1 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 s < 0.01 as compared to control. NS, not significant. not NS, control. to compared as < 0.01 s NS , µ d 1 M; M; receptors contribute contribute receptors ). High concentrations of HU210 and HU-biot , b n ). Notably, the extracellular antagonist antagonist extracellular the Notably, ). = 8 cells) on = 8 cells) Rotenone a at 0mV at 0mV Vehicl ) Effects of ) Effects , b 1 1 Fig. 6 Fig. b Fig. 6 s s ). However, the peptide antagonist antagonist peptide the However, ). µ e Pre-DSI M) did not alter evoked inhibi evoked alter not did M) n n b = 7 = 6 cells) on normalized eIPSCs in CA1 pyramidal hippocampal neurons. Arrow, time of application of application of time Arrow, neurons. hippocampal pyramidal CA1 in eIPSCs normalized on = 6 cells) c n and and , = 9 b Fig. 6 Fig. ), suggesting that intracel that suggesting ), Supplementary Fig. 6a Fig. Supplementary Supplementary Supplementary Fig. 6d DSI a ). The application of of application The ).

∆elPSC a ## 50 ms

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1 Hemo ## P n 6 ). ). ), ). - - n , = 6 cells) or rotenone (2.5 (2.5 rotenone or = 6 cells) < 0.001. **

2 1 ∆eIPSC ( nM 500 of concentrations at HU-biot with or = 8 cells) for the respective full blockade and occlusion of DSI induced by 5-s 5-s by induced DSI of occlusion and blockade full respective the for CB of penetration cell Thus, 5 and nM 500 both of amplitude (%) CB presynaptic by mediated is DSI levels. endocannabinoid 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 ofa alteration direct ( endocannabinoids ( 7b Fig. CB ( depolarization ( amplitude DSI enhanced reliably depolarization rotenone step, long depolarization a 1-s by induced s; DSI (5 period strong on effect no had inhibitor. The application of rotenone to slices hippocampal (2.5 Rotenone is a well known specific and potent mitochondrial complex I DSI to contributes activity mitochondrial of Inhibition neurons. pyramidal postsynaptic of depolarization Supplementary Fig. 7c Fig. Supplementary Fig. 7 Fig. 1 –80 –70 –60 –50 –40 –30 –20 –10 2 10 1 0 −/−

VOLUME 15 | NUMBER 4 | APRIL 2012 APRIL | 4 NUMBER | 15 VOLUME Vehicle mice in the presence of the depolarization ( depolarization the of presence the in mice b b

), to alter the inhibitory effects of HU-biot on eIPSCs eIPSCs on HU-biot of effects inhibitory the alter to ), eIPSCs amplitude ). Rotenone was unable to affect eIPSCs in the absence of of absence the in eIPSCs affect to unable was Rotenone ). (% of baseline) DSI 1 100 150

Rotenone 50 Fig. 7 Fig. 0 s Supplementary Fig. 7a Supplementary ** 1 5 a 10 Supplementary Fig. 7d Fig. Supplementary ). However, if a weaker DSI was induced by a by induced was DSI weaker a if However, ). HU210 µ 15 Time (min) M; M; 2 1 µ –60 –50 –40 –30 –20 –10 c ), or to modify the levels of mitochondrial mitochondrial of levels the modify to or ), 20 10 M ( M n 0 = 6 cells). ( = 6 cells). 25 1

Vehicle 6 Fig. antagonists and agonists is necessary necessary is agonists and antagonists 30 HU-biot 1 2 DSI 1 35 Rotenone c 1 and and 40 receptors 2 s d NS ), ), to induce DSI in slices from ) Effect of hemopressin (Hemo, (Hemo, hemopressin of ) Effect c

Supplementary Fig. 6d Fig. Supplementary

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© 2012 Nature America, Inc. All rights reserved. to underlie many functions of endocannabinoid signaling in the the in signaling endocannabinoid of functions many underlie to key roles in both physiological and pathological brain processes. a new mechanism of modulation of neuronal activity, which might have tionand functional characterization of brain mtCB regulating synaptic plasticity and possibly behavior However, mitochondria also influence mitochondrialthe normal alterations functioning are ofthought the tobrain, be oftions etiopathological relevance view.of point functional a from amount negligible a considered be cannot membranes drial Therefore, the 15% of hippocampal CB naling are mainly exerted by glutamatergic-neuron CB sig of endocannabinoid or properties the orexigenic totoxic seizures limited of percentage receptors. For instance, protection against exci this by exerted are system endocannabinoid the of functions the of neurons glutamatergic in expressed are proteins receptor tions. Although a minimal amount (less than 10%) of total cortical CB CB of subset organelles expressing mtCB a receptors, suggesting strong impact of on cannabinoid signaling the Notably, approximately 30% of neuronal mitochondriamitochondria. brain do contain purified CB in respiration of reduction dependent low.seem However,CB this expression accounts for up cell to 30% of mtCB total of 15% (approximately membrane DSI. of to expression the contributes and likely metabolism energy regulates it acutely where of neurons, membranes on mitochondrial is present production modu energy and to and respiration organelle’s mitochondria the in late occur to proposed recently been have which activity, PKA and levels cAMP regulating by activity, complex I of modulation the through exerted be may function This neuronal respiration. brain mitochondrial regulates of directly it where mitochondria, hippocampus) the in 30% (approximately tion CB the that show results present The DISCUSSION DSI. of forms weak, not CB intracellular that confirming ( by a DSI a weak induced abolish 1-s depolarization was able to hemopressin data, fully these with strong DSI. Consistent ing the involvement of in the presynaptic mitochondria expression of ( postsynaptically infused when any effect produce nature nature brain GPCRs and is comparable to the values of several known or recently or recently known of several values the to comparable is and GPCRs ( 2 Table localization mitochondrial of probability 39.3% (ref. II MitoProt proposed recently been has activity plasticity. synaptic bioenergetics, which might be involved in endocannabinoid-dependent mtCB of ence exist the establish data these Thus, direct activity. I by complex of inhibition mimicked is function this and hippocampus, the in sion form dependent of of plasticity short-term neurotransmis inhibitory CB Intracellular experimental data demonstrate that the metabotropic CB metabotropic the that demonstrate data experimental ( proteins mitochondrial identified CB func brain in player key a as emerging isbioenergetics Neuronal h pooto o CB of proportion The mitochondrial regulate directly may GPCRs that hypothesis The 1 receptors can bear crucial functional significance in specific loca 1 15– 1 , 2 -dependent forms of synaptic plasticity such as DSI are likely likely are DSI as such plasticity synaptic of forms -dependent , 4 ). This theoretical value is much higher than that of many many of that than higher much is value theoretical This ). 1 , NEUR 5 7 . This holds true for neurodegenerativewhich fordisorders, in true holds This . . Neurotransmitter release is a very energy-demanding energy-demanding very a is release Neurotransmitter . 1 OSCI 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- 35) predicted the CB the predicted 35) EN C E

1 receptors located on the mitochondrial mitochondrial the on located receptors VOLUME 15 | NUMBER 4 | APRIL 2012 APRIL | 4 NUMBER | 15 VOLUME 1 receptors contribute to strong, but strong, to contribute receptors upeetr Tbe 2 Table Supplementary 1 6 . . Moreover, a low abundance of , 1 3 protein located on mitochon 0 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 1 1 1 , 2 receptors provides receptors) might might receptors) , 4 Fig. 7 Fig. , Fig. 7 Fig. Supplementary Supplementary 5 . The identifica 1 receptors 7 c , 24 8 d ), suggest ), 1 , 11 ), further ), further receptor receptor , 2 7 , 13 , many many , ). Our Our ).

, 14 27 , , 3 2 1 0 8 4 1 1 ------. . .

lized endocannabinoids. As yet, the mechanistic bases of this paradoxi induced by exogenously applied cannabinoids or endogenously mobi results suggest a mechanistic difference between the decrease of eIPSCs mtCB Conversely, our data clearly show that intracellular CB application of cannabinoids does not seem to involve intracellular CB transmission in the hippocampus one and much to are alter longer synaptic applicationneeded periods previous studies have shown that much higher concentrations of roten its application to slices should result into alterations of eIPSCs. Indeed, one were stimulating endocannabinoid mobilization indirectly synaptic depolarization did not alter inhibitory transmission. If roten the application of rotenone or postsynaptically in the absence of post mobilization endocannabinoid glutamate) example, (for mitter neurotrans of stimulation by signaling endocannabinoid potentiate might which rotenone, of effects indirect to due be also could effect latter The DSI. in activity mitochondrial of involvement the gesting plasticity,sug synaptic of form this potentiated rotenone by activity DSI, respectively. In addition, inhibition of presynaptic mitochondrial blocked and occluded partially only hemopressin antagonist the and means process functioning in health and disease. and health in brain functioning understand better to frameworks theoretical new of opment and cellular regulation, energy with unforeseeable oneffect the devel brain the in signaling GPCR between relationship the detail deeper pave will the way ronal in for mitochondria investigating new studies that CB demonstration The system. endocannabinoid of the exploitation therapeutic for the targets pharmacological new add possibly will and functions brain influences signaling endocannabinoid which through mechanisms the range of are extends involved in regulation DSI further chondrial means. by different plasticity and regulation synaptic of forms Thus, mtCB raising the possibility that mtCB to has dria recently proposed been plasticity synaptic also modulate activity and neurotransmitter release calcium levels and redox potential, which can also modulate neuronal neurotransmitters of release ongoing the for ATPneeded of form the in supply the energy altering and respiration mitochondrial decreasing temporarily mtCB presynaptic at acting Endocannabinoids signaling. endocannabinoid intracellular this of mtCB that suggest and DSI in participate remain silent. would otherwise which nabinoid signaling, so far events cellular unknown to favor means additional of endocan able to endogenously mobilize endocannabinoids might trigger other, thereby different triggering mechanisms. Finally, depolarization steps subdomains, other for preference display might cannabinoids enous exog Conversely, subdomains. cellular specific to limited be might stimulation synaptic and/or depolarization by endocannabinoids of example, tolerance) (for effects extra induce might and drug, the of properties cokinetics only duration their areon depending long-lasting, pharma generally cannabinoids) lipophilic of (especially applications Conversely,drug endocannabinoids of degradation endogenous efficient to due aspects spatial and/or temporal on rely might they but unknown, are difference cal The discovery that intracellular CB intracellular that discovery The CB intracellular that show results our together, Taken 1 4 ) participate in strong DSI. These apparently contradictory contradictory apparently These DSI. strong in participate ) . The membrane-impermeant CB membrane-impermeant The . 5 , and mitochondria can modulate its efficiency by different different by efficiency its modulate can mitochondria and , 1 receptors might receptors to contribute DSI and to possibly other 1 7 . DSI is short-lasting (a few tens of seconds), likely likely seconds), of tens few (a short-lasting is DSI . 4 , 2 5 9 . 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 1 3 1 6 3 6 might interfere with these processes. . This is, however, unlikely because because unlikely however, is, This . 7 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 might be the mediator mediator the be might t r a 1 receptors (likely 1 C I receptors receptors e l per per se 15 563 , 1 s 7 1 1 ------ , . . ,

© 2012 Nature America, Inc. All rights reserved. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/natureneuroscience/. The authors declare no competing interests.financial manuscript. All authors edited the manuscript. experiments. G.M. conceived the andwhole studysupervised and wrote the of the work. P.G. the anatomical biochemical supervised studies. supervised R.R. provided forthe viruses re-expressionlocal of CB hemopressin and performed and measured intracellular hemopressin and AM251. J.G. generated fluorescent M.M.-F. and synthesized and M.L.L.-R. HU-biotbiochemically characterized endocannabinoids and endocannabinoid-degrading enzymatic S.O.-G.,activities. M.M.-L., E.H.-C. and A.C. participated in biochemical experiments. I.M. measured E.S.-G. performed the electrophysiological studies. N.P. performed the anatomical studies. L.B. and study. G.B. performed the biochemical experiments. F.M., J.L. and C.M. performed different parts supervised of this work. G.B., P.G., and R.R. G.M. the designed N.P., J.L. and L.B. equally contributed to experiments. P.G. and equally R.R. (FOR926, B.L.) and CONACyT (E.S.-G.). Ramon y Cajal program (S.O.-G.), Deutsche Forschungsgemeinschaft MICINN P.G.;(SAF2009-07065, M.L.L.-R.), SAF2010-22198-C02-01, P.G.),(UFI11/41, Comunidad de Madrid M.L.L.-R.), (S2010/BMD-2353, (GIC07/70-IT-432-07, P.G.), University of the UPV/EHU Basque Country Instituto de Salud Carlos III P.G.),(RD07/0001/2001, GovernmentBasque Country Foundation University(E.S.-G.), of Bordeaux (J.L.), Red de Trastornos Adictivos, Recherche Medicale (G.M. and M.M.-L.), Region Aquitaine (G.M.), Fyssen Research Council (ENDOFOOD, ERC-2010-StG-260515, G.M.), Fondation pour la Program (REPROBESITY, HEALTH-F2-2008-223713, G.M. and B.L.), European Post-Doc ANR-09RDPOC-006-01 (G.B.), European Union 7 AVENIR/INSERM (G.M.), INSERM–Agence Nationale de la Recherche (Retour P.V. Piazza and C. Wotjak for reading critically the manuscript. Supported by G.M.’s laboratory for discussions; and A. Bacci, D. M. Cota, Guzman, U. Pagotto, the NeuroCentre Magendie for mouse care and genotyping; all the members of We thank D. Gonzales, N. Aubailly and all the personnel of the Animal Facility of Note: Supplementary information is available on the online the at http://www.nature.com/natureneuroscience/. paper of the in version available are references associated any and Methods M t r a 564 COM AUTH Acknowledgmen

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© 2012 Nature America, Inc. All rights reserved. ated from P0–P1 C Glu- mitochondria revealed the percentages of axon terminals containing mtCB1 in with mtCB1 versus total asymmetric or symmetric synaptic boutons containing littermate type controls. Normalizedterminalssymmetricorasymmetric CA1 for each parameter, one-way ANOVA), allowing the pooling of respective wild- observed between the wild-type littermates of the different mutant mice ( microscopy. electron for Immunocytochemistry mitochondria, in density of mtCB labeled percentageof in differenceNoelements. dendritic with synapses dria, total CB NormalizedmtCB dria. MtCB determined the proportion of hippocampal CA1 CB where Semiquantification of mt AKAP 149/121, Santa Cruz Biotechnology). FrontierGo-Af450-1,antiserum(2 Ltd); Co.121goatAKAP Science used in 50- scope pre-embedding immunogold labeling as recently described GABA- (one-site binding). The apparent fitting curve nonlinearbyanalyzed were Concentration curvescells. response determined after washing and subtracting of signals from untransfected control (TecanFarcyte, 485-nm excitation and 535-nm emission). Specific binding was bindingbuffer, fluorescence intensities were measured 96-wellain plate reader plastic tubes (30 min, 30 °C). After centrifugation and three washes with ice-cold (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl The peptide was incubated with 1 × 10 hemopressin was determined using CHO-K1 cells expressing C human CB and analyzed with preparative HPLC and ESI-MS. temperature in the dark. The product was reconstituted in DMSO and purified hemopressinofmg dimethylformamide roomat5 inof cysteineh forthe2 to a by covalentdone was coupling peptide of the a 6.25to mgfluorescein ml of introduction The Co.). Bio (CS standard Fmoc solid-phase synthesis chemistry on a CS336S Peptide Synthesizer Synthesis of fluorescent hemopressin. previously described CB The 95%. > analysis) (elemental Purity column chromatography. Spectroscopic data were consistent with the structure. and acid in the presence of dicyclohexylcarbodiimide, (ref. HU210 between condensation and of Synthesis same amounts of solvents (0.1–0.2% ethanol; 0.1% DMSO). Vehicles the containedPBS. inhemopressins DMSO; in below) (see HU-biot and HU210 (WIN), WIN55,252-2 ethanol; in dissolved was THC Scientific. D 22 male mice were used. Experimenters were blind to genotypes and treatments. A total of 312 female respectively)and were as described GABA- electrophysiology. for used were sexes both of old mice (2–4 months old) were used for anatomy and biochemistry. Mice 20–30 d Female conditions.standard under maintained were Mice 3306369). number of INSERM and the French Ministry of Agriculture and Forestry (authorization M ONLINE METHODS doi:10.1038/nn.3053 B ell culture and transfection. and culture ell rugs. ice. 1 binding assay with fluorescent hemopressin. N CB Experiments were approved by the Committee on Animal Health and Care 4 -hydroxybenzotriazolecouplingas reagents (yield:and32%)bypurified 2 Drugs were from Sigma, Tocris, Abbott Laboratories Inc. or Ascent or Inc. Laboratories Abbott Tocris, Sigma, from were Drugs CB CB 1 . The number of labeled mitochondria normalized to total mitochondria 1 −/− . Axon terminals identification: abundant synaptic vesicles, mitochon 1 1 µ 1 −/− and GABA- −/− labeling density was calculated on the area of labeled mitochondria. m-thick hippocampal sections: goat CB mice (elsewhere called and CB1 C 1 CB labeling versus total CB B 4 1 1 −/− 1 . binding assay for HU-biot. for assay binding −/− CB mice. Dorsal skin was excised and minced in PBS and mice ( C 1 −/− B1. 26– mice. The detailed procedure has been described else Mouse primary fibroblasts (MFs) were gener were (MFs) fibroblasts primary Mouse K n −1 28 1 = 3 each) were processed for electron micro d or proportion of mtCB solution of fluorescein-5-maleimide (Pierce) , value for the CB 38 CB 40) and 40) , 3 5 9 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 The N CB 1 1 -immunopositive mitochon 1 antiserum (2 , HU-biot was prepared by prepared was HU-biot N receptor was 119 idtp, Glu- Wild-type, 1 K -dimethylaminopyridine -KO and GABA- CB d value of the fluorescent 1 1 −/− versus total CB 1 proportion versus Glu- , µ l binding buffer 4 µ 2 g ml . Antibodies CB ± 1 CB µ 1 −1 (hCB −/− 11 nM. CB P g ml g ; CB1- > 0.6, 1 1 and -KO, 1 was −/− −1 1 ). - - - - - ; ,

to the chamber. A coupled respiratory state was obtained by addingchamber. 2 mM ADP. Respiratory substrates (10 mM pyruvate nitol,and malate)25 mM weresucrose, added10 mMdirectly KCl, 10 mM Tris-HCl,mitochondria pH 7.4, 50 mM (1 EDTA)mg) inwere the suspended in oxygenchamberClarkglasselectrodeequipped(Hansatech).a1 awith mlPurified of the respiration buffer (75 mMO man in sample buffer and analyzed by western blot. All samples were centrifuged (12,000 ature). The reaction was stopped by adding protease inhibitor cocktail (Roche). Trypsin digestion min). (0.1% 15 w/v)temperature, wasroom w/v,performed (0.25%, fordigitonin 0, with 5, not 10 or or treated 15 were min (room temper Trypsin and digitonin assays. sected and mitochondria were purified as previously described hippocampioforbrains the death, M resuspended in culture medium and experiments were performed 3 d later. 30 using cuvette2-mm-gap a in membrane fraction and purified mitochondria were performed as described described as performed were mitochondria purified and fraction membrane total brain the from oleoylethanolamide and palmitoylethanolamide (2-AG), mides. and endocannabinoids of quantification and Purification THC were performed at 30 °C for 1 h. to the manufacturers’ instructions. Reactions in presence or absence of WIN or (Assay Designs Inc) and an ELISA kit (Enzo Life Science), respectively,were assayedaccording on isolated mitochondria using the Direct Correlate-EIA cAMP kit C by the Bio-Rad Quantity One system. HRP signal was revealed using the ECL plus reagent (Amersham) and detected bated with secondary HRP-coupled antibodies (1 h, room temperature). Finally, receptormembranesThen(Cayman,incu overnight,and°C).werewashed 4 fractions was analyzed using an antiserum directed against the C terminus of the cadherin (Santa Cruz, overnight, 4 °C). The presence of the CB 1 h, room temperature), synaptophysin (Millipore, 2 h, room temperature) and 4 °C), calreticulin (Santa Cruz, overnight, 4 °C), KDEL (ER; Enzo Life Sciences, overnight, Cruz, (Santa GAPDH againstrespectively antibodies using tigated reticulum (ER), synaptosomes and plasma membrane contaminationstemperature)and Tom-20 (Santa overnight,wereCruz, °C).Cytosol,4 endoplasmic inves were proteins immunodetected Mitochondrial using antibodies (0.05%). against complex PBS-Tween20 III (Mitosciences, in 2 h, room milk 5% in soaked 4–16% acrylamide gels and transferred to PVDF membranes. Membranes were experiments ( some in used also wereomitted) DTT (beta-mercaptoethanolconditionsand Nonreducingmin. ~5 for boiled and DTT mM 50 beta-mercaptoethanoland 2% with supplemented were Samples buffer. sample in diluted and (Roche) Immunoblotting. reaction of NADH oxidation into NAD N by adding the drugs directly to the chamber. permeabilized with digitonin (20 respiratorywith substratespyruvate mM (10andADP andmalate)mM and2 was recorded. For experiments with permeabilized cells,respiration and chamberMFs the in weredirectly transferred were cells supplementedIntact medium. Cell Cell were electroporated in Optimem medium (Invitrogen) at 2 × 10 (Harvard apparatus, 175V, 1-ms pulse, five pulses, 0.5-s interval between pulses).Transfectionsflasks. electroporatorwereout carriedBTX by usinga 830 ECM 25-cm onto seeded were Cells (Invitrogen).solutionpenicillin/streptomycin 1% FBS, 10% with DMEM in resuspended and ugation solution.werebycentrifcollected Cells trypsin 0.25% incubated in thenwith yclic A yclic A xygenconsumption assays. itochondrial purification from mouse hippocampi or whole brain. whole or hippocampi mouse from purification itochondrial The effects of hemopressin, WIN, JZL195, HU210 and HU-biot were analyzed The experiments using MFs were performed on 2 × 10 D H-ubiquinone oxidoreductase (complex I) activity assay. Purification and quantification of anandamide, 2-arachidonoylglycerol M P andP P Supplementary Fig. 3b All samples were supplemented with proteases inhibitors proteases with supplemented were samples All K A activityassays.A Oxygen consumption monitoredwas in °Cat30 Mitochondria diluted in isolation buffer (100 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 nature nature 6 l cells ml -glutamine and 2% and -glutamine NEUR 1 in mitochondrial N 4 We followed the 3 -acylethanola . −1 OSCI 7 in growth cells ml After EN µ C 4 g) −1 3 E 2 ------.

© 2012 Nature America, Inc. All rights reserved. wereaspreviously described E to corresponding standards. Fractions were quantified by measuring the area under the peak and compared B (95:5 methanol:water) with 0.1% ammonium hydroxide and 0.1% formic acid. ESI source in SIM mode. The mobile phases were A (95:5 water:methanol) and 1200LC-MSDinstrumentVL anEclipsewithXDB-C18 column coupled anto 50 and argonstreamofreconstitutedand a under 100 dried in was in reconstituted and 100 argon of stream a under dried was phase aqueous the and the organic and aqueous layers separated by centrifugation.centrifuged, ForDounce-homogenized inhemopressin, chloroform:methanol:water (2:1:1; 2 ml), (sameconditions electrophysiologyinas experiments) were withwater,rinsed spectrography. M D software. Nikon TS100 fluorescence microscope and video camera using the NIS Elementsbeforeincubation.NP-40PBSImagesa 0.5% wereacquiredwith in with lized was measured by flow cytometry (FACScan, Cytek). Some cells were permeabi detached with trypsin, and fixed with 0.01% formaldehyde different(10 times min).( Fluorescence MgCl mM 5 EDTAand mM 2.5 7.4, Tris-HCl,pH wereincubatedCHO-K110cells with C tively). Animals were used for experiments five weeks after viral injection −2.0 mm DV; −3.5 mm AP, 150 nl min AAV-GFP or AAV-CB1 (6 × 10 described as were titers genomic of determination and intoAAVanGFP) expression cassette,packagingAAV1/2 of vectorschimeric CB (HA)-taggedrat Viral rescue of pmol of hydrolyzed anandamide or 2-AG per min per mg protein. volumesCHClof2 incubationmixturethe ofwithextraction aqueousafter countingphase the of FAAH)[ or (20 min, 37 °C) for FAAH and MAGL, respectively. The [ 50 mM Tris-HCl, pH 9 (30 min, 37 °C) for FAAH and in Tris-HCl buffer, pH 7 1.0 mCi mmol mmol mCi 5.0 fraction membrane (50 mitochondria total purified or brain using by detected assays. were activities lipase MAGL or monoacylglycerol and hydrolase amide acid Fatty reliable quantification. Therefore, only data relating to 2-AG are presented. tively. Anandamide was detected in the samples, but at levels below the limit of calibration curve and were expressed as nmol or pmol per mg of protein, respec previously nature nature lectrophysiology. ellular localization of fluorescent hemopressin. fluorescent of localization ellular easurement of hemopressin and A hemopressinand ofeasurement µ L H µ 2 wr aaye b L/S L/S a crid u i a Agilent an in out carried was LC/MS LC/MS. by analyzed were L NEUR O, and 60 −1 4 4 3 into the dorsal and ventral hippocampus (−2.0 mm AP, . The amountsThe anandamide. of 2-AG andwerea usingdetermined H]glycerol (for MAGL) produced were measured by scintillationby measuredwere produced MAGL) (forH]glycerol −1 C Slices previously incubated with 10 −1 Supplementary Fig. 4a–c OSCI , 25 B1 expression in the hippocampus. , 1.8 1.8 , Preparation of hippocampal slices and recording of eIPSCs 3 µ 1 /CH µ receptor(AAV-CB L were analyzed by LC/MS. For AM251, the organic phase M, ARC Inc.) as substrates. Reactions were carried out in EN µ , R Ic) r 2-oleoyl-[ or Inc.) ARC M, 3 C OH (1:1 v/v). Enzymatic activities wereexpressedEnzymaticasactivities v/v). (1:1 OH E ± 28 3.4 mm ML, −3.0 mm DV from bregma, respec , µ 4 11 6 g protein), and [ and protein), g . After. reaching stable baseline (~10minafter viral genomes ml M µ M fluorescentM hemopressinmM 50 in 251 by liquid chromatography/massliquid by 251 1 ) or green fluorescentprotein(AAV-green or ) ) in incubation buffer plus 0.5% BSA, 14 −1 µ C]anandamide (for FAAH,(for C]anandamide 2 1 × 10 × 1 ) was injected bilaterally at M hemopressin or AM251 Cloning of hemagglutinin (30 min, 30 °C), washed °C), 30 min, (30 3 ]lcrl fr MAGL, (for H]glycerol 14 31 C]ethanolamine (for , 4 5 6 . One . µ hCB L of acetonitrile,of L ± 1 2.0 mm ML, µ -expressing l of either of l FAAH 3 1 . - - -

of postsynaptic rotenone on DSI was studied by adding the drug (2.5 and a new DSI protocol was applied after 15 min of baseline recording.obtained Thea stableeffect baseline, a DSI protocol was applied. Then, drugs were applied pairwise schedule was used: CA1 pyramidal neurons were patched and, after we ( experiments other In applied.protocolwas DSI a obtained, was baseline stable a after and,patched then was cell pyramidal CA1 A min. 30 forvehicle) (or drugs with incubated ( experiments some In DSI. on drugs ferent calculated as described voltage step from −70 to 0 mV applied to the postsynaptic pyramidal neuron and was then applied every 10 min after drug applications up to 30 min. applied and patched cells were left undisturbed. The same stimulation protocol werevehicle) (or drugs Then, 0). (time recordedwas baseline stable a and s 3 afferent stimulation as during DSI protocols (see below),rotenone IPSCs on werebasal inhibitory evokedsynaptic transmissionevery using the same conditions of 10–15 s. To determine the effects of HU210, HU-biot, hemopressin (10 (2.5 no effect on following recorded DSI (not shown). depolarization protocol 5-s a with experiment each starting before checked was expressionlayer.DSI stratumtionpositionedthe in radiatum, 100 were evoked using a patch pipette filled with a HEPES-based extracellular solu reachingwhole-cell configuration for infusion of intracellular solution), IPSCs 46. 45. 44. 43. 42. 41. 40. 39. 38. ANOVA (followed by Bonferroni’s t ent data points GraphPad software (version 5.0). Results were expressed as means of independ analyses. Statistical intracellular solution in the patch pipette. -test, one-way ANOVA (followed by Newman-Keuls Twodifferentdif the of treatment evaluate effect tothe schemeswere used For DSI, IPSCs were evoked 3 s,every and DSI was induced by a 1-s or a 5-s To determine the effects of HU210 (500nM), HU-biot (500nM) and rotenone

Lourenço, J. Lourenço, Klugmann,M. of activation Pharmacological C. Mulle, & G. Marsicano, I., Matias, J., Lourenço, G. Benard, N. Puente, & M.L. López-Rodríguez, S., Capolicchio, M., Martín-Fontecha, L., Martín-Couce, Lander,R., Mechoulam, University,N., individual, the of Synthesis J. Zahalka, & A. K. Monory, G. Marsicano, signaling at GABAergic synapses. GABAergic at signaling Neurosci. rats. adult in activity seizure and cognition affect differentially proteins mobilization. endocannabinoid drives (2011). 3243–3248 receptors kainate Physiol. Cell Physiol. J. Am. amygdala. extended receptors. cannabinoid of study for the probes chemical endocannabinoid-based of Development S. Ortega-Gutiérrez, derivative. tetrahydrocannabinol a of TetrahedronAsymmetry enantiomers distinct, pharmacologically mice. in Delta(9)-tetrahydrocannabinol excitotoxicity. µ M) on basal inhibitory synaptic transmission, IPSCs were evoked every every evoked were 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 unpaired Student’s etal. t al. et Physiological diversity of mitochondrial oxidative phosphorylation. oxidative mitochondrial of diversity Physiological 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 long Homer 1 B cnaiod eetr ad ndmn dfne against defense on-demand and receptors cannabinoid CB1 Nat. Neurosci. Nat. 4 4 Fig. 7a Fig.

6 6 302 .

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

291 – c 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

54 , 197–204 (2010). 197–204 , , e269 (2007). e269 , , c post hoc , 5265–5269 (2011). 5265–5269 , and 7 doi:10.1038/nn.3053 d ), slices were first were slices ), . Neurosci. J. test), or two-way – c and µ M) to the µ 7b Mol. Cell Mol. M) and

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CORRIGENDA

Corrigendum: Mitochondrial CB1 receptors regulate neuronal energy metabolism Giovanni Bénard, Federico Massa, Nagore Puente, Joana Lourenço, Luigi Bellocchio, Edgar Soria-Gómez, Isabel Matias, Anna Delamarre, Mathilde Metna-Laurent, Astrid Cannich, Etienne Hebert-Chatelain, Christophe Mulle, Silvia Ortega-Gutiérrez, Mar Martín-Fontecha, Matthias Klugmann, Stephan Guggenhuber, Beat Lutz, Jürg Gertsch, Francis Chaouloff, María Luz López-Rodríguez, Pedro Grandes, Rodrigue Rossignol & Giovanni Marsicano Nat. Neurosci.; doi:10.1038/nn.3053; corrected online 27 March 2012

In the version of this article initially published online, the description of the procedure given in the Online Methods for synthesis of fluorescent hemopressin, taken from different protocol not used in this work, was incorrect. The entire paragraph has been replaced. The error has been corrected in the PDF and HTML versions of this article. © 2012 Nature America, Inc. All rights reserved. America, Inc. © 2012 Nature npg

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