UC Irvine UC Irvine Previously Published Works Title Molecular composition of the endocannabinoid system at glutamatergic synapses. Permalink https://escholarship.ora/uc/item/6ks444r1, Journal The Journal of neuroscience : the official journal of the Society for Neuroscience, 26(21) ISSN 0270-6474 Authors Katona, Istvan Urban, Gabriella M Wallace, Matthew etal, 2006-05-01 bol 10,1523/jneurosci.0309-06.2006 License https://creativecommons.org/licenses/by/4.0/ 4. Peer reviewed WenaS428 TeleunaleMewsdece May 24,206 2601)504-S657 Development/Plasticity/Repair Molecular Composition of the Endocannabinoid System at Glutamatergic Synapses Istvan Katona,’ Gabriella M. Urban! Matthew Wallace,? Catherine Ledent,’ Kwang-Mook Jung,‘ Daniele Piomelli Ken Mackie,* and Tamés F. Freund! tnstute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary, Department of Anesthesiology, University of Washington, Settle, Washington 98185, "Universit Libre de Brueles,Istiut de Recherche Inedscpiaie en Boogie Humaine et Molecule, 1070 Bruxelles, Belgium, and ‘Department of Pharmacology and Center for Dog Discovery, University of Caornia, vin, Ievne, California 82697 Endocannabinoids play central roles in retrograde signaling ata wide varity of synapses throughout the CNS. Although several molec ‘ular components of the endocannabinoid system have been identified recenty, their precise location and contribution to retrograde synaptic signaling is essentially unknown, Here we show, by using two independent riboprobes, that principal cell populations of the hippocampus express high levels of diacyglycerol lipase a (DGL-a), the enzyme involved in generation of the endocannabinoid 2-arachidonoyi-glycerol(2-AG). Immunostaining with two independent antibodies against DGL~-c revealed that this lipase was concen- ‘tated in heads of dendritic spines throughout the hippocampal formation. Furthermore, quantification of high-resolution immunoclec- ‘ron microscopic data showed that this enzyme was highly compartmentalized into a wide perisynaptic annulus around the postsynaptic density of axospinous contacts but did not occur intrasynaptially. On the opposite side of the synapse, the axon terminal forming these excitatory contacts were found tobe equipped with presynaptic CB, cannabinoid receptors. This precise anatomical positioning suggests that 2-AG produced by DGL- on spine heads may be involved in retrograde synaptic signaling at glutamatergic synapses, whereas CB, receptors located on the afferent terminals are in an ideal position to bind 2-AG and thereby adjust presynaptic glutamate release asa function of postsynaptic activity. We propose that this molecular composition of the endocannabinoid system may bea general feature of most glutamatergic synapses throughout the brain and may contribute to homosynaptic plasticity of excitatory synapses and to het- cexosynaptic plasticity between excitatory and inhibitory contacts. Key words: mGluR5; DSI; GABA; interneuron; LTD: lipid MGL Introduction Molecular, anatomical, and physiological evidence has con- {Grmed critical involvement of the endogenous cannabinoid sys- tem in physiological and pathophysiological processes, shedding new light on its molecular components (Piomelli, 2003), Three lipase enzymes were recently identified, waich may contribute to the biosynthesis of lipid-derived endocannabinoid substances in the brain. An_N-acyl-phosphatidylethanolamine-hydralyzing phospholipase D was suggested to be responsible for the synthesis of the firstly discovered endocannabinoid anandamide (Devane ect 23,206 eed p20 cee, 288, ‘Mowshinsagpedby eva es Mette 45). Og dene te pronase es PE HOT hams 006] ED Dans ep an Cara. 1 2 is ‘oto. Weegee ae Nae te et elt ated ‘siempre Stason whet Reatthnt neg ly annessonsmnseptrkthdp sans Bim Da seb wih nea epee aint ni teem ac, Gppsh owes eneane OoeERCARSESISI0 et al, 1992; Okamoto et al, 2004), whereas two closely related nL-specific diacylglycerol lipases (DGL-a and DGL-) were proposed to mediate the formation of another endocannabinoid, 2-arachidonoyl-glycerol (2-AG) (Mechoulam et al, 1995; Sug- jura et al, 1995; Bisogno et a., 2003), Among several molecular targets potentially activated by endocannabinoids (Begg et al. 2005), two G-protein-coupled receptors, CB, and CB, cannabi- noid receptors, have emerged as Key elements of the endocan- ‘nabinotd system (Matsuda et al, 1990; Munro et al, 1993). Fi- nally, elimination of the endocannabinoids is performed by two-step process consisting of carrier-mediated internalization (Beltramo et al, 1997; Hillard etal, 1997; Fegley et al, 2004), {followed by intracellular hydzolysie catalyzed by faty-acid amide hydrolase and monoacyiglyceral lipase (MGI) for anandamide and 2-AG, respectively (Cravatt et al, 1996; Dink etal, 2002), ‘A major physiological ole of the endocannabinoid system is ‘the regulation of ncurotransmitter release at various types of syn- apses throughout the brain (Freund et al, 2003). Endocannabi- roids are lipid-derived messengers that are thought to be pro- duced posteynaptically on demand and evoked by specific physiological stimuli, but they are proposed to act presynaptically ‘on cannabinoid receptors (Alger, 2002; Wilson and Nicol, 2002). ‘This reverse mode of action makes them ideal candidates as ret~ ograde signals in several paradigms of short- and long-term syn-Kata eal «Theta Sem atthe tana Sape aptic plasticity (Chevaleyre eta, 2006). Most forms of synaptic plasticity require precise timing on the millisecond timescale and are strictly localized into subcellular microdomains such as den- dsitic spines in the case of glutamatergic synapses. It is widely believed that several forms of synaptic plasticity in this ater type ‘of synapse use endocannabinoids (for review, see Gerdeman and Lovinger, 2003; Diana and Marty, 2004); however, the underlying ‘molecular composition of the endocannabinoid system and its spatial organization remain speculative. To understand how the endocannabinoid system contributes to synaptic plasticity at glutamatergic synapses, itis essential to localize the exact site of synthesis of these compounds, identify the enzymes responsible for their formation, deline their molec- ular substrates, and, finally, determine their primary sites of ac- tion. Inthe hippocampus, 2-AG may be the main endocannabi- noid involved in synaptic plasticity (Stella eta, 1997; Makara et al, 2005; Straiker and Mackie, 2005). Here we show that DGL-a, a synthetic enzyme for 2-AG, is expressed by hippocampal prin- cipal cells and is strikingly concentrated in dendritic spine heads within a perisynaptic annulus encircling the postsynaptic density of excitatory synapses, Furthermore, we provide direct anatomi- cal evidence that these glutamatergic synapses are formed by axon terminals bearing presynaptic CB, receptors. This specific ‘molecular anatomical azchitectute provides the basis for 2-AG as a retrograde signaling molecule at glutamatergic synapses, Materials and Methods Perfusion and preparation of tinsue rections, Experiments were performed according othe guidelines ofthe institutional ethical code and the Hun {stan Act of Animal Care and Experimentation (1998, XVII, Section 2343/1998). Adult male CS7BLI6H mice (12 wild ype, 61 = 13 dela) and {CDI mice (Ghee wild type and three CB, knock-out all 57 dold) (Ledent tal, 1998) were deeply anesthetized with Equithesn (4.2% wv coral hydrate, 2.128 wiv MgSO, 16.29% w)w Nembutal, 39.686 wie propylene yea, and 1096 wiw ethanol in 1,0; 03 mif100 g, ip.) then perfused ‘with Zambons's fixative containing 4% paraformaldehyde in 0.1 phos: phate bufer(PB),pH74 eight CS7BL/6H mice, and the three wilde nd these knock-out CDI mice). Animals were perfsed transardilly, fist with 0.9% saline for 2 min, followed by 100 ml of Zamboni’ fixative for 20 min. An additional two mice were perfuced using the same fixative but with 0.1% glutaraldehyde. Another two mice were proceesed for a ‘eqenial low pHihigh pl! pesfasio (Berod's Sxative), In these cases, ‘he saline was followed bythe frst component of Berd fixative, pH 6 for S min and the second component of Berod's fixative for 50 min, PE 485. After perfusion, the brain was removed from the skull, and coronal sections (40 jms thick for m sit hybridization and 50 jum thick for Jimminocytochemistry) containing the hippocampus and the entire forebrain a the level ofthe dorsl hippocampus were cut with Leiea (Nussloch, Germany) VI5-1000 vibratome ‘Synthese of riboprobes for DGL-a. Twe nonoverlapping ectons of the mouse DGL-a coding sequence (see Fig. 14) (GenBank acession num: ber 4538590900) were amplified by reverse transeription-PCR from DNA derived from (otal C57BL/6H mouse frontal cortex mRNA, The length and the sequence of primers are listed below for both probes numbering of the nucleotide positions stars from the Beginning ofthe robe 1, $98 bp from 1184 to 1782 (forward prim. (CA ATA AG; reverse primer, 3'-CTA Gt ‘GCCGA GAT GAC CA): prabe 2, 1169 bp from 1967 0 135 (forward primer, 5-TCA GTA TCC GGG GAA CACTG; reverse primer, 5-AGG GCG ATG GIC AAA TCA Cr). The primers were designed using the Primers software (Rozen and Skaletsky, 2000). PCR products were cloned into the Smal ste of pBluescript If SK (Fermentas UAB, Vilsivs, Lithwania). The integrity and orientation af elanes were verified by 2 ‘quencing. Probe Iwas linearized by BamBIl and Ee32I digestion forthe antisense and sense probe, respectively. Probe 2 was linearized by HcoRE snd Bull digestion forthe antisense and sense probe, respectively. The Nees ay 24,2008» 2600188-567 + $29 lineatized template DNA was gel extracted, precipitated, resuspended in diethylpyrocarbonate (DEPC)-tested 11,0 ata concentration of ugh tl and stored at ~20°C, Invitro transcription was performed for 2h at ip a total volume of 20 pl containing 1 4g of template DNA, 1 tion butler, 1X digonigenin RNA labeling mixture, 40 U of Nave inhibitor, and 20 U of 13 or 17 RNA polymerase, which was juste 020 using DEPC-fre double-dstilled 0, Allcomponents ‘were from Roche Molecslar Diagnostics (Mannheim, Germany). L3- beled riboprobes were DNase treated and purified using the RNeasy MinFlute Cleanup kit (Qiagen, Hilden, Germany). Finally the integrity and quantity of the boprober were determined using gs! lectrophoresis. In sit hybridization All slutions used for in stu hybridization were fest treated with 0.1% DEPC for 1 h and shen autoclaved. Chemicals ‘were purchased from Sigma Aldrich (Budapest, Hungary) if otherwise not indicated. Incubation ofthe 40-ym-thick bain slices ws performed ina free-floating manner in RNasefre sterile culture well forall steps. Fist, the sections were washed in PBST (containing 137 mai NaCl 27 Lom NsIPOy 2 mai KiL#O, and01% Tween 20 174) night at ral of hybridization aller containing the digoxigenin-labeled ss°Cin riboprobe (2.5 ug/ml). Hybridization buffer consisted of 50% form amide, $x SSC, 96 SDS, 50 g/ml yeast RNA, and $0 g/ml heparin in DEPC-treated 1,0. During the overnight incubation and the following ‘three washing steps, che sections were continuously incubated on 2 cubation, the sections were Fist 2 ining 50% for amide, 5x SSC, and 196 SDS in DEPC-treated H,0) and then twice for 45 min a 65°C in wash Solution 2 (containing 50% formamide and 2 [SSC in DEPC ‘tested H,0). The section were next washed for 5 min in 0.05 u Tris-buffered saline (78S) containing(0.1% Tween-20 (TBST). pH 7.6, and then blocked in TBST containing 10% normal goat serum (TB- STN) for 1h, both at room temperature. Next, sections were incubated ‘overnight at 4°C with sheep anti-digoxigenin Fab fragment conjugated to alkaline phosphatase (Roche Molecular Disgnostis) diluted a 1:1000 in, "TBSIN. The next day, thesections were washed three times for20 min in "TBST and then developed with feshly prepared chromogen solution na total volume of 10m containing 3.5 ul of 5-bromo-4-chloro-3-indolyl- phosphate and 35 jl of nitroblue-tetazolium-chloride dissolved in ‘chromogen ber (contsining m¥ 100 NaCl, 100 me Tet-C, pH 9.5, 50 sma MgCl, 2 mi (~)tetramiole hydrochloride, and 0.196 Tween 20) “The sections were gently rineed in| ml ofthe above developing solution inthe dazk fo 4~6h, and the reaction wa stopped using PBST. Finally, the sections were washedin 0.1 sr PB three times for 10 min and mounted in Vectashield (Vector Laboratories Burlingame, CA} ont glass slides, and the coverslips were sealed with nal polish ‘Preparation of antibodies for DGL- a, Two polyclonal antibodies were raised in rabiteaguinst glutathione S-transferase (GST) fasion proteins containing residues 790-908 oF 10161042 of human DGL-a (see Fig 2A), Rabbits were immunized, and serum was collected at 3 week ‘val. mmune serum was purified by sequential afinity chromatography: the flow-through from 2 GST coliran wae applied to a fasion protein «column, and the antibody was eluted with 0.2m glycine, After neutral- ination with I neTris base, antibodies wer dialyzed against PBS contain~ ing 50% glycerol and stored st 20°C until ute. Antibody epecificty wae ‘stablished by staining HEK295 cell transiently expressing 83 epitope- lagged DGL-a with either purified antibody or purified antibody prein- ‘abated with 5 glnl immunizing protein. Preincubation with the i rmunizing protein strongly attenuated staining by the DGL-c antibody but notby the epitope tg antibody. In brain sections, the two antibodies revealed 2 similar immunostaining pattern (see Results and Figs. 2C.D, 3B,C. 48,0), which was ciminated by pretreatment with the corre- sponding immunizing protcin Thumumocytochemiry Ale slicing and extensive washing in.1 PB, the 50-ymcthick sections were incubated in 30% suerose overnight fol- lowed by freeze thawing ove liquid nitogen four times. ARerward, the sections were processed for immunoperoxidase, immunogold, or preemisedding immunogold stsining combined with 2 gecond immino- peroxidase staining. Subsequently all washing steps and dilutions ofthe630 News ay 2,206. 26828-5617 antibodies were done in 0.05 TBS, pH 7.4, After extensive washing in BS, the sections were blocked in $96 normal goat serum for 45min and len incubated in one ofthe two affinty-purfied rabbit anti-DGL-a (1:1000-1:3000; ~0.3-1 ug/ml) antibodies or guinea pig ant-CB, (1 il) (a gift ffom Prof. M, Watanabe, Hokkaiso University, Sapporo, Tapan) (deseribed by Fukudome et al, 2008) fora minimum of 48 hat °C. The specificity ofthe latter antibody was confirmed by the lack of Jmmusnostaining in CB, knock-out mice (Ledent etal, 1999). In this Control experiment, sections from wikdtype and knock-out animals ‘were mixed inthe incubation wells and processed together throughout chereaction. In theimmunoperoxidase staining procedute, alter primary body incubations, the sections were treated with bioinyated ant abit gG (1300) or with biotinylated anti-gunea pigIgG (1:30), both raised in goat, for 2 hand then with avidin biotinylated-horseradish peroxidase complex (1:50; Elite ABC; Vector Laboratories) for 13 b The immunoperoxidase reaction was developed using 3,3" diaminobenzidine (DAB) asthe chromogen. Inthe immunogold stain Jing procedure, the sections were incubated in 0.8 nm gold-conjugated fgontanterabbit of goat anci-guinea pig anbody for CB, of DGL-0, respectively (1:50 dition; Aurion, Wageningen, The Netherland), ‘overnight 4°C. Then the sections were silver intensified using the iver fenhancement system R-GENT SE-EM according tothe kt protocol (Au sion). Inthe double-immunostaining experiments, the sections were fist developed for immunogold and then for immunoperoxidase staining Lack of crose-eactivity of the secondary antibodies in the sequential Aetection scheme was verified by omission of ether primary antibody, hich eliminated labeling by the irelevant secondary antibody. Aer development of the immunostaining, the sections were seated with 19 0:0, in 0. 4 PB for 20 min, dehydrated in an ascending series of ethanol and propylene oxide, and embedded in Durcupen (ACM; Fluka, Buchs, Switzerland). During dehydration, the sections vere treated with 19 uranyl acetate in 70% ethanol for 20; embedded in Durcupan, areas of interest tioned for electron microscopy. Sections were collected on coated sngledot grids, stained with lead citrate, and examined with a Hitachi (Yokohama, Japan) 7100 electron microscope. Quantitative analysis ofthe distibution of DGL-v in the head of den Aric spines: To establish the precise subsynaptic or extrasynapic dis: sibution of DGL-a within the pyramidal spines, we performed a high-resolution quantitative evaluation in a population of 300 Jmmunogol-labeled dendtitic spine heads from three animals, Samples for electon microscopic analysis were taken from the stratum radiatum of the CA subfield ofthe hippocampus. Superficial ultrathin sections were collected (rst 5-10 jr) Because immunoreactivity decreased with {epth Tobe able to compare the mean distribution of DGL-«along the plasma membrane surface of dendritic spine heads withthe mean distri bution of metabotropic glutamate receptor aublype5 (mGluR), we fo: lowed the analysis procedure of Lujan and colleagues (fr details, see Txjan etal, 1996, 1997). Brey, the length of spine membrane from the ‘edges ofthe synaptic junction wae measured for every DGL-a-pasiive pine and vat divided into 60 nm bine. The localization of the gold particle representing DGL-a was measured a the distance between the closest edge of the postsynaptic density and the center ofthe imimino- parties present onthe pasta membrane ofthe spins. The three stm- ples were compared using Kruskal-Wallis nonparametric est, and data bd not iter significant (see Results), data were pooled and expresced asthe proportion of gold patile-contaning plasma membrane divi sions. In addition, we also analyzed the same dataset after normalization for the frequency of plasma membrane sents measured inthe ame population of spine heads Tujan and colleagues performed their thorough analysis in rats, whereas we performed our experiments in CS7BLIGH mice. The pub: ished mean synaptic membrane specialization length (meassred slong ‘the largest exten in the plane of sections that randomly eu the synapse) fo the spine heads in rats (189.6 = 52.1 nm) was similar tothe ange of values we obtained in mice (223.5 + 47 nm). Therefor, we believe that the twa spine populations used for analyst in the two studies alloss ‘comparing the distribution ofthese two functionally related molecules Kara eta Thedocnabini Spam athe rate Sse Figure 1, incall expres ih ees of Ge mRNA nthe ppocampes A, Ste anicrepreserain of he postions he we complementary arene mbprbes anche sequence themosseDS-c nN Thelengthofth open reading fae (RF ntheDGLa sis 3125 peng The AGeedeniicats theta inition site on the sequence 2a eoresentspston 3 inthe number, whereas TA the stap codon pston 133- 3135) Theda ovbaborethe cplexscemenscatthepredce equng of dgxgen- Ieednadestiesnteratiftheoalenghot the probes, which wert and 165 pin ‘he ase of pode 1 and poe 2, especie B,C nt hybsizatn by ung the tW0 tense probesilstted in uaesthe incall ayes ofthe mause pecs Ieexpesicleveis ver hgh inthe C8 and pyramidal neurons and smeubat wea, buts igh nthe gana cel ofthe centages Berge ros al cs ae ley the proes nde hse rain anions, indetng much ‘ester exes 3 eamplt ence of GLa in thee cel pes. at the etal Leng the worberbes, which cof thespety lth Econ stuybrizatin wing te ses ropes derived forthe conependig Da seu ano resin any abe, Seba, 200 ner BB. along the surface ofthe spines andi rl Results DGL-a mRNA is highly expressed by principal cells in the hippocampus Previous work suggested that DGL-o may be the main synthetic ‘enzyme for 2-AG in the adult brain (Bisogno et a, 2003). To determine the cellular expression pattern of DGL-aiin the mouse hippocampus, we prepared two independent digoxigenin- labeled riboprobes against the mouse DGL-a sequence corre- sponding to two nonoverlapping sequences (Fig, 1A). Nonradio- active free-floating in situ hybridization on mouse forebrain sections revealed a similar distribution pattern with both anti- sense riboprobes but showed no significant labeling with two ‘control sense probes (Fig, 1B-E). Highest DGL-a expression was observed in the hippocampus, in which the principal cell layers ‘were characteristically visualized by the staining (Fig, 18,C). Py- ramidal neurons in the CA3 and CAL subfields were always more strongly labeled than dentate gyrus granule cells. Weakly labeled cells were scattered in the hilus of the dentate gyrus; these cells ‘may correspond tothe so-called mossy cells, glutamatergic inter- neurons of the deniate gyrus, or GABAergic interneurons, Nei- ther interneurons in other layers nor glial cells were found to ‘express DGL-a, Conversely, we must note that the in st hybrid- ization reactions were performed under highly stringent condi- tions to avoid any nonspecific labeling, which may have resulted in reduced sensitivity. Nevertheless, we can conclude that gluta- muatergic principal cll types express DGL-ce at a very high level, DGL-a mRNA expression was also observed in other principal ‘ell types ofthe forebrain ata Iower level. A more detailed char- acterization of the regional and cellular expression pattern of a toneurotransmitterrelenteKata eal «Theta Ser atthe tana Srape Nees ay 24,2008 2620 S08-565 +5 (Fig. 2B). Similar to the mRNA distribu tion, the CAL and CAS subfields were gen- erally more strongly labeled, especialy the stratum oriens and stratum radiatum, whereas the labeling was somewhat fainter in the stratum lacunosum-moleculare, In contrast, the dentate gyrus showed some- what fainter immunostaining, except the inner third of the molecular layer, which was also strongly immunoreactive for DGL-a (Fig. 2B). At higher magnifica ‘don, a dense punctuated immunostaining was visible throughout the neuropil (Fig. 2D,). This characteristic staining pattern, outlined major dendritic shafts and cell bodies, which were only very faintly im- munopositive, if at all. Neither interneu- ronal nor glial processes were observed to ‘be immunostained in these sections. Im- munostaining for DGI-e in other fore- brain areas, including the neocortex and the basolateral amygdala, revealed a simi- Jar punctate staining pattern (data not shown). ‘To determine which cubcellular do- mains might underlie this characteristic staining pattern at the light microscopic level, we performed a detailed electron mi- pinta Figure2. _Lealzatn of GL ten inthe hppocnps A, Senate he preted tansmenbrane teplgy tthe DC rae, Pree SMART imple moda aciecure esearch ol ana sugges th GLa fturvansmemtrane domains anda long intracllr terial al whch cots pase oman taught be craton 2-Asynthss The woantbos gust we onovelppng eget cf heel ae iceatedy the hot shapes Te eptepe randy NT isa TB ese tech whereasorL26tisthe ls. 26 ane ose theCerios ard label he exaceoan incase eh psa ena, eect 8 Inansctchrt fer GL ‘etinetestheayered tucurf the hppcanps, hii determined te opgapy of ear pty. The spor tlie ine inner th ofstatum molec (sm), but gerry hestaingisdeserinthe CA sl than inthe remainder ote exate yas Sata ores ca ardradatim abbas ahigh dest fc immune, whereas immunostain nthe satu ust-nleare (Lm) apes more modes. Renata neast the eselabelaginthedenticayescelloesotbuthihe pyramidal esnstrtu pyramidal Jasweasthe grees instar ranlosum sg) aoa beled, ncatg ta te Dl poe istargee out one preceses ae sgihessinthe tls. On the who asenareut sian weaker abi pattern vse wing theL26 arbor inunostaning. 0. Athigheragifcan the mina dendies! (A pyran eweasappeartebeimsuncnegative ad ae cated by as dese punctuated inmeosanng pater, ete nea etal alg byte wo abodes, croscopic analysis. Samples taken from ‘most layers of all three major subfields of the hippocampus revealed the same stain- ing pattern with either antibody. The DAB end product of the immunoperoxidase staining procedure, which indicates the subcellular localization of DGL-a, was concentrated in a large number of den- dritic spine heads (Fig. 3A). Notably, al- though DAB gives rise to a diffusible reac tion end product, it did not fill the entire spine head (Fig. 3B,C). Instead, in most cases, it was unevenly distributed along the plasma membrane. Although we tried several fixation protocols and antibody di Confngthespeiy f h abodes Sears B, 60 n , ,20 un, DGL-a is currently underway (1. Katona et al, unpublished observations) DGL-is concentrated in dendritic spine heads of principal cells in the hippocampus ‘To study the precise subcellular localization of DG, we devel- coped two independent polyclonal antibodies against nonoverlap- ping epitopes on the C terminus of DGL-« (Fig. 2). The first antibody recognized sarge intracelilar loop (ab-INT), whereas the second antibody was raised against the lst 26 amino acids of| DGl-a (ab-126) (Fig 2A). The pattern of immunostaining with the two antibodies was similar at both the light microscopic and electron microscopic levels (Fig. 2B-D), although the general density of staining was much stronger for ab-INT and the labeled profiles were much sparse for the ab-126. Atlow magnification, the layered structure of the hippocampus corresponding to the termination zone of certain glutamatergic pathways was evident Iutions and our ultrathin sections were collected from the upper 5 um of the stained sections, we could not achieve the labeling of every spine head in our samples. This may either re- fect the existence af dendritic spines that lack DGL-a or itcan be simply explained by the possibility that the level of DGL=< in these immunonegative spines is below the detection threshold of ‘our antibodies, This second possibility is supported by the obser- vation that the moze sensitive ab-INT always visualized a higher ratio of DGL-a-positive dendritic spines than ab-126, Neverthe- less, in most cases, the ratio of DGI-a-conlaining spines was above 50% with cither antibody, and, in random samples from ‘the strata radiatum and oriens, >80% of spines were positive for DGL-a. Because the ratio of immunopositive spines is strongly «dependent on the success of fixation, the penetration ofthe anti- body and several other unknown factors such as masking the ‘epitope by other interacting proteins, a precise quantification is not feasible, Nevertheless, although we cannot exclude the possi- bility that every spine contains some DGL-a, we consider that the 50% ratio should be regarded as the absolute minimum estimate,632 Lew May 2,206. 26028-5817 ure 3. DGL-ainmanchbelng is conenate inthe ead of dent spas nthe Nppscampus. Low-power elton miceraph ef Da immanostaing inthe statu ves af the CA sil vsles rumerus eit pins dpi) cemtaning the ese nd produc of themmnoperose reaction (98). These DSL-a-cataing pes reve aati saps om Dl-a-rgatie oun (Note the abe seine Teal finnanoencity within the pne ants absence inher neuronal an gl recess B Incase of tage seine spies, which sh ek th base, nthe Conespanng eit safe abe inthe sane plane asthe head, he incre tieriteralindating tellin of L-ahavs highly amparmentale stb thn itet tothe spe head. These sie] abo eee asl asymeial apse cto thei ofthe spine ead by Dl-ccngative aan teri). Noe he sear dstbten prefles he mmanereacive material by th two abodes," and "128" coniring theispect'y. Salebar: A-C02 pm. In contrast to the strong labeling at glutamatergic synapses, none of the antibodies revealed consistent labeling at sites postsynaptic to GABAergic boutons or in other postsynaptic subcellular do- mains, However, these negative findings may be subject to the same limitations discussed above, DGL-avis concentrated in a characteristic perisynaptic annulus around the postsynaptic density at aslutamatergic synapses Although dendritic spines ate specialized microdomains them- selves, recent studies have revealed that they are subdivided into discrete morphological and functional units, which contribute to distinct aspects of synaptic signaling and plasticity. Therefore, we used the resolving power of the silver-eabanced immunogold technique to obtain additional insights and predictions about the potential functional role of DGI-«vat the subsynapticand molec- Figured. 06L-aspresrtonthe plasma mentran inthe eae! ipecampaldedic spies. A, igh-esetonpreembedingirmanogalé staining fr Dl demonstrates tha ‘hs Ips presen te plasma meran af entices. In thigh power eleran ncogrn, the three dene spines (ese an asymm saps rm two aan ‘erin. Thechaacerisicelecren dense postsynaptic oft srape spel ‘on inate tht hee rear Gatergyapes. 8G High-power etn 80% of axon terminals with asym- ‘metrical synapses were unequivocally positive for CB, in the in- ner third of stratum moleculare (Fig. 7A), the most strongly la- beled layer atthe light microscopic level. In other layers of the bippocampus, we typically obtained a ratio of ~30-30%%, High- resolution silver-enhanced immunogold staining further con- firmed the validity ofthe findings, because immunogold particles representing the precise subcellular localization of CB, were al-634 ews May 2,206. 26028-5817 Figure 6. Leathe, caoatinal ceptor oe inthe ppaconpus eo ‘atest opyapialamangenetofguamater paths A ght micegraph ist ing, mmunoeaciiyintehppocnasefwlé ype (WT nose Bywinganorelguines gantody,heimmersainghighghs te dfeentayersatheipescampusacading tothe spatial arangerent ofthe eva pathway ncaa rmunatnng inthe opscampu of Bada mewe shows no rmapete res, eenstating arubody pect. Athigher maga, the rauncreacie Aer tee ransadsheebaraceritichshihe avon ars lente satura) ‘he HS suid, eer he ese nol abligtaughou the sataradatu (2) adovens (a), nwichlclaxon alters! the C3 pyranidalneuons ari, peas Instron cna wih the immunonegatn sratumluiu (the terminationzoneathe ‘xan terial ofthe ane cell the CA uel the sare Bt sarees rearop abl eb ses thenteearonal profes Inte deta gs (0), ‘hemos stkin beng appease inner hid of sven malar 6m), white terinaton anf haar olen, he gatamatercinereon afte det ‘rs Mode sang ioundin the ote parts f tatu mle, wees ny inte earn semata an ates are vilein the statu rls (9) Sale as: A,B, 300 un 650 m0, 75 wm, ‘ways attached to the intracellular side ofthe plasma membrane as predicted by the spatial localization of the epitope (Fig. 78D). “To determine whether the presence of CB, receptors on gli tamatergic axon terminals was species ot strain dependent, we repeated the experiments in both C57BL/6 and CDI mice and found no differences. Furthermore, immunostaining for CB, us- ing the novel antibody visualizes glutamatergic axon terminals in both rat and human hippocampus (Katona et al, unpublished observations) DGL-a and CB, receptors are colocalized on the postsynaptic and presynaptic sides of glutamatergic contacts, respectively Because all three antibodies used inthis study resulted in incom- pletelabeling of the corresponding neuronal profiles, the colocal- iuation ofthese postsynaptic and presynaptic proteins within the same spine population cannot be inferred from single immuno- staining experiments. Exploiting the fact that the primary anti- bodies against DGL-a or CB, were raised in rabbit and guinea pig, respectively, we performed double immunostainings in which DGL- was visualized using the immunogold procedure and CB, was visualized using the immunoperoxidase (DAB) technique. Extensive electron microscopic analysis confirmed in _most layers ofall three subfields ofthe hippocampus that DGL-« is localized on postsynaptic spine heads receiving an asymmetr cal synapse from CB, bearing axon terminals (Fig. 8). Kana eta Thedocnabii Spam ate raat Sse Discussion “According to the current dogma, endocannabinoids are derived ‘rom postsynaptic elements; hence, they may be retrograde mod- ulatorsin a number of synaptic plasticity paradigms. Conversely, ‘the precise source of endocannabinoids and the enzymes respon- sible for ther on-demand synthesis at diferent types of synapses zemain unknown. Inthe present study, we found the following: (1) DGL-a, a primary synthesizing enzyme for the endocannabi- ‘noid 2-AG, is highly expressed by the glutamatergic principal ell populations ofthe hippocampus; (2) DGL-« is concentrated on the head of dendsitc spines, the specialized postsynaptic mi- ‘rodomains receiving glutamatergic synaptic input; (3) in rla- tion to glutamate release sites, DGI-a is strikingly concentrated in the perisynaptic annulus around the synaptic specialization with a decrement along the extrasynaptic membrane surface, but itis almost entirely excluded from the synaptic junction itself and (4) on the opposite side ofthe glutamatergic synapse, CB, ‘cannabinoid receptors are localized presynaptically on glutama- tergic axon terminals on most excitatory pathways in the Iippocampus Postsynaptic DGL-a at glutamatergic synapses ‘The most important finding of the present study is the provoca- tive gradient of DGL-a within the head of dendritic spines of, pyramidal neurons and granule cells. Three experimental find- ings support the validity of this immunocytochemical result. ‘First, n situ hybridization using two independent riboprobes 1e- vealed very high expression levels of DGl-a in the three major ‘el types bearing dendritic spines, namely inthe granule eels of the dentate gyrus and in pyremidal cells of the CA3 and CAL subfields, Second, the strong DGL- immunoreactivity in spine heads was observed using two distinct antibodies raised against ‘two independent epitopes on DGL-« Third, high-resolution im- ‘munogold labeling by both antibodies resulted ina labeling pat- tern corresponding tothe predicted topology of DGL-a, ie im- ‘munogold particles were always attached to the intracellular surface ofthe spine plasma membrane. Dendsitic spines are highly versatile suructures. It is widely accepted that their activity-dependent reorganization reflects, cexperience-dependent changes in neuronal function (Segal, 2005). The head of the spine is usually innervated by a single ‘excitatory axon terminal, which forms characteristic asymmet- rical synapse with a pronounced postsynaptic density. The na row spine neck serves asa barrier for most signaling pathways to ‘ensure synapse-specifc plasticity mechanisms. From this aspect, itis interesting to note that current models identify endocannabi- ;oids asthe most probable candidates to serve as the retrograde signal in homosynapticlong-term plasticity at glutamatergic syn apses (Gerdeman etal, 2002; Robbe et a, 2002; Sjostrom etal, 2003). The finding that the endocannabinoid synthesizing en- zyme DGL-vis localized on the head of the spine isin complete ‘agreement with this proposed model Furthermore, several find- ings point to 2-AG as the main endocannabinoid involved in hippocampal synaptic plasticity at both the glutamatergic (Stella, etal, 1997; Striker and Mackie, 2005) and GABAergic (Cheva- leyre and Castillo, 2003; Kim and Ager, 2004; Makara eal, 2005) synapses, Because DGL.-a maybe a key enzyme for 2-AG synthe sis in the postnatal brain (Bisogno etal, 2003), the demonstra- tion of its presence postsynapticaly at glutamatergic synapses provides anatomical support forthe conclusion of previous phys iological experiments obtained by pharmacological tool, Remarkably, we did not find DGL-a labeling at symmetrical synapses formed by GABAergic boutons despite focused search-Kata eal «Theta Sem atthe tana Sape Figure. ing ‘Thismay simply reflect alevel of DGL-thatisbelow the thresh- cof detection in our experiments, Conversely, two laboratories recently reported thatthe exclusively postomaptic depolariza- tion- dependent form of endocannabinoid-mediated symaptic depression at GABAergic synapses “conventional” depolarization-induced suppression of inhibition (DSD)] is not blocked by DG. inhibi- tors (Chevaleyre and Calo, 2003; wards ota, 2006). In contrat, inhibitors ofthe 2-AG degrading enzyme, MGI, pro- Jong DSi, suggesting 2-AG involvement in this form of synaptic plasticity (Makara eal, 2005) I wil be important to determine twhetheraltemative biochemical pathways for 2-AG synthesis ‘eg involving phospholipase A, and lyso-phospholipase C (PLC), operate at GABAergic synapses, Simularly, the lack of DGL-a in GABAergic interneurons may reflect an expression level below our detection threshold, However, it has been te- ported that retrograde endocannabinoid signaling is absent from these cells (Hfotiman et a, 2008). Together, thse findings pro- vide lear evidence that distinct endocannabinoid signaling path- ‘ways exis in parallel inthe brain, and, to understand tht phys Jologiesl significance (eg, bow they are recraited and a which types of synapses they operate), the precise molecular and spatial properties of each of their components mest be carefully determined, Perisynaptic DGL-a pool underlies a functional link to -mGIURS receptors Ample evidence is available that the endocannabinoid system is also involved in heterosynaptic plasticity (for review, see Cheva- (Bcanabinid reps areca presyaptaly an tth gtamatergiandABReniaon emia be Napcamous A Te elcron nicogaphdenentaes the stking accrltion fang mrarorectywiinasan terminals inthe inethed of esta molec ote dentate gs. Two pes of botos thee fo, Ecaary _2antemial which frm asymealsyapes dpi arawbeasanaiitary rote, wich formset al yapss indicated by Single sow. ABRer aon temas which ek pesyapti CB, ree ae labled by double atows. BD Inenaogall labeling eels achnacersticpresyaptcleauaton fC, whch sata tte tae sufecftbepsmamentraeinbth psf axon emia, )inacordance with eptopelcaatin. Te elecran cogs ae taken fom satu leave, Basel scons stratum ies ofthe AS abel (ado stata atu of he RT bl (0 age aos st rune parce reser te aatan of, wheres arowketspont sexo spapesardsmallaronspittainhiy sane SalebrsA 05 wr B-D.02 um. Nees ay 24,2008 2600 $8-56 +538 leyre etal, 2006). The best example ishet- crosynaptic long-term depression of inhi- bition (LTD) in CAL pyramidal cells. ‘This phenomenon is expressed presynap- tically on GABAergic axon terminals, but induced postsynaptically by stimulation of the glutamatergic Schaffer collaterals, and is dependent on the activation of postsyn~ aptic type I mGluRs (Chevaleyre et al, 2003; Chevaleyze and Castillo, 2004), CAL pyramidal cells express mainly the mGluR5 subtype of type I mGluRs, whereas mGluR] is found in selected types of interneurons (Baude etal, 1993; Lujan etal, 1996). A precise analysis ofthe sub- cellular distribution of mGluRS on CAL pyramidal cells revealed that these recep- torsare segregated into perisynaptic pool around the postsynaptic specialization on, the head of dendritic spines (Lujan et al, 1996, 1997). Using the same criteria for DGL‘a on a large population of CAI py- ramidal cell dendeitie spines, we found a similar perisynaptic accumulation within 60 nm of the edge of the synaptic junction asreported for mGIuRS (Lujan etal., 1997, their Fig. 5B,B'), along with a similar gra- dient of decreasing extrasynaptic distibu- tion, Importantly, mGluR5 activates PLC-B, which produces certain DAG spe- cies, including those with arachidonic acid at the sn-2 position that serve as precur~ sors for 2-AG synthesis. Indeed, pharma- cological activation of mGIuRS induces a ‘considerable amount of 2-AG release in striatal and hippocampal ‘cultures, which can be blocked by PLC-B and DGL inhibitors (Jung et al, 2005). The inhibition of I-LTD by DGL inhibitors (Chevaleyre etal, 2003; Edwards et al, 2006) and the perisynap- Lic colocalization of mGlURS with DGL-a suggest that mGluRS and DGL-a cooperate to produce 2-AG at glutamatergic syn- apses during heterosynaptic long-term depression. Iti notewor- thy that type 'mGIUR activation also contributes to other, short- term forms of endocannabinoid-dependent synaptic plasticity at hippocampal GABAergic synapses (Varma et al, 2001; Ohno- Shosaku etal, 2002a), which are also dependent on DGL activity (Edwards et al, 2006). In contrast, mGluRs are not involved in DSI, because DSI cannot be blocked by DGL inhibitors (Cheva- leyre and Castillo, 2003; Edwards et al, 2006) and persists in PLC-BI knock-out animals (Hashimotodani et al, 2005). This ‘stiking heterogencity in the biochemical signaling pathways and the spatial segregation of mGluR5 and DGL-a suggest that 2-AG- ‘mediated endocannabinoid signaling may differ between distinet types of synapses and contribute differently to homosynaptic and heterosynaptic plasticity. Presynaptic CB, cannabinoid receptors at glutamatergic synapses ‘The molecular identification of presynaptic cannabinoid recep- tors at glutamatergic synapses in the hippocampus has been a ‘controversial issue since the frst description of cannabinoid ef- fects on excitatory neurotransmission (Shen et al, 1996). Al- ‘though the specificity of weak CB, mRNA signal in hippocampal pyramidal ces was confirmed by using an elegant mouse model