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Neuroscience 204 (2012) 38–52

REVIEW

ENDOCANNABINOID SIGNALING IN THE AMYGDALA: ANATOMY,
SYNAPTIC SIGNALING, BEHAVIOR, AND ADAPTATIONS TO STRESS
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T. S. RAMIKIEa AND S. PATELb,c*aNeuroscience Graduate Program, Vanderbilt University School of

edicine, Nashville, TN 37232, USAbDepartment of Psychiatry, Vanderbilt University School of Medicine,

ashville, TN 37232, USAcDepartment of Molecular Physiology and Biophysics, Vanderbilt Uni-ersity School of Medicine, Nashville, TN 37232, USA

Abstract—The molecular constituents of endocannabinoid(eCB) signaling are abundantly expressed within the mamma-lian amygdaloid complex, consistent with the robust role of eCBsignaling in the modulation of emotional behavior, learning, andstress-response physiology. Here, we detail the anatomical dis-tribution of eCB signaling machinery in the amygdala and therole of this system in the modulation of excitatory and inhibitoryneuroplasticity in this region. We also summarize recent find-ings demonstrating dynamic alternations in eCB signaling thatoccur in response to stress exposure, as well as known behav-ioral consequences of eCB-mediated modulation of amygdalafunction. Finally, we discuss how integrating anatomical andphysiological data regarding eCB signaling in the amygdalacould help elucidate common functional motifs of this systemin relation to broader forebrain function.

This article is part of a Special Issue entitled: Stress, Emo-tional Behavior and the Endocannabinoid System. © 2011IBRO. Published by Elsevier Ltd. All rights reserved.

*Correspondence to: S. Patel, Departments of Psychiatry and Molec-ular Physiology and Biophysics, Robinson, Research Building Room724B, Vanderbilt University Medical Center, Nashville, TN 37232,USA. Tel: �1-615-936-7768.E-mail address: [email protected] (S. Patel).Abbreviations: AC, adenylate cyclase; AEA, anandamide; BA, basalamygdala; BLA, basolateral amygdaloid complex; BMA, basomedialamygdala; CB1, cannabinoid receptor type-1; CB1ir, cannabinoid re-ceptor type-1 immunoreactive; CCK, cholecystokinin; CeA, centralamygdala; CeAL, central amygdala lateral; CeAM, central amygdalamedial; CRHR1, corticotrophin releasing hormone receptor type-1;DAGL�, diacylglycerol lipase alpha; DHPG, dihydroxyphenylglycine;dLA, dorsal lateral amygdala; DSE, depolarization-induced suppressionof excitation; DSI, depolarization-induced suppression of inhibition; EC,external capsule; eCB, endocannabinoid; EM, electron microscopy;eIPSC, evoked inhibitory postsynaptic current; EPSC, excitatory postsyn-aptic current; EPSP, excitatory postsynaptic potential; FAAH, fatty acidamide hydrolase; GAD65, glutamic acid decarboxylase 65; GPCR, G-protein coupled receptor; HFS, high-frequency stimulation; ICMs, inter-calated cell masses; IPSC, inhibitory postsynaptic current; IPSP, inhibi-tory postsynaptic potential; ISH, in situ hybridization; LA, lateral amygdala;LFS, low-frequency stimulation; LTD, long-term depression; LTDi, long-term depression of inhibitory transmission; LTP, long-term potentiation;MeA, medial amygdala; MGL, monoacylglycerol lipase; mGluR, metabo-tropic glutamate receptors; mPFC, medial prefrontal cortex; PKA, proteinkinase A; PLC, phospholipase C; PPR, paired pulse ratio; sIPSC, spon-taneous inhibitory postsynaptic current; THC, �9-tetrahydrocannabinol;

hLA, ventral lateral amygdala; 2-AG, arachidonoylglycerol; 5-HT 3, sero-onin type 3 receptor.

0306-4522/12 $36.00 © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2011.08.037

38

Key words: marijuana, CB1 receptor, anxiety, fear condition-ing, anatomy, cannabis.

ContentsEndocannabinoids 38The amygdala 39

Anatomy of eCB signaling in the amygdala 39CB1 receptor 39FAAH 41MGL 41DAGL 41Summary 41

Synaptic modulation by eCBs in the amygdala 41eCBs modulate GABAergic transmission in the BLA 41eCBs modulate glutamatergic transmission in the BLA 43eCB modulation of synaptic signaling in the CeA 44In vivo electrophysiological studies 45Summary 46

Stress-induced adaptations in eCB signaling in the amygdala 46Biochemical adaptations 46Synaptic adaptations 47

Behavioral consequences of cannabinoid-mediatedmodulation of amygdala function 47

Behavioral effects of eCB signaling in the BLA 47Behavioral effects of eCB signaling in the CeA 48Summary 48

Future directions 48Comparing eCB modulation of amygdala neurocircuitry with

other forebrain circuits 49References 49

Endocannabinoids

Endocannabinoids (eCBs) comprise a family of arachidonicacid-derived lipid molecules synthesized by vertebrate andinvertebrate animals (Piomelli, 2003; Di Marzo et al., 2005).eCBs include 2-arachidonoylglycerol (2-AG), anandamide(AEA), noladin ether, and several other candidate molecules.2-AG is synthesized by conversion of sn-2 arachidonic acidcontaining diacylglycerols to the monoacylglycerol 2-AG, viaan sn-1-specific diacylglycerol lipase (DAGL) (Bisogno et al.,2003) and is degraded primarily by monoacylglycerol lipase(MGL) (Dinh et al., 2002b; Blankman et al., 2007). On theother hand, synthesis of AEA has been demonstrated tooccur via multiple pathways but is degraded primarily by fattyacid amide hydrolase (FAAH) (Cravatt and Lichtman, 2002;Ahn et al., 2008; Ueda et al., 2010).

Although many molecular targets of eCBs have beenescribed, the primary targets of these compounds are theannabinoid receptor types 1 and 2 (CB1 and CB2, re-pectively). eCBs are produced by almost all cell types that
ave been examined and the CB1 and CB2 receptors are
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T. S. Ramikie and S. Patel / Neuroscience 204 (2012) 38–52 39

present in both nervous and peripheral tissues. Here, wewill primarily focus on CB1 receptor-mediated eCB signal-ing, as it is most heavily implicated in the synaptic andbehavioral functions of cannabinoids (Chevaleyre et al.,2006; Hashimotodani et al., 2007; Heifets and Castillo,2009; Kano et al., 2009).

Within neurons of the CNS, initiation of eCB signalingresults in retrograde inhibition of afferent neurotransmission.Specifically, postsynaptic eCB synthesis, initiated via voltage-dependent calcium channel activation or G-protein-coupledreceptor (GPCR)-dependent mechanisms, results in releaseand diffusion of eCBs into the synaptic cleft (Kano et al.,2009). Thereafter, eCBs bind to the CB1 receptor localized toaxon terminals of glutamatergic and GABAergic neurons,thus activating intracellular signaling downstream of Gi/o pro-eins and causing either short-term or long-term suppressionf vesicular neurotransmitter release (Chevaleyre et al.,006). This signaling motif has been described in numerousrain regions, including the brainstem, midbrain, cerebellum,ippocampus, striatum, amygdala, and neocortex. The wide-pread distribution of eCB-mediated synaptic signaling un-erscores the pleiotropic functions of eCBs in the regulationf motor control, learning and memory, pain, motivation, met-bolic integration, and affect regulation (see Kano et al.,009).

he amygdala

he amygdaloid complex comprises several interconnectedegions which can be classified into cortical-like nuclei includ-ng the basolateral complex (basolateral amygdaloid complexBLA]; which comprises the lateral [LA], basal [BA]), andasomedial [BM]) nuclei), as well as the centromedial nuclei,hich include the central amygdala (CeA; which is subdi-ided into the lateral [CeAL] and medial [CeAM] compart-ents) and the medial amygdala (MeA) (see Sah et al., 2003;ape and Pare, 2010 for review).

The BLA exhibits a cortical-like structure (i.e. being com-rised of principal projection neurons and local interneurons),hereas the CeAL exhibits a striatal-like cytoarchitecture

medium spiny-like, predominantly GABAergic neurons).urthermore, it has been suggested that cells of the CeAMhare significant homology to pallidal neurons based oneurochemical, connectional, and cytoarchitectural evi-ence (Cassell et al., 1999; McDonald, 2003). Thus, theoncept of a cortico-striato-pallidal motif, traditionally con-eptualized as a functional unit of forebrain organizationelevant to motor/cognitive control, may also be relevanto the fundamental organization of the amygdala (Cassellt al., 1999). There also exists a population of identifiedmygdalar neurons known as intercalated cell massesICMs). These are groups of tightly packed GABAergicnterneurons that form islands at the anatomical interfaceetween the BLA and CeA (Royer et al., 1999; Paré et al.,004). Functionally, these cells are thought to provideeed-forward inhibition onto CeM neurons in response toLA input (Samson and Paré, 2006; Amano et al., 2010).

The BLA receives massive cortical and thalamic inputarrying monomodal and highly processed polymodal sen-
ory information (Sah et al., 2003; Pape and Pare, 2010).
he BLA contains glutamatergic pyramidal projection neu-ons that exhibit very low rates of spontaneous activity dueo a high level of tonic GABAergic input supplied by localnterneurons. There are also extensive reciprocal, predom-nantly excitatory, connections between various nucleiithin the BLA (Sah et al., 2003; Knapska et al., 2007).urthermore, the BLA projects to the nucleus accumbens,ippocampus, prefrontal cortex, and the CeA (Sah et al.,003; Knapska et al., 2007).

The CeAL contains predominantly GABAergic neuronshat receive excitatory input from the BLA, cortex, thala-us, and brainstem autonomic nuclei carrying visceral, asell as nociceptive information, and in turn, project to thearabrachial nucleus, the bed nucleus of the stria termina-

is, basal forebrain, and CeAM (Knapska et al., 2007). TheeAM is considered the output structure of the amygdala,nd receives excitatory input from the cortex, BLA, brain-tem, and hypothalamus, and tonic inhibitory input fromhe CeAL (Sah et al., 2003; Knapska et al., 2007; Ciocchit al., 2010).

From this anatomical organization, it is clear that theLA is positioned to receive information regarding salientnvironmental stimuli and orchestrate instrumental re-ponses to these stimuli via projections to the prefrontalortex and nucleus accumbens, as well as to the CeAM,hich in turn targets hypothalamic, brainstem, and reticu-

ar structures. Thus, the BLA and CeA are often consid-red to comprise a serial pathway, where information fromhe BLA is transferred to the CeA to modulate specificndocrine, autonomic, and arousal-related responses (Da-is, 1997; Davis and Whalen, 2001). However, a parallelrocessing system, where the BLA and CeA serve distinctunctions that operate independently has also been sug-ested (see Knapska et al., 2007 for review).

Considerable work has established the amygdala as aey site involved in the generation of fear and anxiety re-ponses, the assignment of emotional salience to externaltimuli, the coordination of affective, autonomic, and behav-

oral responses to such stimuli, as well as Pavlovian learningrocesses (Davis, 1997; Paré et al., 2004; Pape and Pare,010). Significant recent progress has been made to eluci-ate the complex synaptic connectivity between amygdaloiduclei and the means by which afferent information is pro-essed within the amygdala to affect behavioral outcomes.ver the past decade, eCB signaling has emerged as a keyeuromodulatory system that regulates synaptic efficacyithin and between amygdaloid subnuclei. Here, we summa-

ize data elaborating the role of eCB signaling in amygdala,he adaptations in this system that occur in response totress, and the behavioral consequences of eCB-mediatedodulation of amygdala function.

ANATOMY OF eCB SIGNALING IN THEAMYGDALA

CB1 receptor

The CB1 receptor is a membrane-associated GPCR that isexpressed widely, but not exclusively, within the CNS. The
CB1 receptor is activated by the principle psychoactive

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T. S. Ramikie and S. Patel / Neuroscience 204 (2012) 38–5240

constituent of Cannabis sativa, �9-tetrahydrocannabinolTHC), as well as synthetic and endogenous ligandsPertwee, 2005). In situ hybridization (ISH) studies in de-eloping and adult rodents and humans have repeatedlylassified CB1-expressing neurons into two groups: low-xpressing and high-expressing. High CB1-expressingells are sparsely distributed within the BLA and otherortical structures, whereas low CB1-expressing cells areore evenly distributed and found within both the BLA and

entromedial nuclei (Mailleux and Vanderhaeghen, 1992;atsuda et al., 1993; Marsicano and Lutz, 1999; Chhatwalt al., 2005; Hermann and Lutz, 2005; Yoshida et al.,011).

Marsicano and Lutz provided the first detailed descrip-ion of CB1 receptor mRNA expression within the mousemygdala (Marsicano and Lutz, 1999). These authors re-orted the presence of both high CB1- and low CB1-xpressing cells within the BLA and low levels of CB1RNA in the central amygdala. These authors showed

hat �95% of high CB1-expressing cells co-expressed theABAergic marker glutamic acid decarboxylase 65

GAD65). Furthermore, almost all high CB1-expressingells, and 90% of low CB1-expressing cells, co-expresshe peptide cholecystokinin (CCK). Subsequent work byhis group demonstrated that 38% of CB1-expressing neu-ons within the BLA co-expresses corticotrophin releas-ng hormone receptor type-1 (CRHR1) mRNA, and allRHR1-expressing neurons within the BLA co-expressB1 mRNA (Hermann and Lutz, 2005).

Co-expression of serotonin type 3 receptor (5-HT3)nd CB1 has been demonstrated within the BLA (Hermannt al., 2002; Morales and Bäckman, 2002; Morales et al.,004). Between 16% and 36% of CB1-expressing neu-ons, depending on the subregion of the BLA, expressranscript for 5-HT3 receptors. Conversely, 37%–55% of-HT3 receptor-expressing neurons also express CB1 re-eptor transcript. These co-expressing neurons corre-pond to the GABAergic, high CB1-expressing populationithin the BLA (Morales et al., 2004).

Within the CeA, CB1 mRNA expression has generallyeen described as low but present (Matsuda et al., 1993;arsicano and Lutz, 1999; Chhatwal et al., 2005; Hermannnd Lutz, 2005). However, it is unclear from these studieshether there are differences in CB1 mRNA expressionithin subregions of the CeA (Chhatwal et al., 2005).

Immunohistochemical studies have also revealed theresence of CB1 receptor immunoreactivity within the rodentmygdala. The first detailed description by Tsou and co-orkers, using an antibody directed against the N-terminal

of the CB1 receptor, revealed CB1-immunoreactive(CB1-ir) neurons within both the centromedial nuclei andthe BLA (Tsou et al., 1998a). Using this antibody, Mc-Donald and Mascagni found light staining in principal neu-rons of the BLA, other cortical-like amygdaloid nuclei,CeAL, and anteroventral division of the MeA. In addition,light CB1-ir dendrites of pyramidal cells were also ob-served in all BLA nuclei. Double-labeling studies revealedthat between 60% and 81% of high-CB1 expressing neu-
rons within the BLA co-expressed CCK. Furthermore, all
medium- to large-sized CCK neurons (type L) co-ex-presses CB1 (100% co-expression of CB1 and CCK inL-type CCK-positive neurons), whereas only a small pop-ulation of the small CCK-expressing neurons (type S) co-expresses CB1 (10%–14% colocalization depending onanatomical subregion) (McDonald and Mascagni, 2001).

Freund and co-workers used a CB1 receptor antibodyraised against the C-terminal intracellular tail of CB1 re-ceptor to explore its immunohistochemical distributionwithin the mouse and rat amygdala (Katona et al., 2001). Ingeneral, the densest immunoreactivity was found withinthe BLA and related cortical-like nuclei, whereas the CeA,MeA, and ICMs were not immunoreactive for CB1. Themost prominent feature of the CB1 immunostaining in thisstudy was a dense meshwork of varicose axon collaterals.These axon collaterals were observed to form pericellulararrays around immunonegative cell bodies, whereas nodendritic staining was observed using this antibody. Thispattern of staining was also observed by Elphick and co-workers in rats and mice using a C-terminal antibody(Egertová et al., 2003). In line with ISH data, double-labeled immunofluorescence experiments revealed that88% of CB1-ir neurons co-expressed CCK with only thelarge CCK expressing neurons co-expressing CB1 (Ka-tona et al., 2001).

These authors also investigated the subcellular distri-bution of the CB1 receptor using electron microscopy (EM)(Katona et al., 2001). Within the BLA, immunogold labelingwas observed within intracellular membrane compart-ments including rough endoplasmic reticulum and golgi. Inaddition, multivesicular bodies were densely labeled. Fur-thermore, no staining was observed on either the plasmamembrane of cell bodies or proximal dendrites. Denselylabeled axon terminals were also observed using immuno-gold labeling on the intracellular surface. These axon ter-minals formed symmetric, presumably GABAergic, con-tacts with somata and dendritic shafts. CB1-expressingterminals were also found to co-express CCK and formexclusively symmetric synapses (Katona et al., 2001).

Ong and Mackie, using an N-terminal CB1 antibody,provided a description of CB1 receptor distribution withinthe primate brain (Macaca fascicularis) (Ong and Mackie,1999). Their studies also reported that the amygdala wasdensely labeled for the CB1 receptor. Within the BLA, bothputative pyramidal projection and nonprojection neuronswere labeled. EM studies revealed large, CB1-ir cell bod-ies with features of pyramidal projection neurons, as wellas labeled smaller neurons with features of GABAergicinterneurons. Many densely labeled dendrites were ob-served in the neuropil of the LA, which formed symmetriccontacts with unlabeled axon terminals. In addition, occa-sionally labeled axon terminals forming asymmetric syn-apses were also observed (Ong and Mackie, 1999).

Eggan and Lewis used C-terminal antibodies to ex-plore the distribution of CB1 receptors within the amygdalaof this species as well (Eggan and Lewis, 2007). Theirfindings were in qualitative agreement with those of Ongand Mackie as they observed a dense meshwork of CB1-ir
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the BLA; although some differences in the intensity ofstaining was seen within various subregions of the BLA.No CB1-ir axons were observed within the centromedialnuclei (CeA and MeA). However, light staining above thatof the white matter was observed within these nuclei,although this labeling was noted to be morphologicallyindistinct (Eggan and Lewis, 2007). Although CB1 receptorexpression within the amygdala may shed light onto itspotential modulatory mechanisms, exploring the expres-sion of the enzymes that are integral to eCB metabolismmay further unveil the role of eCB signaling within theamygdala.

FAAH

FAAH is a serine hydrolase that catalyzes the degradationof AEA into arachidonic acid and ethanolamine (Ahn et al.,2008). Initial ISH studies by Cravatt and co-workers re-vealed an intense ISH signal within the amygdala, andspecifically within the BLA (Thomas et al., 1997). Thesestudies were closely followed by immunohistochemical lo-calization of FAAH within the CNS using a C-terminalantibody (Tsou et al., 1998b). This study found intracellularpunctate staining preferentially localized to large principalneurons of the rat brain, a pattern consistent with intracel-lular membrane localization of the protein. Within the BLA,FAAH immunoreactivity was described as moderate tostrong. However, within the CeA, smaller round neuronsalso expressed cytoplasmic FAAH immunoreactivity, al-though to a lesser degree than that of the BLA.

Similarly, Elphick and coworkers described FAAH im-munoreactivity within the somata of neurons throughoutthe BLA (Egertová et al., 2003). Furthermore, Freund andcoworkers, using an antibody generated against a native6X-His tagged truncation of FAAH (Bracey et al., 2002),also published detailed light and EM descriptions of FAAHwithin the rat and mouse amygdala (Gulyas et al., 2004).At the light microscopy level, a strong cellular (cytoplasmicand proximal dendritic) and granular/reticular neuropilstaining was observed within the BLA. In the CeA, onlyoccasional neurons were FAAH immunoreactive (Gulyaset al., 2004). At the EM level, FAAH was present postsyn-aptically within dendrites and somata of BLA neurons.FAAH was localized to dendritic shafts and spine heads,but not to axon terminals (Gulyas et al., 2004).

MGL

In contrast to FAAH, MGL is a serine hydrolase that de-grades 2-AG into arachidonic acid and glycerol (Dinh et al.,2002a). MGL has been shown to be an important regulatorof brain 2-AG signaling in vivo (Long et al., 2009). ISHtudies did not reveal a prominent hybridization signalithin the rat amygdala (Dinh et al., 2002a). However,

mmunohistochemical studies using an N-terminal anti-ody against rat MGL revealed punctuate staining withinhe BLA, but not the CeA, of the rat amygdala (Gulyas etl., 2004). This punctuate staining was observed within theeuropil and often surrounded by unstained neurons withinhe BLA; furthermore, no cytoplasmic staining was ob-
erved. At the EM level, MGL was localized to a subset of
xon terminals that formed both symmetric and asymmet-ic synapses onto MGL-negative postsynaptic dendritesGulyas et al., 2004). MGL expression in inhibitory axonerminals is higher in the BA than LA (Yoshida et al., 2011).

AGL

AGL catalyzes the second step of 2-AG synthesis, liber-ting 2-AG from diacylglycerol (see earlier in the text).ecent molecular characterization of Sn-1 selective DAGLas shed considerable light on the 2-AG-CB1 signalingystem (Bisogno et al., 2003). Two isoforms, � and �

DAGL, were cloned and appear to have similar enzymaticproperties. During embryonic development, DAGL� is lo-calized to axon terminals, whereas during adulthood, asomatic/dendritic localization seems to predominate (Bi-sogno et al., 2003).

Two recent studies have elucidated the expressionpattern of DAGL� in the amygdala. We initially demon-trated a significant heterogeneity in DAGL� expression

within the amygdala (Patel et al., 2009). High levels ofDAGL� were observed in the BA and dorsal lateralamygdala (dLA), whereas lower levels were observed inthe ventral lateral amygdala (vLA). Within the CeA, theCeAL was more heavily stained than the CeAM. In addi-tion, ICMs were noticeably immunoreactive (Patel et al.,2009). Subsequent studies by Yoshida et al. demonstrateda similar subregional pattern (Yoshida et al., 2011). Theseauthors also showed postsynaptic localization of DAGL� inclose apposition to CCK and CB1-ir terminals forminginvaginating synapses (Yoshida et al., 2011). These au-thors suggest that invaginating synapses on BA principalneurons are subject to robust modulation by 2-AG signal-ing, relative to flat (non-CB1/DAGL�-expressing) syn-apses.

Summary

A summary of the regional and subcellular expression ofvarious eCB signaling components is shown in Fig. 1a, b.In general, the BLA expresses eCB signaling molecules ina distribution pattern common to other cortical brain re-gions. Specifically, the BLA exhibits high CB1-expressionby a subset of large CCK�GABAergic interneurons andlow levels of expression by principal neurons. FAAH andMGL are expressed postsynaptically and presynaptically,respectively. DAGL� is expressed by principal neurons,nd appears to cluster at invaginating synapses that re-eive GABAergic inputs from CB1�/CCK� interneurons.he CeA exhibits staining more similar to that of the stria-

um, although more detailed subcellular studies are lackingn this brain region.

SYNAPTIC MODULATION BY eCBs IN THEAMYGDALA

eCBs modulate GABAergic transmission in the BLA

Cannabinoids have been shown to decrease evokedand spontaneous GABAergic synaptic transmission onto
BLA principal neurons. Within BA principal neurons,

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Katona et al. demonstrated that the amplitude of evokedGABAA receptor-mediated inhibitory postsynaptic cur-rents (eIPSC) are depressed by application of the CB1agonists Win 55212-2 and CP 55,940; effects that werereversed by the CB1 receptor antagonist SR141716(Katona et al., 2001). Similarly, Win 55212-2 was unableto suppress GABAergic transmission in CB1�/� mice.Moreover, Win 55212-2 also decreased the frequencyand conductance of spontaneous, action potential-

Fig. 1. (a) Schematic diagram depicting the regional heterogeneity in tamygdala subnuclei. The localization of DAGL�, CB1 receptors, FAAHiagram of the amygdala with simplified cellular organization. BLA pr

nputs in gray. Boxed area is expanded in the main figure. Synaptic locynaptic inputs from a GABAergic interneuron and glutamatergic input these synapses are overlaid and specific eCB ligands noted where

driven IPSCs (sIPSC). Azad et al. found a similar affect

of CB1 receptor activation on eIPSCs recorded frommouse LA principal neurons (Azad et al., 2003).

Zuh and Lovinger used an isolated neuron/synapticbouton preparation to examine the effects of CB1 receptoractivation on the regulation of GABAergic transmission inBLA principal neurons (Zhu and Lovinger, 2005). Applica-tion of Win 55212-2 to this preparation produced a rapidlyreversible reduction in the frequency and amplitude ofsIPSCs. These authors also found that blockade of the

ssion of the molecular components of eCB signaling between differentare depicted based on data summarized in the text. (b) Left top inset:euron in black, GABAergic interneuron in yellow, and glutamatergic

of eCB signaling components in a schematized BLA neuron receivingexternal capsule. Known forms of eCB-mediated synaptic signalingavailable.

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sIPSC frequency. This effect was eliminated when thepostsynaptic neuron was filled with a calcium chelator orextracellular calcium concentrations were reduced, suggest-ing a tonic CB1 receptor activation via calcium-dependent,postsynaptic eCB production in this preparation. Recentwork by Yoshida et al. demonstrated that depolarization-induced suppression of inhibition (DSI) in the BA is absentin DAGL� knockout mice, implicating 2-AG as the relevantCB ligand mediating this form of eCB signaling (Yoshidat al., 2011).

In addition to the modulation of IPSCs by direct CB1eceptor activation, eCB signaling has been demonstratedo mediate long-term depression (LTDi) and DSI of inhib-tory synaptic transmission onto principal neurons of theLA. Initial studies by Marsicano et al. within the BA,howed that LTDi was induced by low-frequency (1 Hz)timulation (LFS) of the external capsule (100 pulses),hich produced a decrease in isolated GABAA receptor-

mediated eIPSC amplitude to 66% of control. This reduc-tion in IPSC amplitude was accompanied by an increase inthe paired pulse ratio (PPR), indicating a presynaptic ex-pression mechanism. In addition, LFS reduced sIPSC fre-quency but not amplitude, further suggesting a presynapticmechanism of action (Azad et al., 2004). Furthermore,LFS-induced LTDi was mediated by eCB signaling, as itwas absent in CB1�/� mice and prevented by the CB1eceptor antagonist SR141716.

Subsequent studies by the same authors shed consid-rable light on the molecular mechanisms subservingCB-mediated LTDi in the BA. These authors found thatTDi was occluded by Win 55212-2 and did not depend onostsynaptic elevations in intracellular calcium (Azad et al.,004). A role for metabotropic glutamate receptorsmGluR) in LTDi was also suggested. The mGluR1/5 ag-nist, dihydroxyphenylglycine (DHGP), suppressed eIPSCmplitude in a manner similar to LTDi in wild-type, but notB1�/� mice. DHPG also occluded LFS-LTDi, and did notffect eIPSC amplitude after LTDi had been induced. Apecific role for mGluR1, rather than mGluR5, was sug-ested based on pharmacological evidence. LTDi was also

ound to be dependent on postsynaptic G-protein activa-ion and the adenylate cyclase (AC)-protein kinase APKA) pathway (Azad et al., 2004). However, LTDi was notependent on PLC or DAGL activity. Finally, FAAH�/�

mice (which exhibit elevated brain AEA levels) showed amore pronounced LTDi than wild-type mice. These authorssuggest that the G-protein coupled mGluR1, acting via anAC-PKA pathway, is important for LTDi in the BA, and AEArather than 2-AG may be the relevant eCB subserving thisprocess (Azad et al., 2004).

A role for the presynaptic protein RIM1� has also beenemonstrated in BLA LTDi. Specifically, LFS-LTDi andHPG-LTDi are both absent in RIM1��/� mice (Cheva-

eyre et al., 2007). Interestingly, Win 55212–2-inducedTDi is also absent in RIM1��/� mice; instead, it yielded a

reversible depression. These data implicate RIM1� as animportant synaptic protein mediating long-term synapticeffects of eCBs in the BLA, and other brain regions such as
the hippocampus (Chevaleyre et al., 2007).
eCB signaling has also been explored at the circuitlevel in vitro. Specifically, eCB-mediated LTDi (see detailsmentioned earlier in text) is expected to increase the ex-citability of BLA projection neurons by decreasing inhibi-tory tone, and thus increase the activity of BLA targetneurons in the CeA, nucleus accumbens, and prefrontalcortex. Using extracellular field potential recordings, Azadet al. found that high-frequency stimulation (HFS; 100pulses 50 Hz) of the external capsule (EC) produced along-term potentiation (LTP) of field potentials recordedfrom the BLA (Azad et al., 2004). Interestingly, preinduc-tion of LTDi enhanced the magnitude of HFS-induced LTP;an effect that was abolished by the CB1 receptor antago-nist SR141716. To determine whether this enhanced ex-citability of BLA neurons in response to eCB-mediatedLTDi affects the activity of their target neurons, EPSCsfrom neurons within the CeA were recorded in response toLA stimulation, before and after HFS-LTP induction. Pre-induction of eCB-dependent LTDi enhanced LTP in theLA-CeA pathway, an effect abolished by SR141716 (Azadet al., 2004). These findings suggest eCB-mediated LTDienhances the excitability of BLA neurons in response toafferent stimulation and thus, increases the activity of theBLA-CeA pathway.

Zhu and Lovinger have also demonstrated a role foreCB signaling in DSI at inhibitory synapses onto BLAprincipal neurons in mechanically dissociated postsynapticneuron/synaptic bouton preparations and in acute rat brainslices (Zhu and Lovinger, 2005). In the acute brain slicepreparation, postsynaptic depolarization (�60 to 0 mV) ofBLA principal neurons for 4 s produced a 44% decrease insIPSC frequency and a 34% decrease in sIPSC amplitude.Chelation of postsynaptic calcium eliminated DSI; how-ever, a slowly developing decrease in sIPSC frequencywas still present under these conditions. The mGluR5receptor antagonist MPEP did not eliminate DSI but didsignificantly shorten its duration, suggesting that the lon-ger-lasting component of DSI requires mGluR5 receptoractivation, rather than increases in intracellular calcium(Zhu and Lovinger, 2005). These data further suggest thatmGluR-dependent long-term eCB-mediated depression ofGABAergic transmission is calcium independent.

Recently, heterogeneity in the responsivity of GABAergictransmission to eCB signaling within the LA and BA divi-sions of the BLA has been demonstrated. Specifically, Win55212-2-induced depression of eIPSCs is significantlygreater in the BA than the LA, an effect related to higherexpression of the CB1 receptor on inhibitory axon termi-nals in the BA relative to the LA (Yoshida et al., 2011). Assuch, this difference in CB1 receptor expression results inan increase in DSI magnitude in the BA compared with LA(Yoshida et al., 2011).

eCBs modulate glutamatergic transmission in theBLA

The effect of CB1 receptor activation on glutamatergictransmission in the BLA has also been extensively studied.When recording from neurons in the LA, and stimulating
glutamatergic afferents arising from the EC, Win 55212-2

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reduced AMPA-dependent eEPSC amplitude to 52% ofcontrol while significantly increasing the PPR, an effectabsent in CB1�/� mice. Win 55212-2 also reduced therequency of sEPSCs and action potential-independentEPSCs without affecting the amplitude of either re-

ponse. These data indicate that CB1 receptor activationecreases EC-evoked excitatory glutamatergic transmis-ion onto LA principal neurons via a presynaptic mecha-ism (Azad et al., 2003). This effect was subsequentlyonfirmed to be CB1 receptor-dependent using mutantice with selective deletion of CB1 receptors from fore-rain glutamatergic neurons (Domenici et al., 2006).

Short-term eCB mediated synaptic plasticity in the formf depolarization-induced suppression of excitation (DSE),as also been described in the LA (Kodirov et al., 2009).utz and coworkers found that 10 s of depolarizationroduced a slowly developing, low magnitude depres-ion of EPSCs recorded from LA pyramidal neurons.SE of sEPSCs was also observed in this study, both ofhich were mediated via activation of CB1 receptors

Kodirov et al., 2009).In addition to direct modulation of EPSCs by cannabi-

oids within the LA, Huang et al. have demonstrated a roleor eCB signaling in amphetamine-induced long-term de-ression (LTD) of EC-evoked excitatory postsynaptic po-ential (EPSPs) in LA principal neurons (Huang et al.,003). Application of amphetamine caused a rapid, dose-ependent reduction in eEPSP slope with an early com-onent (acute depression) and late component (LTD). In-erestingly, the CB1 receptor antagonist, AM251, blockedhe amphetamine-induced acute depression and LTD,hile having no effect on eEPSP slope alone. In thisreparation, Win 5512-2 produced an initial depressionnd LTD similar to amphetamine, an effect abolished byM 251. A presynaptic mechanism of action for both am-hetamine and Win 55212-2 was demonstrated by showingn increase in PPR and a reduction in mEPSPs frequency,ut not amplitude, for both compounds. Furthermore, Win5212-2 occluded amphetamine-induced LTD, whereasmphetamine occluded Win 55212-2-induced LTD. Simi-

arly, blockade of eCB transport with AM404, at low con-entrations, mimicked amphetamine and Win 55212-2 in-uced LTD, whereas at high concentrations occluded am-hetamine-induced LTD. eCB-mediated amphetamineTD was dependent on postsynaptic calcium influx and/Q-type calcium channel activity. Taken together, theseata strongly suggest that amphetamine-induced LTD isediated by eCBs acting at presynaptic CB1 receptors

hat regulate glutamate release onto LA principle neuronsHuang et al., 2003).

Recently, a role for eCB signaling at thalamic inputs tohe LA has been demonstrated (Shin et al., 2010). In thistudy, LFS of thalamic inputs to the LA combined withostsynaptic depolarization resulted in LTP that is ex-ressed postsynaptically. This post LTP requires postsyn-ptic calcium and CB1 activation to suppress a form ofainate-mediated presynaptic LTP. These authors con-lude that combined LFS and postsynaptic depolarization
eleases eCBs that counteract LFS-induced presynaptic p
TP, resulting in an exclusively postsynaptic LTP. Theseata suggest a hierarchical order of pre- and postsynapticTP at thalamo-LA synapses that is regulated by eCBignaling (Shin et al., 2010).

The effects of cannabinoid signaling on neuronal pop-lation activity and lateral amygdalar circuits in vivo and initro have been explored. Azad et al. explored the effectsf cannabinoid signaling on EC stimulation-induced localeld potentials within the LA. Win 55212-2 reduced fieldotential amplitudes to 52% of control (Azad et al., 2003).his effect was eliminated by the application of SR141716which had no effect on field potential amplitude alone), asell as the Gi/o protein inhibitor pertussis toxin (PTX), and

absent in CB1�/� mice. Furthermore, inhibition of PKA didot affect Win 55212-2-induced reductions in LA field po-ential amplitude (Azad et al., 2003). These data suggesthe AC-PKA pathway does not significantly contribute tohe CB1 receptor-mediate reductions in field potential am-litude within the LA.

A role for eCB signaling in synaptic plasticity at theeuronal population level has also been examined. HFS (5rains of 100 Hz for 1 s) of the EC induced a LTP ofopulation spike amplitude within the LA (Marsicano et al.,002). This HFS-induced LTP was significantly enhanced

n CB1�/� mice, but not affected by SR141716, suggestinghat long-term neural adaptations in these mice couldnderlie these effects, rather than an acute involvementf eCB signaling. Similarly, LFS (900 pulses at 1 Hz)roduced an LTD of population spike amplitude within

he LA, which was similar in both wild-type and CB1�/�

mice, suggesting no role for eCB signaling in theseforms of synaptic plasticity (Marsicano et al., 2002).

owever, LFS-LTD was prevented by activation of theB1 receptor specifically located on GABAergic termi-als (Azad et al., 2008). Preapplication of either CB1eceptor agonist, Win 55212-2 or THC, prevented LFS-TD via a mechanisms involving Gi/o proteins, inwardly-ectifying potassium channels, and PKA-dependent reg-lation of N-type calcium channels. These data providenovel link between eCB signaling at GABAergic syn-

pses and long-term plasticity at excitatory synapses inhe LA (Azad et al., 2008).

CB modulation of synaptic signaling in the CeA

he first study that examined CB1 receptor signaling in theeA found no effect of Win 55212-2 on GABAergic trans-ission in this region (Katona et al., 2001). However, a

ubsequent study by Roberto et al. examined eIPSPs fromeurons within the CeAM, and found that Win 55212aused a �50% depression in eIPSP amplitude (Robertot al., 2010). Interestingly, blockade of CB1 receptors withR141716 caused �130% increase in eIPSP amplitude,uggesting a significant tonic eCB-mediated suppressionABA release at these synapses. The SR141716-induced

ncrease in eIPSP amplitude was abolished by postsynap-ic calcium chelation, supporting the notion of enhancedCB tone, rather than effects due to the inverse-agonist
roperties of SR141716. In this study, Win 55212-2 was

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In addition to CB1 agonist-induced synaptic suppres-sion, DSI and DSE have both been demonstrated withinthe CeAM. Kamprath et al. recently demonstrated thatGABAergic inputs from the CeAL to the CeAM were sus-ceptible to DSI (Kamprath et al., 2011). In fact, depolariza-tions as short as 100 ms induced DSI in these neurons.Similarly, excitatory BLA inputs to CeAM were susceptibleto DSE: however, much longer depolarization times wererequired (up to 10 s). Moreover, both DSI and DSE wereblocked by CB1 receptor antagonists and absent inCB1�/� mice, and showed rapid adaptations to acute foot-hock stress as will be discussed later in the text (Kam-rath et al., 2011). Taken together, these data indicate aigh level of eCB tone regulating GABAergic transmission

n the CeAM, and excitatory synapses are less susceptiblehan GABAergic synapses to eCB-mediated retrogradeignaling. Whether these differences are due to reducedB1 receptor function at glutamatergic synapses (relative

o GABAergic synapses) or differences in eCB productiont these different synapses remains to be determined.

n vivo electrophysiological studies

everal studies have investigated the effects of cannabi-oid signaling on amygdalar neurocircuitry in vivo. Pistis etl. studied the effect of systemically administered CB1eceptor agonists on the activity of nucleus accumbenseurons receiving monosynaptic inputs from BLA projec-ion neurons, using single-unit extracellular recordingsPistis et al., 2002). Stimulation currents were adjusteduch that 50% of the stimuli delivered to the BLA (at 1 Hz)esulted in an action potential in the nucleus accumbenseuron being recorded. Systemic administration of theB1 receptor agonists Win 55212-2, HU-210, and THC allecreased BLA-stimulation evoked spike probability in nu-leus accumbens neurons, an effect reversed by the CB1eceptor antagonist SR141716, which when administeredlone, had no effect (Pistis et al., 2002). These data sug-est that CB1 receptor activation modulates glutamateelease from BLA projection neurons, likely at the level ofxon terminals that innervate target structures such as theucleus accumbens.

Studies by Pistis et al., using single-unit extracellularecordings, explored the effect of CB1 receptor activationn the spontaneous activity of BLA principal neurons and

ocal GABAergic interneurons within the BLA (Pistis et al.,004). Administration of the CB1 receptor agonist, HU-10, decreased BLA interneuron spike frequency; an effecteversed by the CB1 receptor antagonists AM251 andR141716. Win 55212-2 had a biphasic effect on interneu-

on firing rate, as a low dose (0.125 mg/kg i.v.) stimulatedring, whereas higher doses (0.5–1 mg/kg i.v.) inhibitedring. Both effects were blocked by the CB1 receptor an-agonists AM251 and SR141716. Furthermore, both Win5212-2 and HU-210 inhibited the firing rate of antidromi-ally identified BLA projection neurons. This effect of Win5212-2 was reversed by the CB1 receptor antagonist
R141716 and the vanilloid receptor antagonist, capsaz-
pine, but not the CB1 receptor antagonist AM251 (Pistist al., 2004). Interestingly, AM251 not only failed to reversehe inhibitory effect of Win 55212–2, it strongly potentiatedt. Neither SR141716 nor AM251 administered alone af-ected BLA projection neuron firing rate (Pistis et al., 2004).

These authors also studied the effect of cannabinoideceptor activity on medial prefrontal cortex (mPFC) stim-lation-induced BLA neuron responses (Pistis et al., 2004).timulation current was adjusted such that 50% of stimuliroduced spiking activity in BLA neurons. Win 55212-2educed mPFC stimulation-induced spike probability inLA neurons to 48% of control; an effect that was reversedy SR141716, but not AM251. HU-210 did not affectPFC stimulation-induced spike probability in BLA neu-

ons (Pistis et al., 2004).Recently, CB1 receptor-mediated signaling within the

LA-mPFC circuit was explored in the context of a Pav-ovian conditioning paradigm. The mPFC–amygdala circuitas been shown to play a critical role in the processing and

ntegration of emotionally salient sensory information andearning (Milad and Quirk, 2002); however, a mechanismy which cannabinoid signaling within this circuit could

nfluence emotional associative learning at the level of theingle neuron was unknown. To this end, Laviolette andrace carried out single unit extracellular recordings ofyramidal, BLA-responsive mPFC neurons during a Pav-

ovian odor fear-conditioning procedure in anesthetizedodents (Laviolette and Grace, 2006b). This procedurenvolved the acquisition of an association between an aver-ive foot-shock and an odor cue, via repeated contiguousresentation. During the testing phase, animals were pre-ented with either a footshock-paired odor (CS�) or aonpaired odor (CS�).

In this paradigm, postconditioning CS� presentationcaused a stimulus-locked discharge in BLA-responsivemPFC neurons, which was absent on CS� presentation.Systemic CB1 receptor activation by WIN 55,212-2 (0.5m/kg i.v.) before conditioning dramatically potentiated neu-ronal associative learning to the CS� as demonstrated byboth increased neuronal burst frequency and spikes withineach burst event compared with saline treatment (Lavio-lette and Grace, 2006b). This effect was blocked by sys-temic co-administration of the CB1 receptor antagonist,AM251 (1.0 mg/kg i.v.). Given that cortical neuronal burst-ing has been hypothesized to represent the processing ofreward-related learning cues, memory encoding, as wellas decision-making, these results suggest that CB1 recep-tor signaling modulates the active encoding and expres-sion of associative learning in BLA-responsive mPFC neu-rons (Laviolette and Grace, 2006b).

Laviolette and coworkers also investigated the role ofcannabinoid signaling in LTP. LTP was examined usingthe functional connections between the BA and the prelim-bic cortex (Tan et al., 2010). In vivo, tetanic stimulation(HFS, three sets of 10 trains, 100 Hz) of the ipsilateral BAproduced a robust LTP of field potentials within the prelim-bic cortex of control rats. This effect was dose-dependentlyblocked by systemic pretreatment with the CB1 antagonist,
AM251, thereby demonstrating that long-term synaptic


plasticity along the BA-prelimbic cortex pathway is depen-dent on CB1 receptor signaling (Tan et al., 2010).

To further explore CB1 receptor-mediated signalingwith the BA-prelimbic cortex circuit, this group also usedsingle-unit in vivo extracellular recordings to determinehow intra BA CB1-receptor activity could remotely controlneuronal activity patterns within subpopulations of prelim-bic neurons (Tan et al., 2011). These data indicate thatintra-BA microinfusion of WIN 55,212-2 caused an in-crease in neuronal activity (as measured by the spontane-ous firing rate, neuronal bursting activity, and interspikeintervals) of recorded prelimbic neurons. Conversely, in-tra-BA CB1 receptor blockade by treatment with AM251caused a decrease in spontaneous prelimbic cortex neu-ronal activity. Interestingly, under the highest tested dosesof both intra-BA treatment conditions, a small population ofprelimbic cortex neurons demonstrated the opposite re-sponse patterns, that is, a decrease or increase in prelim-bic cortex neuronal activity in response to intra-BA CB1receptor activation or inhibition respectively; possibly dem-onstrating the bidirectional functional connectivity betweenthese two regions (Tan et al., 2011). These authors dem-onstrate that intra-BLA CB1 receptor activity can stronglymodulate neuronal activity within a subpopulation of pre-limbic cortex neurons (Tan et al., 2011).

A role for eCB signaling in alcohol-induced suppres-sion of BA stimulated activation of nucleus accumbensneurons has also been demonstrated (Perra et al., 2005).Alcohol was shown to depressed BA-evoked spike proba-bility in nucleus accumbens neurons, an effect reversed bySR141716, which, when administered alone had no effecton BA-evoked spike probability in nucleus accumbensneurons. The FAAH inhibitor URB597 also inhibited thealcohol-induced suppression of BLA-evoked spike proba-bility in nucleus accumbens neurons. Interestingly, treat-ment with the CB1 receptor agonist Win 55212-2 15–20 hbefore alcohol treatment reduced the alcohol-induced sup-pression of BLA-stimulated spike probability in nucleusaccumbens neurons. This effect was not due to a func-tional tolerance as the dose–response curves for Win55212-2-induced suppression of BLA-evoked spike prob-ability in nucleus accumbens neurons was not differentbetween control rats and rats treated with Win 55212-215–20 h earlier (Perra et al., 2005). These data suggest arole for eCB signaling in alcohol-induced suppression ofBLA-evoked spike probability in nucleus accumbens neu-rons and further suggest that CB1 receptor activity canmodulate BA efferent neurotransmission.

Summary

eCB signaling robustly modulates GABAergic signaling inthe BLA (see Fig. 1b). DSI and LTDi are expressed by BLApyramidal neurons. Although DSI is mediated via 2-AG,the identity of the eCB ligand subserving LTDi remainscontroversial. Circuit levels analyses reveal that LTDi inthe BA facilitates activation of BA-CeA pathway. At gluta-matergic synapses onto BLA neurons, 2-AG mediatesDSE, but the eCB ligand mediating amphetamine-LTD is
not known. In general, the BA exhibits more robust eCB
signaling than the LA. eCB signaling also modulates ex-citatory transmission between the BA and efferent targets,including the nucleus accumbens and prefrontal cortex.Within the CeAM, eCB-mediated DSI and DSE have alsobeen demonstrated.

STRESS-INDUCED ADAPTATIONS IN eCBSIGNALING IN THE AMYGDALA

Biochemical adaptations

We initially investigated the effects of acute and repeatedrestraint stress on levels of 2-AG and AEA in the amygdalaof mice (Patel et al., 2004, 2005; Patel and Hillard, 2008).With regard to stress effects on 2-AG levels, we observeda progressive increase in tissue 2-AG levels, which wasgreater after 10, as compared with 5 or 1 consecutive daysof restraint stress for 30 min/d. We subsequently showedthat this increase in 2-AG was observable within the BLAspecifically and was also associated with increases in twodiacylglycerol precursors of 2-AG (Patel et al., 2009). Sim-ilar effects were observed in rats exposed to 10 days ofconsecutive restraint stress (Hill et al., 2010b). The stress-induced increase in 2-AG clearly demonstrates sensitiza-tion; however, the increase in 2-AG levels in response torepeated restraint stress is short-lived. Indeed, 2-AG levelspeak at 20 min following the start of the 10th restraintexposure and, by 60 min, are almost back to control levels(Patel et al., 2009). Similar effects were observed in rats,where 24 h after the 10th restraint exposure, 2-AG levelsare not different from control (Hill et al., 2010b). These datasuggest that levels of 2-AG surge in response to stress,and that if the stress is repeated in a predictable manner,each surge gets progressively larger. However, betweensurges, 2-AG levels return to baseline. In addition to re-straint stress, administration of chronic corticosterone torats increases levels of 2-AG in the amygdala (Hill et al.,2005), nonetheless, acute corticosterone treatment pro-duced only a nonsignificant trend toward 2-AG elevation(Hill et al., 2010a).

Another consistent finding in rats and mice is thatrestraint stress and chronic unpredictable stress produce aprofound and sustained reduction in AEA levels within theamygdala (Patel et al., 2005; Hill et al., 2009). In contrastto 2-AG, reductions in AEA are not related to subsequentstress exposure as 24 h after the last stress exposure,AEA levels are still profoundly reduced in many brainregions (Hill et al., 2009). In some cases, chronic stressproduces an upregulation of FAAH activity, suggestingincreased tonic AEA degradation as a mechanism sub-serving the sustained reductions in AEA levels after re-peated stress (Rademacher et al., 2008; Hill et al., 2009).In contrast to 2-AG, acute corticosterone produces a rapid(within 10 min) elevation of AEA in the amygdala.

With regard to changes in CB1 receptor expression,chronic corticosterone does not alter number or affinity ofCB1 receptors in the amygdala of rats (Hill et al., 2005).Furthermore, neither chronic restraint stress in mice
(Rademacher et al., 2008) nor chronic unpredictable stress

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Synaptic adaptations

Several recent studies have begun to describe synapticadaptations in eCB signaling in the BLA and CeA in re-sponse to both acute and chronic stress. We found thatCB1-mediated suppression of GABAergic transmission isreduced in the BA after 10 days of restraint stress, sug-gesting a small but significant desensitization of CB1 re-ceptors by chronic stress (Patel et al., 2009). In contrast,DSI is prolonged in mice exposed to 10, but not 1 day, ofrestraint stress (Patel et al., 2009). These data indicateeCB signaling at GABAergic synapses in the BLA is en-hanced by chronic stress exposure, whereas CB1 recep-tors exhibit a small measure of functional desensitization.

Within the CeA, Pape and coworkers showed that 24 hafter a single tone-foot shock pairing, the magnitude of DSIand DSE was enhanced in neurons from the CeAM (Kam-prath et al., 2011). This effect was not observed 3 daysafter foot-shock stress. These findings suggest that strongacute stressors can cause transient enhancements inshort-term eCB signaling in the CeAM (Kamprath et al.,2011). The biochemical mechanisms subserving these ad-aptations require further investigation.

BEHAVIORAL CONSEQUENCES OFCANNABINOID-MEDIATED MODULATION OF

AMYGDALA FUNCTION

Behavioral effects of eCB signaling in the BLA

eCB signaling within the BLA has been implicated instress-induced analgesia, stress-induced neuroendocrineactivation, and associative learning. This section will high-light some of these key behavioral roles subserved by eCBsignaling in the BLA.

Bilateral microinjection of the CB1 agonist, Win55212-2, produced antinociceptive effects in the tail-flickassay, which was blocked by a CB1, but not a CB2 recep-tor antagonist, suggesting that activation of CB1 receptorsin this region may be an important anatomical site for theproduction of cannabinoid antinociception (Hasanein et al.,2007). Interestingly, stress-induced analgesia is partiallyblocked by administration of the CB1 receptor antagonistSR141716 into the BLA (Connell et al., 2006). However,blockade of CB1 receptors in the right BLA did not alterfear-conditioning-induced analgesia in rats (Roche et al.,2007).

eCB signaling in the BLA has also been demonstratedto play an important role in stress-induced neuroendocrineactivation. In response to acute restraint stress, AEA levelsare robustly decreased in the BLA, and levels of AEA areinversely correlated with plasma corticosterone levelsmeasures after stress exposure (Hill et al., 2009). Intra-BLA infusion of a FAAH inhibitor, or a direct CB1 agonist,reduced stress-induced corticosterone release, whereasinfusion of the CB1 antagonist AM251 increased cortico-sterone release in response to stress (Hill et al., 2009). In
addition, intra-BLA infusions of Win 55212-2 blocked ele- i
vated platform-stress-induced increase in plasma cortico-sterone in rats (Ganon-Elazar and Akirav, 2009). Thesestudies suggest that decreased AEA signaling in the BLAfacilitates stress-induced neuroendocrine activation. Withregard to long-term adaptation of neuroendocrine stressresponses, Hill et al. showed that 2-AG levels in theamygdala, following repeated restraint stress, are in-versely correlated with plasma corticosterone levels mea-sured after the last restraint session (Hill et al., 2010b).Importantly, bilateral intra-BLA infusions of the CB1 antag-onist, AM251, before the last restraint session preventedthe normal habituation of the corticosterone response (Hillet al., 2010b). These data link 2-AG-CB1 signaling in theBLA with functional habituation of the neuroendocrine re-sponse to repeated homotypic stressors.

With regard to unconditioned anxiety behaviors, unilat-eral intra-BLA infusions of THC produced an anxiogeniceffect in rats measured using the elevated plus-maze (Ru-bino et al., 2008). In contrast, neither bilateral intra-BLAWin 55212-2, nor intra-BLA AM251 had any effect onlocomotor behavior or anxiety measures in the open-fieldassay (Ganon-Elazar and Akirav, 2009).

Using aversive associative learning paradigms, it haseen shown that posttraining blockade of CB1 receptors in

he right BLA increased freezing behavior and ultrasonicocalizations in a contextual fear-conditioning paradigm inats (Roche et al., 2007). Along similar lines, Kamprath et al.ecently showed that posttraining intra-BLA infusions of theB1 antagonist SR141716 impaired long-term extinction ofonditioned freezing in a cue fear-conditioning paradigm inice (Kamprath et al., 2011). Similarly, Ganon-Elazar andkirav recently showed that intra-BLA infusion of AM251ither before conditioning or before extinction training, im-aired extinction of inhibitory avoidance learning in ratsGanon-Elazar and Akirav, 2009). In contrast, these authorsound that intra-BLA infusion of Win 55212-2, before condi-ioning, did not affect acquisition or extinction of inhibitoryvoidance learning in rats (Ganon-Elazar and Akirav, 2009).

nterestingly, intra-BLA Win 55212-2 infusion did prevent theollowing: (1) the enhancement of platform stress on thecquisition of inhibitory avoidance learning when adminis-

ered before conditioning and (2) the impairment in extinctionaused by preextinction stress exposure. By means of aimilar olfactory-fear conditioning paradigm, Laviolette andoworkers also demonstrated that asymmetrical, interhemi-pheric injection of AM251 in the BLA-mPFC pathway beforeonditioning, dose dependently prevented the encoding ofssociative fear memory as demonstrated by decreased per-entage time spent freezing and increased spontaneous ex-loratory activity upon CS� presentation (Laviolette andrace, 2006a). These data indicate that eCB signaling in theLA is critical for extinction of aversive conditioned re-ponses but could also be important for the acquisition ofhese responses.

In contrast to aversive associative learning, little isnown regarding the role of BLA eCB signaling in appeti-ively motivated learning. Parsons and coworkers investi-ated the role of eCB signaling within the BLA in appetitive

nstrumental learning (Alvarez-Jaimes et al., 2008). They


used a heroine self-administration paradigm to study therole eCB signaling in cue-induced relapse behavior andfound that blockade of CB1 receptors in the prefrontalcortex and nucleus accumbens prevented cue-induceddrug seeking behavior, whereas blockade of CB1 recep-tors in the BLA had no effect (Alvarez-Jaimes et al., 2008).

Behavioral effects of eCB signaling in the CeA

Initial studies into the role of cannabinoid signaling in theCeA by Onaivi and coworkers described an anxiogeniceffect of THC (100 and 150 �g per injection) bilaterallyadministered into the CeA of mice, using the elevatedplus-maze test (Onaivi et al., 1995). However, a morerecent study demonstrated that the CB1 agonist ACPA(1.25 and 5 ng/rat) caused an anxiolytic effect in rats whenadministered directly into the CeA using this same assay(Zarrindast et al., 2008). The differences in these studiescould be related to the well-known biphasic effects ofcannabinoids on anxiety, with low doses producing anxio-lytic effects, whereas higher doses producing anxiogeniceffects, as the former study used doses of THC signifi-cantly higher than doses of ACPA used in the latter. Inter-estingly, intra-CeA blockade of the CB1 receptor, usingAM251, caused a decrease in exploratory activity withoutchanging any anxiety measures (Zarrindast et al., 2008). Inaddition to these studies on unconditioned fear, the ex-pression of conditioned fear is also modulated by eCBsignaling in the CeA. Specifically, posttraining infusion ofthe CB1 antagonist SR141716 into the CeA causes im-pairment in within session (short-term) extinction of condi-tioned freezing responses to CS� presentation (Kamprathet al., 2011). Taken together, these data suggest that eCBsignaling in the CeA serves to facilitate active/exploratorybehaviors, and suppress passive/freezing coping re-sponses to salient aversive stimuli.

With regard to motivational behavior, intra-CeA admin-istration of the CB1 antagonist, AM251, produced placeaversion and blocked the place preference induced bymorphine (Rezayof et al., 2011). In contrast, activation ofCB1 receptors with ACPA produced place preferencewhen administered alone and enhanced place preferenceinduced by morphine (Rezayof et al., 2011). One limitationof these studies is the lack of distinction between the CeALand CeAM; because of the close proximity of these nuclei,it will be challenging to dissociate functional roles of theseregions using microinjection techniques.

Summary

Taken together, these data indicate a prominent role foreCB signaling in the BLA in the acquisition/expression ofaversive associative memories, as well as in the long-term(over period of days) extinction and/or habituation of con-ditioned fear responses. In addition, eCB signaling withinthe BLA is important for the deleterious effect of stress onassociative learning and extinction. eCB signaling in theBLA is also important for stress-induced neuroendocrineactivation and for the habituation of neuroendocrine re-sponse to repeated homotypic stress. Finally, eCB signal-
ing in the BLA contributes to descending pain modulation.
Within the CeA, available data suggest that eCB signalingis important for short-term extinction of conditioned fearresponses, facilitates exploratory behavior, and producesa reinforcing motivational state.

FUTURE DIRECTIONS

Although the past work summarized here has provided sub-stantial insights into the role of eCB signaling in theamygdala, as well as the behavioral consequences of phar-macological and genetic modulation of this system, severalkey questions remain open for experimental analysis.

One important question is whether there are conditionsunder which physiologically released AEA and 2-AG exertactions over distinct synapses in the amygdala, especiallybecause some types of stress appear to differentially reg-ulate these two ligands (see earlier in the text). For exam-ple, do AEA and 2-AG regulate retrograde signaling atdifferent synapses based on chemical characteristics, thatis, AEA modulates glutamate release while 2-AG modu-lates GABA release? Alternatively, is differential signalingspatially regulated with perisomatic synapses being regu-lated by one ligand regardless of the neurotransmitterbeing affected, while distal dendritic synapses being reg-ulated by the other or both? Additionally, determiningwhether there are pathological conditions under which thistype of regulation is perturbed could facilitate rational de-velopment of eCB-based treatment for these conditions.

Determining the adaptations in eCB synaptic signalingthat occur after in vivo treatment with eCB modulating ligandswith anxiolytic/antidepressant actions are important experi-ments that could shed light on fundamental aspects of thepathology of anxiety and depressive disorders. Some initialstudies have suggested that chronic MGL inhibition withJZL184 treatment (which increases 2-AG levels and hasanxiolytic effects in animal models) downregulates CB1 re-ceptor function after chronic treatment and thus impairs eCBretrograde signaling in some brain regions (Schlosburg et al.,2010). Therefore, if pathological conditions exist where eCBsignaling is deleteriously enhanced, chronic, high-doseJZL184 administration may provide therapeutic benefits by“tuning down” eCB signaling in brain regions with high 2-AGturnover. In contrast, if pathological conditions can be identi-fied where eCB signaling is reduced, low-doses of eCB deg-radation inhibitors could be beneficial by enhancing or pro-longing eCB-mediated synaptic signaling in brain regionswith eCB deficits. This hypothesis suggests that determiningthe relationship between tissue content of eCBs and retro-grade signaling capacity is a critical question, as is determin-ing the relationship between eCB signaling at different syn-apses in the amygdala and the expression of anxiety-relatedbehavior and stress responses. This could allow us to deter-mine whether eCB modulation of glutamatergic signaling inthe amygdala is relevant for some aspects of behavior inde-pendent of modulation of GABAergic transmission, and viceversa, or are the coordinated effects of eCBs on both neu-rotransmitters more relevant?

Moreover, understanding the relationship between adap-
tations in eCB signaling in models of affective disorders and


during behavioral dysregulation will be critical to developmentof eCB-based therapeutics. Along these lines, Kamparath etal., have recently shown that fear-conditioned mice exhibitincreased DSI/DSE in the central amygdala (see earlier in thetext), and that blockade of the CB1 receptor in this regionimpairs short-term behavioral adaptation to conditioned fearstimuli, suggesting these synaptic adaptations are a benefi-cial compensatory response aimed to moderate fear re-sponses. Additional work, conceptually modeled after thesetypes of studies, will be of significant benefit in developing amore detailed scheme illuminating the role of eCB signalingin the modulation of anxiety behaviors.

COMPARING eCB MODULATION OFAMYGDALA NEUROCIRCUITRY WITH OTHER

FOREBRAIN CIRCUITS

Although significant progress has been made in elucidat-ing the molecular determinants of eCB signing in the brain(Kano et al., 2009), global functional properties have beenscantly discussed in the literature. Here, we suggest astructural framework, derived from both anatomical andphysiological data, for describing what we consider to be afundamental functional motif of the eCB signaling systemthat modulates forebrain cortico-striato-pallidal circuits(Fig. 2).

Within neocortical brain regions, eCB signaling modu-lates GABA release from a subset of GABAergic interneu-rons, and modulates cortico-cortico and some thalamocor-

Fig. 2. A highly simplified schematic diagram of a common eCBsignaling motif expressed across different cortical territories and theirassociated parallel cortico-striato-pallidal circuits. In “motor” circuits,cortical glutamatergic pyramidal cells (red), synapse onto striatal neu-rons, and are subject to eCB-mediated retrograde suppression ofglutamate release (indicated by green line). GABAergic (black) striatalneurons send projections to the globus pallidus, which are also subjectto eCB-mediated retrograde suppression of GABA release. Thus,each stage of the motor circuit is modulated by eCB signaling. A similarmotif is evident within “limbic” cortical regions such as the PFC, and wesuggest a similar motif may exist within the amygdala circuit. Theselimbic parallel circuits also exhibit a high degree of interaction, asindicated by the cross projections between and from the PFC and the
BLA. (?) indicates lack of experimental data.
tical glutamatergic inputs to cortical pyramidal neurons(Auclair et al., 2000; Bodor et al., 2005; Hill et al., 2007;Lafourcade et al., 2007). Within the striatum, eCB signalingmodulates cortical glutamatergic inputs to medium spinyneurons, as well as recurrent collateral GABAergic trans-mission onto these cells (Gerdeman and Lovinger, 2001;Uchigashima et al., 2007). Within the globus pallidus, eCBsignaling modulates GABA release from striatal neuronterminals onto pallidal neurons (Engler et al., 2006). Basedon the anatomical and physiological data on the role ofeCB signaling within the amygdala summarized earlier, asimilar pattern can be observed in the amygdaloid com-plex. The BLA, a cortical-like structure (see Introduction),exhibits functional eCB signaling essentially identical toother cortical regions, that is, eCB modulation of localGABAergic transmission and afferent glutamatergic inputs.Within the CeAL, which exhibits homology to the striatum,eCBs modulate glutamatergic inputs to this region,whereas eCB signaling modulates GABAergic transmis-sion from the CeAL to the CeAM (which shows homologyto the globus pallidus). From these anatomical and phys-iological similarities arise several testable hypotheses.First, the functional role of eCB signaling, from an informa-tion processing perspective, is likely to be similar acrosshomologous structures (i.e. PFC, sensory cortex, and BLA;striatum and CeAL; globus pallidus and CeAM). Second,the net modulation of circuit output is a function of thecombined modulation at each step of the cortico-striato-pallidal circuit. Third, regulatory mechanisms that governadaptations of eCB signaling may be similar across ana-tomical domains (i.e. cortex and BLA, striatum and CeAL).

Conceptualization of eCB signaling within the forebrainas being overlaid on parallel but interactive cortico-striato-pallidal loops, with each node in the circuit subject to thesame functional modulation across cortical subdomains(motor and limbic; see Fig. 2) could allow for a moreintegrated understanding of the role of eCB signaling inbehavioral output. This conceptualization yields testablehypotheses that could drive future research into the role ofeCB signaling in the modulation of forebrain function fromboth a systems and computational perspective.

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(Accepted 17 August 2011)(Available online 22 August 2011)

endocannabinoid signaling in the amygdala: anatomy, synaptic signaling, behavior, and adaptations to...

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