synaptic targets of d9-tetrahydrocannabinol in the...

Synaptic Targets of D9-Tetrahydrocannabinolin the Central Nervous System

Alexander F. Hoffman and Carl R. Lupica

U.S. Department of Health and Human Services, National Institutes of Health, National Institute on DrugAbuse Intramural Research Program, Electrophysiology Research Section, Baltimore, Maryland 21224

Correspondence: [email protected]

The availability of potent synthetic agonists for cannabinoid receptors has facilitated ourunderstanding of cannabinoid actions on synaptic transmission in the central nervoussystem. Moreover, the ability of these compounds to inhibit neurotransmitter release atmany central synapses is thought to underlie most of the behavioral effects of cannabinoidagonists. However, despite the widespread use and misuse of marijuana, and recognition ofits potential adverse psychological effects in humans, comparatively few studies have exam-ined the actions of its primary psychoactive constituent, D9-tetrahydrocannabinol (THC), atwell-defined synaptic pathways. Here we examine the recent literature describing the effectsof acute and repeated THC exposure on synaptic function in several brain regions andexplore the importance of these neurobiological actions of THC in drug addiction.

Marijuana (Cannabis sativa) is the mostcommonly used illicit drug in the United

States, and its prevalence and abuse potentialamong adolescents continues to increase (Sub-stance Abuse and Mental Health Services Ad-ministration 2002). Cannabis dependence iscurrently recognized in the Diagnostic and Sta-tistical Manual of Mental Health Disorders(American Psychiatric Association 2000) and isessentially defined by three majorcriteria: (1) theexpenditure of considerable time and resourcesto acquire cannabis, (2) continued use of canna-bis despite significant use-related negative phys-ical and/or psychological consequences, and (3)the need for increasing amounts of cannabis tomaintain the desired level of intoxication. Ad-ditionally, there is now strong evidence for can-nabis withdrawal symptoms in humans that

include insomnia, cognitive impairment, emo-tional lability, psychiatric depression, irritabili-ty, and anger. However, despite its widespreaduse compared with other drugs and its growingavailability to larger segments of the popula-tion, our knowledge of the effects of acute andlong-term use of cannabis on the brain remainsrelatively poor.

The major psychoactive component of mar-ijuana, D9-tetrahydrocannabinol (THC), wasisolated nearly 50 years ago by Mechoulamand colleagues (Gaoni and Mechoulam 1964;Mechoulam and Gaoni 1965). Many subse-quent studies have established that most of thepsychoactive effects of cannabis, such as eupho-ria, relaxation, anxiety, confusion, memory loss,and paranoia, are mediated by THC (Adamsand Martin 1996; Wachtel et al. 2002; Hall and

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Degenhardt 2009). These actions of THC, andthose of synthetic and endogenous cannabinoidmolecules, occur through the activation of dis-tinct cannabinoid receptors (Devane et al. 1988;Howlett et al. 1990), and it is through specificantagonism of cannabinoid receptors that theCB1 receptor subtype has been identified as me-diating the subjective “high” described by hu-man marijuana users (Huestis et al. 2001). Al-though it is well established that THC mediatesmost of the psychoactive actions of cannabis,there are also a large number of additional can-nabinoid molecules present in the marijuanaplant (Mechoulam 1970; Adams and Martin1996). However, our understanding of the phar-macological actions of most of these com-pounds is limited, particularly with regard toputative synaptic function. Therefore, our dis-cussion here will necessarily remain confined tothe actions of THC.

Whereas the behavioral effects of THC arewell-established by many studies, comparativelyfew physiological investigations have examinedthe actions of this drug on synaptic function(Foy et al. 1982; Nowicky et al. 1987)—studiesthat are best conducted in vitro. The relativepaucity of studies examining synaptic effectsof THC may reflect the extremely poor aqueoussolubility of this compound and the limited ex-tent to which this lipophilic molecule can accesssynaptic cannabinoid receptors in brain-slicepreparations (Laaris et al. 2010). Consequently,much of the mechanistic information regardingthe effects of cannabinoids on synaptic func-tion has been obtained using more soluble syn-thetic agonists, rather than THC (Mackie andHille 1992; Sullivan 1999; Hoffman and Lupica2000, 2001; Gerdeman and Lovinger 2001).These studies have been critical in defining theprimary physiological role of cannabinoid re-ceptors as presynaptic modulators of excitatoryand inhibitory neurotransmitter release in thecentral nervous system (CNS) (Fig. 1), and haveidentified these same sites as mediating the ef-fects of endogenous cannabinoids that are pro-duced by neurons during physiological activa-tion (Wilson and Nicoll 2001; Alger 2002).However, given the widespread use of THC inhumans, its proposed involvement in addiction

and mental illness (Leweke and Koethe 2008),the potential expansion of its use in humantherapeutic contexts, and the experimental evi-dence that it can act at non-cannabinoid recep-tor sites (Hejazi et al. 2006; De Petrocellis and DiMarzo 2010; Xiong et al. 2011), we believe that itis important to define its capacity for alteringbrain activity, with a particularemphasis on syn-aptic function. Therefore, our intention here isto review the recent literature to examine theneurobiological substrates acted on by THC, tohighlight its effects on specific synaptic process-es following acute and long-term exposure, andto examine its potential roles in drug addiction.

LOCALIZATION AND FUNCTIONALPROPERTIES OF CANNABINOIDRECEPTORS IN THE CNS

Two receptor subtypes are known to mediatethe effects of synthetic and natural cannabinoidagonists in the CNS. Both CB1 and CB2 canna-binoid receptors belong to the larger class of

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Figure 1. Generalized scheme of the sites of THCaction throughout the mammalian central nervoussystem. CB1 receptors are shown on both excitatoryglutamatergic (Glut.) and inhibitory (GABA) axonterminals, and in many cases these synapses areformed with neurons providing output (Output neu-ron) of central nuclei (e.g., hippocampal pyramidalneurons and MSNs in the NAc and dorsal striatum).These presynaptic CB1 receptors can be activated bythe release of endocannabinoids (eCB) on depolari-zation of postsynaptic neurons or by exogenous ago-nists such as THC. Also shown is the chemical struc-ture of THC (inset).

A.F. Hoffman and C.R. Lupica

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G-protein-coupled receptors (GPCRs) andhave been designated as either “central” or “pe-ripheral,” based on their localization in the CNSor in peripheral nonneuronal tissues, respec-tively (Pertwee 1997). This designation has re-cently been challenged by several reports dem-onstrating CB2 receptors in the brain, albeit atmuch lower levels than CB1, and these receptorsmay mediate some of the central actions of THCas well as synthetic and endogenous CB agonists(Van Sickle et al. 2005; Atwood and Mackie2010; Xi et al. 2011). However, all physiologicalstudies to date using CB1 selective antagonistsand CB1 receptor “knockout” mice in intactneural circuits have shown that the CB1 recep-tor mediates the inhibition of inhibitory (Hoff-man and Lupica 2000; Manzoni and Bockaert2001; Wilson et al. 2001; Kreitzer et al. 2002;Chevaleyre and Castillo 2003) and excitatory(Sullivan 1999; Gerdeman and Lovinger 2001;Robbe et al. 2002; Melis et al. 2004; Straiker andMackie 2005; Takahashi and Castillo 2006) neu-rotransmitter release from axon terminals (Fig.1). Thus, although it is possible that CB2 recep-tors may influence synaptic transmission in theCNS, the existing literature supports a predom-inant, if not exclusive, role for CB1 receptors asthe major target for endogenous and exogenouscannabinoids at axon terminals.

The activation of synaptic CB1 receptors byendogenous and exogenous agonists inhibitsneurotransmitter release directly, via inhibitorycoupling to voltage-dependent calcium chan-nels (Mackie and Hille 1992; Sullivan 1999;Hoffman and Lupica 2000), or through activa-tion of potassium channels, which shortens ac-tion potential duration and lessens the amountof neurotransmitter release per action potential(Mu et al. 1999; Schweitzer 2000; Robbe et al.2001). Both CB1-dependent mechanisms ap-pear to occur through the direct interaction ofbg G-protein subunits with the ion channels,rather than through a second messenger (Hoff-man and Lupica 2000; Robbe et al. 2001), al-though other effectors have been shown to beinvolved in some of the presynaptic effects ofcannabinoids (Pan et al. 2008; Benard et al.2012). Endocannabinoids that are released byneurons during heightened neuronal activity

also interact with axon terminal CB1 receptorsto inhibit the release of GABA or glutamate(Alger 2002; Melis et al. 2004; Riegel and Lupica2004). These transient endocannabinoid-de-pendent phenomena have been labeled depolar-ization-induced suppression of inhibition orexcitation (DSI/DSE), respectively. The activa-tion of CB1 receptors by endocannabinoids canalso initiate long-term forms of synaptic plas-ticity, including long-term depression (LTD),and can modify the strength of long-term po-tentiaton (LTP) (Carlson et al. 2002; Kortlevenet al. 2011). The reader is referred to severalreviews on endocannabinoid-mediated plastic-ity for further detail (Alger 2002; Gerdemanet al. 2003; Chevaleyre et al. 2006; Kano et al.2009; Alger and Kim 2011).

ACUTE ACTIONS OF THC ON SYNAPTICTRANSMISSION IN THE HIPPOCAMPUS

Excitatory Synaptic Transmission

The well-described actions of marijuana on cog-nitive function and memory (Abel 1970; Drewand Miller 1974), together with the establishedrole of the hippocampus in various forms oflearning and memory (Squire 1992; Burgesset al. 2002; Bird and Burgess 2008), led inves-tigators to evaluate the effects of THC in hippo-campal brain slices. Early studies describedrelatively complex bimodal effects on extracellu-larly recorded field potentials in the hippo-campus (Foy et al. 1982; Nowicky et al. 1987).However, these studies were performed beforeidentification of CB1 receptors as mediatorsof cannabinoid actions and before the devel-opment of cannabinoid receptor antagonistsand transgenic animals. Additionally, the effec-tive THC concentrations in these studies arenot consistent with more contemporary studiesin which the aqueous insolubility of THC inbrain slices is specifically addressed (Hoffmanet al. 2010; Laaris et al. 2010). Several studieshave circumvented the difficulty of low levelsof passive diffusion of THC into brain slicesthrough the use of synaptically coupled hip-pocampal neurons in culture. One of these stud-ies showed that THC inhibited glutamatergic

THC and Synaptic Transmission

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synaptic responses onto neighboring culturedhippocampal pyramidal neurons with a 50% ef-fective concentration (EC50) of 20 nM (Shen andThayer 1999). Because the efficacy of THC wasless than that of the synthetic aminoalkylindolecannabinoid agonist, WIN55,212-2, the investi-gators concluded that THC was a partial agonistat CB1 receptors located on excitatory axon ter-minals in the hippocampus (Shen and Thayer1999). We have also found that THC can act asa partial agonist at presynaptic CB1 receptors toinhibit glutamate-mediated synaptic responsesin hippocampal slices (Hoffman et al. 2010).However, the effects of THCon glutamate releasewere only observed when THC was solubilizedin b-cyclodextrin (Hoffman et al. 2010), whichgreatly improves the penetration of the drug intobrain slices (Hazekamp and Verpoorte 2006). Incontrast to these studies, other work in hippo-campal cultures has showed that, whereas THChad no effect on glutamatergic synaptic trans-mission, it blocked the effects of both syntheticand endogenous cannabinoid agonists at thesesamesynapses (StraikerandMackie 2005).How-ever, prolonged (�19 h) application of THC tocultured hippocampal neurons desensitizedCB1 receptors on glutamate terminals, suggest-ing that THC can antagonize the effects of fullagonists at CB1 receptors acutely, but can alsoeffectively down-regulate CB1 receptor functionfollowing longer exposure. This observation ledto the provocative hypothesis that some of thepsychoactive effects of THC may be mediatedby antagonism of endocannabinoid actions,rather than through direct activation of CB1 re-ceptors in the CNS (Straiker and Mackie 2005).

Inhibitory Synaptic Transmission

Together, the above studies examining the ef-fects of THC on glutamatergic synaptic trans-mission showed that the phytocannabinoidacts either as a partial agonist at CB1 receptorsor as an antagonist of exogenous and endoge-nous cannabinoids at these receptors (Shen andThayer 1999; Straiker and Mackie 2005). How-ever, in contrast to these studies, much less at-tention has been given to the effects of THC onGABAergic synaptic transmission in the CNS,

despite the much higher density of CB1 recep-tors on these axon terminals compared withglutamate terminals in the hippocampus (Mar-sicano and Lutz 1999; Kawamura et al. 2006). Toaddress this we investigated the effects of THCon GABAergic synaptic transmission in hippo-campal brain slices using THC solubilized in b-cyclodextrin, as described previously. We found,similar to studies with synthetic and endoge-nous cannabinoids, that THC inhibits synapticGABA release via activation of presynaptic CB1receptors. However, in contrast to the partialagonist properties of THC at excitatory synaps-es, we found that synaptic GABA currents wereinhibited with an efficacy that was not distin-guishable from the full agonist, WIN55,212-2(Fig. 2) (Laaris et al. 2010). This difference inagonist efficacy at excitatory versus inhibitorysynapses in the hippocampus likely reflectsthe lower level of CB1 receptor expression onglutamatergic axon terminals compared withGABAergic terminals (Kawamura et al. 2006),because partial agonist activity is often observedwhere “receptor reserve” is low (Hoyer and Bod-deke 1993). The differential efficacy of THC atexcitatory versus inhibitory hippocampal syn-apses is likely important for the modulation ofhippocampal circuits and for the pronouncedeffects of marijuana on cognitive function andmemory in humans. Thus, inhibitory axon ter-minals would likely be more sensitive to, andmore completely inhibited by, THC, leadingto a disruption of hippocampal network activ-ity that is largely controlled by synchronizedGABA neuron activity and is profoundly dis-rupted by cannabinoids (Hajos et al. 2000;Robbe et al. 2006). In support of this hypothe-sis, targeted deletion of the CB1 receptor fromGABAergic neurons prevented the disruptionof hippocampal-dependent behavior by THC,whereas deletion of this receptor from glutama-tergic neurons was ineffective in abolishingthe effects of THC (Puighermanal et al. 2009).These data, together with those demonstratingfull agonist effects of THC at inhibitory synaps-es in the hippocampus, suggest that the well-known effects of this drug on memory and cog-nition in humans likely occur through the pre-synaptic inhibition of GABA release.



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ACUTE EFFECTS OF THC: NON-CANNABINOID-RECEPTORSITES OF ACTION

To date, there have been few studies examiningthe direct effects of THC on synaptic transmis-sion in other brain areas. Brown and colleaguesreported that THC (10 mM) and WIN55,212-2

(3 mM) produce similar effects on striatal glu-tamate release, although a full dose–responseanalysis was not performed (Brown et al. 2003).Interestingly, these investigators also report thatTHC acts through CB1 receptors to inhibit glu-tamate uptake, which then activated presynapticmetabotropic glutamate receptors (Brown etal. 2003). Effects of THC on neurotransmitter

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Figure 2. Inhibition of synaptic GABA release from inhibitory interneuron axon terminals in the mousehippocampus by THC. (A) Illustration of a whole-cell voltage clamp configuration to record GABA-mediatedsynaptic currents evoked by electrical stimulation of GABA neuron axon terminals, and the recording of non-synaptic GABAA currents activated by ultraviolet (UV) light uncaging of GABA in the same hippocampalpyramidal neurons. UV light was focused into a �45-mm spot near the pyramidal neuron soma to releasefree GABA from a-carboxy-2-nitrobenzyl (CNB)-caged GABA. (B) Synaptic and nonsynaptic GABAA receptorcurrents are unaffected by THC (10 mM), suspended in b-cyclodextrin (see text) when applied to hippocampalslices from CB12/2 mice. (C) THC inhibition of synaptic GABAA receptor-mediated responses evoked byelectrical stimulation (squares) and absence of THC effects on nonsynaptic GABAA currents activated by UVuncaging of GABA (black circles) in hippocampal slices from CB1þ/þmice. (D) Comparison of concentration–response curves for the aminoalkylindole synthetic cannabinoid agonist WIN55,212-2 and THC. Note thatwhereas THC is less potent than WIN55,212-2, the maximal inhibition of IPSCs by both cannabinoids is similaron GABAergic synaptic responses. (Adapted from Laaris et al. 2010; reprinted, with permission, from theauthor.)



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uptake have been observed previously (Maneufet al. 1996; Coull et al. 1997). However, the ki-netics of directly activated nonsynaptic GABAA

receptor currents by GABA released with ul-traviolet (UV) laser uncaging (Fig. 2) were in-sensitive to THC, suggesting that GABA uptakeis not affected by THC in the hippocampus(Laaris et al. 2010). More recently, it has beenshown that THC can impair cellular metabo-lism through activation of CB1 receptors lo-cated on mitochondria, and that these recep-tors regulate hippocampal DSI (Benard et al.2012). Another potential target that has beendescribed for THC is the glycine receptor-chlo-ride channel (Hejazi et al. 2006). This groupshowed that THC potentiates glycine currentsin cultured ventral tegmental area (VTA) neu-rons, with an EC50 of 115 nM (Hejazi et al.2006). This occurred independently of CB1 re-ceptor activation through interaction with spe-cific sites on the glycine receptor (Xiong et al.2011). Thus, although further study is needed,it appears that both CB1 receptor-dependentand -independent actions of THC may exist inthe CNS.

ENDURING SYNAPTIC EFFECTS OFACUTE IN VIVO THC

Numerous studies have evaluated the effects ofacute in vivo exposure to addictive drugs, suchas cocaine, nicotine, and alcohol, on synapticfunction, using an “ex vivo” strategy in whichelectrophysiological recordings are made fromneurons in brain slices obtained from drug ex-posed animals (Ungless et al. 2001; Saal et al.2003; Niehaus et al. 2010). However, compara-tively few studies have used this strategy withTHC exposure. Mato and colleagues examinedthe effects of a single, acute injection of THC(3 mg/kg, i.p.) in mice (Mato et al. 2004) andfound a CB1-dependent impairment of LTDof synaptic transmission at both inhibitorysynapses in the hippocampus and at excitatorysynapses on medium spiny neurons (MSNs)in the nucleus accumbens (NAc). Each formof LTD is dependent on the release of endoge-nous cannabinoids during conditioning trainsof electrical stimulation of the brain slices, as

well as the activation of presynaptic CB1 recep-tors (Robbe et al. 2002; Chevaleyre and Castillo2003). Although there was no change in CB1receptor expression or G-protein coupling tothese receptors following single injections ofTHC, a reduction in cannabinoid agonist-me-diated inhibition of both GABA and glutamaterelease suggested the development of rapid tol-erance. Whereas behavioral studies have shownrapid tolerance to the behavioral effects of THC(Hutcheson et al. 1998), this was the first studyto show synaptic changes following an acutedosing paradigm. It is not clear how such arapid loss of CB1 receptor sensitivity develops,although there is evidence to suggest that CB1receptors are highly mobile within the plasmamembrane of cortical neurons and that this mo-bility can be dramatically reduced by short-termagonist treatment (Mikasova et al. 2008).

A single exposure to THC can also cause theselective remodeling of a population of gluta-matergic synaptic inputs to midbrain dopamine(DA) neurons in the ventral tegmental area(VTA) (Good and Lupica 2010). In this study,a single in vivo exposure to THC (10 mg/kg,i.p.) rapidly increased the expression of AMPAreceptors containing the GluA1 subunit at sub-cortical pedunculopontine nucleus (PPN) glu-tamatergic inputs to VTA DA neurons in rats(Good and Lupica 2010). Furthermore, thissynaptic remodeling was prevented by pretreat-ment with the CB1 antagonist AM251. Becausethe GluA1 subunit confers calcium permeabil-ity and increases the single-channel conduc-tance of AMPA receptors, this THC-inducedmodification is thought to result in long-termpotentiation (LTP) of these synapses and there-by increase the subcortical drive of VTA DAneurons by the PPN (Argilli et al. 2008; Goodand Lupica 2010). This modification in AMPAreceptor function also renders these glutamatesynapses susceptible to subsequent LTD, whichoccurs through the insertion of calcium-im-permeable GluA2 AMPA receptor subunitsand the removal of GluA1 (Good and Lupica2010). Thus, a single exposure to THC can pro-duce an enduring increase in the sensitivity ofDA neurons to subcortical excitatory drive at aspecific set of synapses, and this can be modified



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by subsequent activation of this circuit. Thissuggests that one of the early neurobiologicalresponses to THC might be a change in VTADA neuron sensitivity to glutamatergic synapticinputs, and this may subsequently play a rolein the long-term modification in mesolimbo-cortical DA output. In contrast, to the effectsof a single exposure to THC, a single exposureto cocaine increased GluA1 function at bothPPN and non-PPN glutamatergic synapsesonto VTA DA neurons (Good and Lupica2010). Although previous data have suggestedthat many commonly abused drugs affect syn-aptic plasticity in VTA DA neurons in a similarfashion (Saal et al. 2003), it would appear thatTHC may have somewhat unique, pathway-de-pendent effects in this brain reward circuit. Thisfurther suggests that different abused drugs mayhave the capacity to alter distinct excitatory in-puts to midbrain DA neurons.

ENDURING SYNAPTIC EFFECTS OFLONG-TERM IN VIVO THC

In contrast to the limited number of studiesexamining the ability of acute THC to affectsynaptic function, a number of laboratorieshave evaluated the physiological consequencesof repeated THC exposure. Many of these stud-ies were based on the observation that long-term THC treatment (usually 5–10 d) causesadaptations of CB1 receptors and/or theirdownstream cellular effectors (Breivogel et al.1999; Rubino et al. 2000; Tonini et al. 2006).

NUCLEUS ACCUMBENS

The NAc, also known as the ventral striatum,is involved in motivational behavior, plays apivotal role in mediating the effects of abuseddrugs, and undergoes significant molecular,structural, and physiological remodeling dur-ing passive and volitional drug administration(Lupica et al. 2004; Chen et al. 2010; Russo et al.2010; Luscher and Malenka 2011). Both theNAc and the dorsal striatum (discussed in alater section) receive extensive cortical glutama-tergic input onto the primary GABAergic out-put neurons, known as medium spiny neurons

(MSNs) that also receive input from ventralmidbrain DA neurons. Because of its centralrole in the brain’s motivation and reward cir-cuitry, the actions of abused drugs on synapticprocess in the NAc have received considerableattention. Early studies in our laboratory andothers showed that, similar to the hippocam-pus, CB1 receptors are located on both gluta-matergic and GABAergic axon terminals inthe NAc, and that activation of these receptorscould inhibit neurotransmitter release (Hoff-man and Lupica 2001; Robbe et al. 2001). Sub-sequent studies found that repeated exposure toTHC (10 mg/kg, i.p. for 7 d) resulted in toler-ance to the inhibitory effects of WIN55,212-2at both glutamate and GABA synapses in theNAc (Hoffman et al. 2003). Furthermore, be-cause LTD of excitatory synaptic transmission inthe NAc is dependent on the release of the en-docannabinoid 2-arachidonoylglycerol, and itsactivation of CB1 receptors on glutamate axonterminals (Robbe et al. 2002), an early focus wasto determine the effect of chronic THC on thisform of synaptic plasticity. We found that themarked tolerance of CB1 receptors in the NAcfollowing chronic THC exposure was associ-ated with a loss of endocannabinoid-mediatedLTD (Hoffman et al. 2003). Although the behav-ioral roles of endocannabinoid-dependent LTDin the NAc are largely unknown, it has beenspeculated that synaptic remodeling of striatalcircuits by long-term plasticity may representa mechanism through which drug use may pro-gress from casual to impulsive (Gerdeman etal. 2003; Kauer and Malenka 2007). Therefore,from these studies we can conclude that ei-ther short-term (Mato et al. 2004) or long-term (Hoffman et al. 2003) exposure to THCcan limit the degree to which NAc glutamatesynapses undergo LTD, and this appears to re-sult from a down-regulation of CB1 receptorfunction and the ability of endocannabinoidsto initiate this form of synaptic plasticity. Wepropose that this effect of THC may alter moti-vational processes mediated by the NAc andmay play a role in modulating the reinforcingproperties of other abused drugs acting eitherdirectly within the NAc or indirectly by alteringDA function in this brain structure.



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CEREBELLUM

Cannabinoid CB1 receptors are found at veryhigh concentrations in the cerebellum, wherethey are expressed on excitatory parallel fi-ber (PF)–Purkinje cell (PC) synapses (Leveneset al. 1998; Safo and Regehr 2005). The firingpatterns of PCs are thought to be involved insetting motor activity and gait, and cannabi-noids are known to induce ataxia, catalepsy,and hypolocomotion, at least partly throughinteractions with cerebellar CB1 receptors(Patel and Hillard 2001). In addition, toleranceto these acute motoric effects of THC occurswith prolonged administration, and withdrawalfrom THC is associated with alterations in cer-ebellar cyclic AMP/PKA pathway function(Tzavara et al. 2000). For these reasons, Toniniand colleagues examined the effects of repeatedTHC exposure (10 mg/kg, 2� daily, 4.5 d) inmice on PF synapses on cerebellar PCs (Toniniet al. 2006). This THC treatment producedtolerance to the inhibitory effects of the fullCB1 receptor agonist, CP55,940, at PF glutama-tergic synapses. The investigators also reportedthat chronic THC enhanced glutamatergictransmission at this synapse, and that the in-crease in glutamate release activated extrasynap-tic metabotropic glutamate type-1 receptors(mGluR1) located on the cerebellar PCs. En-docannabinoid-dependent LTD and LTP areknown to occur at glutamatergic PF-PC synaps-es, and these forms of plasticity are thought tounderlie cerebellar-dependent motor learning(Edwards and Skosnik 2007). Therefore, theconsequences of chronic THC exposure on cer-ebellar synaptic plasticity are of critical impor-tance to our understanding of cannabis effectson this brain structure. Although LTD appearedto be unaffected by chronic THC, stimuli thatwould normally generate LTP at the PF–PC syn-apse produced LTD instead (Tonini et al. 2006).Furthermore, the investigators found that LTPcould be rescued following blockade of adeno-sine A1 receptors, suggesting that THC treat-ment enhanced endogenous adenosine releasethat limited LTP, via presynaptic inhibitionof glutamate release. These investigators alsofound that the effects of repeated THC on PF-

PC synapses could be prevented by a reduc-tion in extracellular signal regulated kinase(ERK) signaling, through either pharmacologi-cal or genetic approaches (Tonini et al. 2006). Assuggested by the investigators, this “pathologi-cal” shift of cerebellar synaptic plasticity fromLTP to LTD following chronic THC would likelyhave profound consequences for cerebellar in-formation processing (Tonini et al. 2006). Thisstudy shows the breadth of changes in down-stream cellular signaling pathways that can beaffected by chronic THC exposure (Rubinoet al. 2004) and serves to highlight the criticallink between these changes and altered synapticprocesses.

HIPPOCAMPUS

In addition to the disruptive acute effect of THCon hippocampal synaptic function discussedabove, there have been many studies detailingthe deleterious effects of long-term cannabisuse on cognitive executive function in humans,and the completeness of the reversibility of theseimpairments is currently a subject of debate(Bolla et al. 2002; Pope et al. 2002; Crean et al.2011). For these reasons the effects of long-termTHC treatment on synaptic function and long-term synaptic plasticity in the hippocampus invitro have been examined. We compared LTP atSchaffer collateral glutamatergic synapses ontoCA1 pyramidal neurons in brain slices obtainedfrom adolescent rats treated with THC (10 mg/kg, for 1, 3, or 7 d) or vehicle (Hoffman et al.2007). In contrast to the results of Mato et al.(2004), we did not observe an impairment ofLTP 24 h following a single THC injection.However, a reduction in hippocampal LTP wasobserved 24 h following three consecutive daysof THC administration, and LTP was complete-ly absent following 7 d of THC treatment (Hoff-man et al. 2007). The disruptive effects of 7 dTHC on LTP were prevented when each THCinjection was preceded by an injection withAM251, strongly suggesting that chronic THCinterfered with hippocampal LTP via activationof CB1 receptors (Hoffman et al. 2007). Addi-tional experiments showed that at the time ofLTP induction THC was no longer detectable in



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the brains of the chronically treated rats usingliquid chromatography-mass spectrometry (LC-MS), suggesting that the LTP impairment wasnot simply the result of ongoing activation ofCB1 receptors by residual THC, but rather thatchronic exposure to the drug caused enduringneurobiological changes that interfered withsynaptic plasticity.

When the time course for reversal of the LTPimpairment was examined, it was found thatLTP partially recovered beginning 3 d followingTHC treatment cessation, although completerecovery was not observed at time points upto 14 d post THC (Hoffman et al. 2007). Thismay have implications for interpreting the long-lasting effects of cannabis on human memory(Crean et al. 2011).

These data suggest that long-term THC ex-posure can disrupt hippocampal LTP by causingcellular or synaptic changes that endure follow-ing cessation of the drug. Therefore, in an ef-fort to determine the mechanism of the chron-ic THC impairment of hippocampal LTP weexamined glutamatergic and GABAergic synap-tic function following this same 7-d THC treat-ment. Similar to results described in cerebellum(Tonini et al. 2006), we observed an increase inthe probability of glutamate release followingchronic THC, as well as alterations in AMPAreceptor function (Hoffman et al. 2007), thatmight explain the LTP impairment. These re-sults have recently been confirmed and expand-ed by another study, which also showed thatchronic THC blocks LTP at glutamatergic den-tate granule cell perforant path synapses ontoCA3 pyramidal neurons (Fan et al. 2010). Ad-ditionally, these investigators describe increasedsynaptic glutamate release and a reduction inpostsynaptic AMPA and NMDA receptor ex-pression in hippocampal tissue from animalschronically treated with THC. The investigatorssuggest that the alterations in glutamatergic syn-apses likely result from CB1R-dependent reduc-tions in phosphorylation of cAMP response-element binding protein (CREB), as a conse-quence of the inhibition of adenylyl cyclase bythese receptors (Fan et al. 2010). Although base-line GABAergic transmission was not altered by7 d THC treatment, we observed a marked tol-

erance of GABAergic synapses to WIN55,212-2.This is consistent with the full agonist actions ofTHC at GABAergic synapses described previ-ously (Laaris et al. 2010), as well as the criticallydefined role for these synapses in mediating thebehavioral effects of THC (Puighermanal et al.2009). Together these data suggest that long-term use of cannabis and exposure to THC re-sults in specific alterations in both glutamater-gic and GABAergic synaptic function that canprevent a form of synaptic plasticity that is likelycentral to the formation of hippocampal-de-pendent memories (Whitlock et al. 2006). Fur-thermore, it is likely that these changes are notrestricted to the hippocampus, but likely occurthroughout the CNS in brain regions where CB1receptors are highly expressed, and this may ex-plain the extensive effects of long-term cannabisuse on executive cognitive function. The extentto which these cellular changes fully reverse fol-lowing termination of cannabis use and wheth-er there is an interaction between the use of thedrug, the age of the individual, and the revers-ibility of these phenomena is currently underinvestigation.

DORSAL STRIATUM

The dorsal striatum plays an integral role inmotor behavior, and the ability of THC to in-duce hypolocomotion and ataxia is thought toresult from activation of CB1 receptors in thisand other brain areas (Patel and Hillard 2001;Gerdeman et al. 2003). Furthermore, toleranceto the motoric effects of THC is readily observ-able (Rubino et al. 2005). Because the dorsalstriatum processes information received fromcortical, thalamic, and midbrain DA inputs itis also central to many forms of instrumentallearning and habit formation, which is of im-portance to the process of addiction (Gerdemanet al. 2003; Everitt et al. 2008). Recent evidencesuggests that the dorsomedial and dorsolateralstriatum may be involved in instrumental, re-ward-based processing and habit-driven be-havior that persists in the absence of overt re-ward, respectively (Yin et al. 2006, 2008, 2009).Also, similar to the NAc (ventral striatum), LTDof cortical glutamatergic inputs to the dorsal



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striatum is dependent on endocannabinoid ac-tivation of presynaptic CB1 receptors (Gerde-man et al. 2002; Ronesi et al. 2004), whichmay be involved in the establishment of stria-tal-dependent learning and habit formation(Kano et al. 2009). Because of the importanceof the endogenous cannabinoid system in me-diating motor behavior, instrumental learn-ing, and habit formation and its potential in-volvement in compulsive drug seeking, the roleof THC in regulating endogenous cannabinoidfunction has been studied (Nazzaro et al. 2012).This study found that, similar to previous workin other brain areas, treatment of mice withTHC (10 mg/kg, i.p., 5 d) eliminated endoge-nous cannabinoid-dependent LTD of corticalinputs to MSNs in the dorsolateral striatumthat was associated with behavioral toleranceto THC (Nazzaro et al. 2012). Striatal MSNssend their GABAergic output to downstreamtargets via two pathways known as indirect anddirect, which are defined by various postsynap-tically expressed receptors and neuropeptides(Kreitzer 2009). When electrophysiologicallyrecorded dorsolateral striatal MSNs were segre-gated according to indirect and direct pathways,using post-hoc identification with single-cellreverse transcriptase polymerase chain reaction(RT-PCR) or immunohistochemistry, it wasfound that only the DA D2 receptor expressingMSNs associated with the indirect pathwayshowed endogenous cannabinoid-dependentLTD (Nazzaro et al. 2012). Furthermore, chronicTHC treatment blocked LTD only at synapsesonto these indirect pathway MSNs. The in-vestigators also showed that endocannabinoid-independent LTD in the dorsomedial striatumwas not affected by chronic THC (Nazzaro et al.2012).

Depotentiation is another form of synapticplasticity in which previously established LTPis reversed by appropriate subsequent low-frequency stimuli (Wagner and Alger 1996).Long-term potentiation can be induced at cor-ticostriatal synapses onto MSNs in the dorso-lateral striatum using high-frequency electricalstimulation when extracellular magnesium isreduced (Centonze et al. 1999). Nazzaro et al.(2012) found that depotentiation of cortico-

striatal synapses on indirect pathway MSNscould be blocked by the CB1 receptor antago-nist AM-251, demonstrating a role for endo-cannabinoids in the weakening of these previ-ously strengthened synapses. When the effectsof chronic THC were examined on depotentia-tion, it was found that, like LTD, depotentiationwas prevented. Thus, the investigators showedthat both LTD and depotentiation of LTP inthe dorsolateral striatum were mediated by en-dogenous cannabinoids and were both blockedby chronic THC exposure. Because habit for-mation and habitual responding is thoughtto be mediated by the dorsolateral striatum(Yin et al. 2008) and is impaired by cannabinoidreceptor blockade (Hilario et al. 2007), it isimportant to evaluate the behavioral conse-quence of the aforementioned loss of endocan-nabinoid-dependent synaptic plasticity in thedorsolateral striatum on these behaviors. There-fore, Nazzaro (2012) evaluated the effect ofchronic THC on habitual behavior in mice.They found that, in contrast to mice treatedwith vehicle, mice tolerant to THC continuedto perform operant tasks in which the strengthof a food reinforcer was weakened by a deval-uation procedure. Based on these results, andthose of the synaptic plasticity experiments,the investigators conclude that long-term treat-ment with THC results in a switch from goaldirected to habitual behavior, in which micecontinue to emit responses despite having in-formation to suggest that the reinforcer associ-ated with that response is no longer as “valu-able” (Nazzaro 2012).

The investigators also make a strong causalcase for endocannabinoid-dependent LTD anddepotentiation in this behavioral process be-cause both the behavioral change and the lossof synaptic plasticity could be reversed with thesmall conductance calcium-sensitive potassiumchannel (SK) channel blocker, apamin. BecauseSK channels are involved in limiting the dura-tion of action potentials (Louise Faber 2009)blockade of this channel with apamin can in-crease action potential duration and therebyincrease the calcium-dependent release of en-dogenous cannabinoids from neurons (Pio-melli 2003; Riegel and Lupica 2004).



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SUMMARY AND CONCLUSIONS

The studies reviewed above show that bothacute and repeated exposure to THC have de-finable physiological consequences for synapticfunction and several forms of synaptic plasticityin the CNS. Pharmacological and genetic ap-proaches indicate that these effects of THCcan largely be ascribed to activation of CB1 re-ceptors, and although non-CB1 targets for THChave been described, the contribution of theseeffects to synaptic function appears to be small.Based on these findings and the ubiquitous ex-pression of CB1 receptors in the CNS, it is clearthat synaptic alterations caused by chronic THCcan affect a wide variety of brain processes andbehaviors. Future studies will likely focus moreclosely on causally linking the synaptic changescaused by acute or repeated THC exposure onthe behaviors mediated by brain regions inwhich endocannabinoid-dependent plasticityis observed and CB1 receptors are expressed.

ACKNOWLEDGMENTS

This work is supported by the U.S. Departmentof Health and Human Services, The NationalInstitutes of Health, and the National Instituteon Drug Abuse Intramural Research Program,and earlier studies were supported by a grantfrom the National Institute on Drug Abuse(DA14263 to C.R.L.).

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published online December 3, 2012Cold Spring Harb Perspect Med Alexander F. Hoffman and Carl R. Lupica System

-Tetrahydrocannabinol in the Central Nervous9∆Synaptic Targets of

Subject Collection Addiction

DependenceThe Role of the Central Amygdala in Alcohol

Marisa Roberto, Dean Kirson and Sophia Khomof Ventral Tegmental Area Dopamine NeuronsOpioid-Induced Molecular and Cellular Plasticity

Marie A. Doyle and Michelle S. Mazei-Robison

Release in Reward Seeking and AddictionA Brain on Cannabinoids: The Role of Dopamine

Kate Z. Peters, Erik B. Oleson and Joseph F. Cheer

Growth Factors and Alcohol Use DisorderMirit Liran, Nofar Rahamim, Dorit Ron, et al.

Accumbens Glutamatergic TransmissionPsychostimulant-Induced Adaptations in Nucleus

William J. Wright and Yan DongUse DisorderMedications Development for Treatment of Opioid

Matthew L. BanksE. Andrew Townsend, S. Stevens Negus and

the Central Nervous System-Tetrahydrocannabinol in9∆Synaptic Targets of

Alexander F. Hoffman and Carl R. Lupica

Opioid Pharmacogenetics of Alcohol AddictionWade Berrettini

Role of GABA and GlutamateThe ''Stop'' and ''Go'' of Nicotine Dependence:

Manoranjan S. D'Souza and Athina Markou WithdrawalHabenulo-Interpeduncular Pathways in Nicotine Mesolimbic Dopamine and

John A. Dani and Mariella De BiasiSystems Level Neuroplasticity in Drug Addiction

Matthew W. Feltenstein and Ronald E. See Transmission in the Ventral Tegmental AreaCocaine-Evoked Synaptic Plasticity of Excitatory

Christian Lüscher

TerminalisReceptors in the Bed Nucleus of the Stria

-Methyl-d-AspartateNEthanol Effects on

Tiffany A. Wills and Danny G. Winder

Animal Studies of Addictive BehaviorLouk J.M.J. Vanderschuren and Serge H. Ahmed

Translational Research in Nicotine DependenceJill R. Turner, Allison Gold, Robert Schnoll, et al.

Epigenetics and Psychostimulant Addiction

West, et al.Heath D. Schmidt, Jacqueline F. McGinty, Anne E.

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

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