Systemic, But Not Local, Administration ofCannabinoid CB1 Receptor Agonists

Modulate Prefrontal Cortical AcetylcholineEfflux in the Rat

CHRISTOPHER D. VERRICO,1 J. DAVID JENTSCH,3 LAURA DAZZI,4 AND ROBERT H. ROTH1,2*1Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA

2Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, USA3Department of Psychology at University of California at Los Angeles, Los Angeles, California, USA

4Department of Experiment Biology at the University of Cagliari, Cagliari, Italy

KEY WORDS �9-tetrahydrocannabinol (THC); Win 55,212-2; SR 141716A

ABSTRACT Drugs acting on brain cannabinoid CB1 receptors exert complex actionson modulatory transmitters that are involved in attention and cognition; however, littleis known about the precise pharmacological and anatomical mechanisms that governthese effects. Previously demonstrated effects of cannabinoids on acetylcholine (ACh) inthe hippocampus prompted us to evaluate changes in the prefrontal cortex, a siteassociated with mnemonic and attentional functions. We utilized in vivo microdialysis,coupled with direct reverse perfusion of agents, to study the actions on cannabinoidergicdrugs on ACh release within the rat frontal cortex. Systemic administration of the CB1

receptor agonists �9-tetrahydrocannabinol (THC) or WIN 55,212-2 (WIN) dose- andtime-dependently increased ACh release; these effects were blocked by pretreatmentwith the selective CB1 receptor antagonist / partial inverse agonist SR141716A (SR).THC applied by reverse dialysis in the frontal cortex caused no change in ACh release,although intrastriatal infusions of THC decreased ACh efflux. These data indicate thatcannabinoid agonists potentiate ACh release in the frontal cortex by activating canna-binoid receptors in brain regions other than the frontal cortex. Synapse 48:178–183,2003. © 2003 Wiley-Liss, Inc.

INTRODUCTION

�9-tetrahydrocannabinol (THC), one of the psycho-active ingredients in smoked marijuana (Cannabis sa-tiva) produces a variety of behavioral effects in humansand animals. There are extensive reports of severaldeficits of short- and long-term memory, as well asattention, after intake of cannabinoidergic substances,such as THC (Pope and Yurgelun-Todd 1991), effectsthat may be directly mediated by actions of THC oncannabinoid CB1 receptors in brain (Litchman et al.,1995; for review, see Hampson and Deadwyler, 1999).Likewise, CB1 receptor agonists, including THC, areknown to produce alterations of modulatory neuro-transmitters involved in cognition (Acquas et al., 2000,2001; Jentsch et al., 1997), providing an additionalsubstrate by which cannabinoids affect learning, mem-ory, and attention.

Cholinergic neurons innervating the prefrontal cor-tex (PFC), originating in the basal forebrain and thereticular core of the brainstem, are thought to be crit-ically involved in cognitive functions such as working

memory and attention (Broersen et al., 1995; Granon etal., 1995; Muir et al., 1994; Passetti et al., 2000). Stud-ies involving central injections of cholinergic drugshave identified cholinergic activity in the PFC as animportant substrate of short-term memory, (Granon etal., 1995) and visual attention (Muir et al., 1994).These studies are consistent with the overall view ofPFC cholinergic transmission playing an importantrole in short-term memory, in attentional processing ofenvironmental stimuli, and in arousal mechanisms(Everitt and Robbins, 1997; Sarter and Bruno, 2000).As THC, and other cannabinoid agonists, are alsoknown to affect cholinergic transmission (Acquas et al.,

*Correspondence to: Robert H. Roth, Ph.D., Department of Pharmacology,Yale University School of Medicine, PO Box 208066, New Haven, CT 06520-8066. E-mail: robert.roth@yale.edu

Received 6 September 2002; Accepted 11 February 2003

DOI 10.1002/syn.10202

SYNAPSE 48:178–183 (2003)

© 2003 WILEY-LISS, INC.

2000, 2001; Gessa et al., 1998a), the cognitive deficitsassociated with cannabis consumption may be due, inpart, to dysfunction of the cortical cholinergic system.However, current available reports are not congruent,as Gessa et al. (1998a) report a decrease in corticalacetylcholine (ACh) release following cannabinoid ad-ministration, while Acquas et al. (2000, 2001) report anincrease in release.

The immunohistochemical studies by Tsou et al.(1998) indicate that cannabinoid CB1 receptors arelocated in the PFC. These cannabinoid receptors maybe located on cholinergic synaptic nerve terminals inthe PFC, where they could directly influence the re-lease of ACh. This hypothesis is strengthened by therecent findings of Gifford et al. (2000), who reportedthat the synthetic cannabinoid receptor agonist WIN55,212-2WIN inhibits ACh release in cortical synapto-somes.

We therefore set out to characterize the effects of twocannabinoid CB1 receptor agonists, THC and WIN onACh efflux in the freely moving rat. Moreover, giventhe discrepancies between available reports on thissubject, we attempt to resolve a possible underlyingsource of this inconsistency. Furthermore, given thatlittle is known about the anatomical localization of thein vivo effects of the cannabinoids on ACh neurotrans-mission, we utilized reverse dialysis to examinewhether ACh efflux in the prefrontal cortex could bealtered by the direct application of THC into this regionof the rat brain.

MATERIALS AND METHODSAnimals

Male Sprague-Dawley rats (Charles River Labs, Por-tage, MI), 250–350 g, were housed in groups of three tofour per cage and maintained under a 12-h light/darkcycle (lights on 7:00 AM to 7:00 PM). Food and waterwere available ad libitum. After surgery, the rats wereindividually housed in Plexiglas boxes that doubled asthe experimental environment. All experiments wereperformed between 8:00 AM and 5:00 PM. The animalswere maintained under conditions consistent with theNIH Guide for the Care and Use of Laboratory Animalsand all protocols were approved by the Yale UniversityAnimal Care and Use Committee.

Microdialysis/reverse dialysis probeimplantation and experimental procedures

Rats were anesthetized with halothane (HalocarbonProducts Corp., River Edge, NJ) and placed in a ste-reotaxic frame. The skull was exposed and holes weredrilled for skull screws and for a concentric microdialy-sis probe (BAS, West Lafayette, IN). Concentric dialy-sis probes, with an active zone of 2 mm, were implantedinto the medial prefrontal cortex (AP �3.2 mm, ML�0.8 mm, DV –5.3 mm) or into the striatum (AP �1.7

mm, ML �1.2 mm, DV –5.0 mm) according to Paxinosand Watson (1982). In some rats a polyethylene cath-eter (Abbott Laboratories, North Chicago, IL) was in-serted into the intraperitoneal cavity and tunneledsubcutaneously to exit at the nape of the neck. Theprobe, infusion line, catheter, and screws were securedin place with dental cement. Experiments were per-formed at least 24 h after surgery, which has previ-ously been reported as sufficient time to allow theanimals to resume normal feeding, grooming, and mo-tor behaviors (Gessa et al., 1998a; Acquas et al., 2000,2001). The day of experiment, Ringer’s solution (pH7.4) containing 3 mM K�, 150 mM Na�, 1.3 mM Ca��,1.0 mM Mg��, and 155 mM Cl- was pumped via aperfusion pump through the dialysis probe at a con-stant rate of 2 �l/min. Neostigmine bromide (0.1 �M),obtained from Sigma-RBI (Natick, MA), was added tothe Ringer’s solution to increase basal concentrationsof ACh. Samples were collected every 20 min into ahead-mounted loop and analyzed by high-pressure liq-uid chromatography (HPLC) for the determination ofACh. Electrochemical detection of ACh was made ac-cording to the previously published methodology(Damsma and Westerink, 1991). All sample valueswere referenced with regard to external standards, mi-crodialysis samples were taken, and neurotransmittersmeasured for 120–180 min postdrug administration.Baseline was set as the average of the last three pre-treatment samples, not differing more than 20%.

In reverse dialysis experiments, the probes were per-fused with drug-free Ringer’s solution until a stablebaseline was achieved (as described above). After astable baseline was achieved, the perfusion solutionwas changed to a Ringer’s solution containing drug andallowed to flow for 10 min to equilibrate before collec-tion of a new sample started. The animals receivedinfusions of THC in ascending concentrations (10-5 to10-4M) into the medial frontal cortex or striatum. THCwas suspended in a drop of Tween 80, made up in a 10-3

M concentration, and then diluted to the concentra-tions utilized. Three 20-min samples were collected ateach concentration and, as always, a 10-min equilibra-tion period was observed between each concentration.

Drugs

(–)-trans-(6aR,10aR)-6a,7,8,10a-tetrahydro-6,6,9-tri-methyl-3-pentyl-6H-dibenzo-[b,d]pyran-1-ol [(–)-�-9-tetrahydrocannabinol], R-(�)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphtalenyl)methanone mesylate [WIN-55,212-2], and{N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-H-pyrazole-3-carboxamide}HCl [SR141716A]were obtained from Sigma-RBI. RBI, through theNIMH Chemical Synthesis Program, generously pro-vided the SR 141716A. THC (shipped in 100% ethanol)was first evaporated under a stream of purified nitro-gen. All drugs were dissolved in a drop of Tween 80 and

CANNABINOIDS MODULATE CORTICAL ACH 179

then brought to volume in saline. Injections were givenintraperitoneally (via an indwelling catheter) in a vol-ume of 1 ml/kg, or infused into the PFC or striatumthrough the dialysis probe.

Statistical analysis

Values are expressed as percent change with respectto baseline (being 100%). Data were analyzed withrepeated measure analysis of variance (time being therepeated measure). Tukey’s post-hoc test was appliedwhen ANOVA revealed main effects or interactions.

RESULTSBasal prefrontal cortical ACh output and

effects of vehicle on ACh release

The overall mean � SEM baseline concentrations ofACh in the dialysates from the prefrontal cortex was49.14 � 7.8 fmol/�l (n � 70). Intraperitoneal adminis-tration of vehicle (one drop of Tween 80 in saline) didnot significantly affect basal ACh output from the pre-frontal cortex (P � 0.05).

Effects of cannabinoid agonists (via indwellingi.p. catheter) on prefrontal cortical ACh release

Intraperitoneal administration of WIN 55,212-2 (1,5, and 10 mg/kg) produced a dose-dependent increase

in cortical ACh efflux (Fig. 1A; F(3,25) � 3.57, P � 0.05);post-hoc Tukey’s test revealed that this main effect wasdue to significant differences between both 5 and 10mg/kg of WIN and vehicle (P � 0.05). IndividualANOVAs revealed that WIN (5 mg/kg) produced a max-imal increase of 85 � 19% in ACh efflux (relative tovehicle) between the 20-min time point (P � 0.05), withrelease being significantly elevated at the 20, 40, and60-min time points. In addition, WIN (10 mg/kg) max-imally elevated ACh efflux (relative to vehicle) at the40-min time point (P � 0.05), with an increase of 66 �29% over baseline. The dose of 1 mg/kg failed to signif-icantly affect ACh release (P � 0.05) when compared tovehicle alone.

Figure 1B shows the effects of various doses of THC(1–10 mg/kg) vs. vehicle, delivered i.p., on ACh efflux inthe prefrontal cortex. Repeated measures ANOVA re-vealed a main effect of dose (F(3,28) � 8.88, P � 0.0005)for ACh efflux; post-hoc Tukey’s test revealed that thismain effect was due to significant differences between10 mg/kg of THC and vehicle (P � 0.05). THC (10mg/kg) significantly elevated ACh efflux (relative tovehicle) at all time points (P � 0.05), with a maximalincrease of 220 � 103% increase over basal values at 40min postinjection. Repeated measures ANOVA re-vealed a main effect of dose for THC 5 mg/kg (F(1,16) �

Fig. 1. Cannabinoid receptor agonists acutely increase prefrontalcortical acetylcholine (ACh) release; blockade with the cannabinoidreceptor antagonist, SR 141716A (SR). A: Acute effects of vehicle (n �8) and WIN (1, 5, or 10 mg/kg i.p.) (n � 6, 8, and 7, respectively) onprefrontal cortical ACh release of conscious freely moving rats. B:Acute effects of vehicle (n � 8) and THC (1, 5, or 10 mg/kg i.p.) (n �6, 12, and 6, respectively) on prefrontal cortical ACh release of con-

scious freely moving rats. C: Effects of SR (0.1 mg/kg i.p.) pretreat-ment on WIN (5 mg/kg i.p.) induced increases on prefrontal corticalACh release (n � 6). D: Effects of SR (0.1 mg/kg i.p.) pretreatment onTHC (5 mg/kg i.p.) induced increases on prefrontal cortical ACh re-lease (n � 5). Values expressed as means � SEM. Percentages ofpreinjection basal ACh levels. Comparison of all treatment sampleswith WIN or THC-treated rats and vehicle-treated rats (*P � 0.05).

180 C.D. VERRICO ET AL.

9.019, P � 0.01) on ACh efflux when compare to vehiclealone. A significant effect of time (F(1,8) � 6.276, P �0.0001) for THC 5 mg/kg was revealed; post-hoc tests ateach collection interval post-drug administration re-vealed that 5 mg/kg THC significantly elevated AChefflux (relative to vehicle) at the 20, 40, 60, and 80-mintime points, with a maximal increase of 64 � 17% overbasal values at 60 min postinjection. The 1 mg/kg doseof THC failed to significantly affect ACh release (P �0.05).

Effects of SR 141716A on basal and cannabinoidagonist-induced increases in prefrontal

cortical ACh release

Intraperitoneal administration of the CB1 receptorantagonist SR (0.1 mg/kg) alone had no significanteffect on cortical ACh release (P � 0.05). However, SR,given 40 min prior to WIN (5 mg/kg), blocked WIN-induced elevations of ACh release (P � 0.05) (Fig. 1C).Similarly, SR (0.1 mg/kg) attenuated the THC-induced(5 mg/kg) increase in ACh efflux (P � 0.05) (Fig. 1D).

Effect of THC (via i.p. injection) on prefrontalcortical ACh release

Intraperitoneal injection of THC (5 mg/kg) producedan increase in cortical ACh efflux (Fig. 2; F(3,25) � 17.4,P � 0.0001); post-hoc Tukey’s test revealed that thismain effect was due to significant differences betweenvehicle (injected administration) and THC (5 mg/kg;injected administration), significant differences be-tween the THC groups (administration via injection vs.the indwelling catheter), and significant differences be-tween the vehicle (indwelling catheter administration)

and either form of THC administration (injection or theindwelling catheter). Individual ANOVAs revealedthat THC (injection) produced a maximal increase of242 � 30% in ACh efflux (relative to vehicle) betweenthe 40-min time point (P � 0.05), with release beingsignificantly elevated at the 20, 40, 60, and 80-min timepoints. In addition, THC administration, via injection,significantly elevated ACh efflux relative to THC ad-ministration, via the indwelling catheter (P � 0.05), atthe 20, 40, 60, and 80-min time points. The injection ofTHC increased release �79% over THC administrationby catheter (242 � 30% vs. 163 � 16%) at the 40-mintime point.

Effect of intracortical THC perfusionon basal ACh release

The effects of intracortical infusion of THC on corti-cal ACh release is shown in Figure 3A. At the concen-trations tested, THC did not significantly affect basalACh (Fig. 3A) output from the prefrontal cortex (P �0.05). To verify that THC could cross the dialysis probe,we investigated if THC infused into the striatum couldalter ACh release. Figure 3B shows the effect of intra-striatal infusion of THC on ACh release. Infusion ofTHC into the striatum resulted in a decrease in AChefflux (F(6,2) � 7.991, P � 0.007); post-hoc Tukey testsindicated that 10-4 M of THC significantly differed fromthe vehicle baseline samples. A maximal decrease of30 � 6% below basal values at the dose of 10-4 M wasobserved.

DISCUSSION

The present studies demonstrate that the acute i.p.administration of the cannabinoid CB1 receptor ago-nists WIN and THC have activating effects on AChrelease in the rat medial prefrontal cortex, findingsconsistent with previous reports (Acquas et al., 2000,2001). The enhancement in PFC ACh release appearsto be mediated by the cannabinoid CB1 receptor, given

Fig. 2. Comparison of THC-induced effects on prefrontal corticalacetylcholine (ACh) release: administration via an indwelling cathe-ter vs. administration with a needle. Acute effects of vehicle viainjection (n � 3), THC via injection (5 mg/kg i.p.) (n � 6), vehicle viaindwelling catheter (n � 8), and THC via indwelling catheter (5 mg/kgi.p.) (n � 12) on prefrontal cortical ACh release of conscious freelymoving rats. Values expressed as means � SEM. Percentages ofpreinjection basal ACh levels. Comparison of all treatment sampleswith THC-treated rats and vehicle-treated rats (*P � 0.05). Compar-ison of THC via injection with THC via catheter rats (�P � 0.05).

Fig. 3. Effects of unilateral microinfusions of THC on acetylcho-line (ACh) efflux in the prefrontal cortex and the striatum. A: Effect ofunilateral microinfusions of the cannabinoid agonist THC (10-5 M to10-4 M) (n � 6) into the prefrontal cortex on ACh efflux. B: Effect ofunilateral microinfusions of the cannabinoid agonist THC (10-5 M to10-4 M) (n � 7) into the striatum on ACh efflux as measured bymicrodialysis in the conscious freely moving rat. Values expressed asmeans � SEM. Percentages of preinjection basal ACh levels. Com-parison of all treatment samples with vehicle treatments (**P �0.005).

CANNABINOIDS MODULATE CORTICAL ACH 181

that the effects are blocked by the specific CB1 receptorcannabinoid receptor antagonist SR 141716A. Directapplication of THC to the PFC by reverse dialysis failedto alter ACh efflux. By contrast, THC was able toinhibit ACh release when directly infused into the stri-atum.

Our findings are inconsistent with earlier reportsthat systemic administration of cannabinoids decreasePFC ACh release in vivo (Gessa et al., 1998a). How-ever, our findings corroborate recent studies of cholin-ergic responses to systemic cannabinoid administra-tion (Acquas et al., 2000, 2001). Our studies differ inmany aspects from those of Gessa et al. (1998a), suchas the anesthesia utilized for implanting the probe(equithesin vs. halothane), the type of probe used(transverse vs. concentric), and finally, the method ofdrug delivery. While in both studies the drugs concen-trations and the routes of administration were thesame, our rodents were surgically implanted with anindwelling i.p. catheter to reduce stress-induced alter-ations in neurotransmitter release resulting from han-dling, restraint, and injection (Nilsson et al., 1990).Our study is similar to the studies of Acquas et al.(2000, 2001), who reported that stimulation of corticalACh release occurs when cannabinoidergic drugs aredelivered via indwelling catheters. To resolve this is-sue, we employed a technique similar to our own inevery way with the notable exception of the method ofdrug delivery. As such, we are able to conclude that themethod of delivery is not responsible for the discrep-ancy; in fact, we found the THC-induced increase incortical ACh release (via an indwelling catheter) to bepotentiated by handling the animal and injecting itwith THC.

Methodological issue

Careful consideration is required when interpretingresults, as the use of a cholinesterase inhibitor in-creases, nonphysiologically, extracellular ACh thatmay dampen the responsivity of cholinergic neuronsvia autoreceptor stimulation or long-loop feedbackmechanisms, thus increasing local cholinergic tone.This may be of particular relevance when attemptingto interpret and compare the present results with thoseof Gessa et al. (1998a). The concentration of neostig-mine utilized in the experiments by Gessa et al. (1998)was 1,000� greater than the concentration utilized inthe above experiments (0.1 mM vs. 0.1 �M). Studiesthat have examined the stimulated efflux of ACh undertwo neostigmine concentrations reported that stimu-lated efflux (as a change from baseline) did not differ intwo cortical regions (fronto-parietal and medial pre-frontal cortex) under these different concentrations(Himmelheber et al., 1998). Similar results have beenreported following handling-induced increases in hip-pocampal ACh (Moore et al., 1992) and benzodiazepine-induced alterations in cortical ACh efflux (Moore et al.,

1993). Collectively, these data suggest that the use ofneostigmine (at various concentrations) does not limitthe validity of experiments documenting changes incortical ACh efflux. Whether these different concentra-tions of neostigmine are of significance for the observeddisparity of the cannabinoid-induced alterations in cor-tical ACh release is not known. However, the range ofneostigmine concentrations in the aforementionedstudies does not approach the exceedingly high concen-tration of neostigmine (0.1 mM) utilized by Gessa et al.(1998a).

Anatomy of the effects

The mechanism by which cannabinoid agonists act toelicit an increase in cortical ACh release remains to beelucidated. A study in cortical synaptosomes reportedan inhibition of evoked ACh release by the syntheticcannabinoid WIN 55,212 (Gifford et al., 2000), suggest-ing a direct action of cannabinoids on synaptic termi-nals. However, we report here that the intracorticaladministration of THC into the PFC does not alter AChrelease. The discrepancy between these results can beaccounted for by the significantly different methods ofexperimentation employed, which may include the invitro vs. in vivo preparations, cannabinoid adminis-tered (WIN vs. THC), or the end measures of cannabi-noid-induced changes in ACh release, stimulated(Ca��, K�) vs. unstimulated. We thus hypothesize thatcannabinoid-induced stimulation of cortical ACh re-lease in vivo is not a local phenomena mediated withinthe PFC. It has been proposed that dopamine release inthe ventral striatum controls ACh release in the pre-frontal cortex (Moore et al., 1999). Since cannabinoidshave been shown to stimulate both dopamine release inthe ventromedial striatum (Tanda et al., 1997) and thefiring of dopaminergic neurons in the ventral tegmen-tal area that project to the ventral striatum (Gessa etal., 1998b), the possibility that release of ACh is indi-rectly related to the activation of dopamine in subcor-tical structures warrants further investigation.

The consequences of chronically consuming cannabispreparations on human cognition and brain neuronalsystems strongly suggest that the functions of the pre-frontal cortex are persistently perturbed. We reporthere that the acute administration of cannabinoids torats alters cortical ACh release, a neurotransmitterinvolved in the cognitive/attentional processes associ-ated with the prefrontal cortex. It is important to notethat drugs which increase concentrations of ACh, suchas the anticholinesterase physostigmine, do not en-hance or impair cognitive functions in normal subjects(Mirza and Stolerman, 2000). Further research is nec-essary to determine what role a hypercholinergic statein the prefrontal cortex may play in THCs detrimentaleffects on attention and cognitive performance.

182 C.D. VERRICO ET AL.

SUMMARY

In conclusion, the present studies have demon-strated that CB1 receptors regulate ACh efflux in theprefrontal cortex and striatum in vivo. These data arein concert with the observation that other psychotoge-nic drugs, including phencyclidine (Jentsch et al.,1998), ketamine (Kikuchi et al., 1997; Nelson et al.,2002), and d-amphetamine (Nelson et al., 2000), in-crease cortical ACh release and may represent a com-mon mechanism by which these drugs produce variousforms of psychopathology, including impaired cogni-tive/attentional function.

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