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Research Report

Inhibition of spontaneous neurotransmission in the nucleus ofsolitary tract of the rat by the cannabinoid agonist WIN 55212-2is not via CB1 or CB2 receptors

Daniela Accorsi-Mendonça, Carlos E.L. Almado, André L.A. Dagostin,Benedito H. Machado, Ricardo M. Leão⁎

Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo,Av. Bandeirantes, 3900 14049-900 Ribeirão Preto, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Article history:Accepted 4 January 2008Available online 16 January 2008

Cannabinoids have been shown to modulate central autonomic regulation and baroreflexcontrol of blood pressure. Both CB1 and CB2 cannabinoid receptors have been described in thenucleus tractus solitarius (NTS), which receives direct afferent projections of cardiovascularreflexes. In the present study we evaluated the effects of WIN 55212-2 (WIN), a cannabinoidagonist, on fast neurotransmission in the NTS. We recorded spontaneous post-synapticcurrents using the whole-cell configuration in NTS cells in brainstem slices from young rats(25–30 days old). Application of 5 μM WIN inhibited the frequency of both glutamatergic andGABAergic sPSCs, without affecting their amplitudes. Effects of WIN were not blocked byapplication of the CB1 antagonist AM251, the CB2 antagonist AM630 or the vanniloid receptorTRPV1 antagonist AMG9810, suggesting that the effect of WIN is via a non-CB1 non-CB2receptor. Neither the CB1/CB2 agonist HU210 nor the CB1 agonist ACPA affected the frequencyof sPSCs. We conclude WIN inhibits the neurotransmission in the NTS of young rats via areceptor distinct from CB1 or CB2.

© 2008 Elsevier B.V. All rights reserved.

Keywords:Patch-clampNTSCannabinoid receptorNeurotransmission

1. Introduction

Cannabinoids (CBs) produce an array of behavioral and phys-iological effects that have been characterized in several animalspecies including humans (Pertwee, 2006). Besides well-knownbehavioural effects, CBs also affect the autonomic nervous sys-tem. For example, 9-tetrahydrocannabinol (9-THC) produces atransient pressor response followed by a long lasting hypoten-sion and bradycardia in rat and dogs (Jandhyala and Hamed1978; Vireo et al., 1996). Peripherical administration of theendocannabinoid N-arachidonylethanolamine (AEA; ananda-mide), produces a pronounced, transient fall in blood pressure,

followed by a pressor and a subsequent hypotensive response(Kwolek et al., 2005).

Twowell-characterized cannabinoid receptors have been id-entified: CB1 and CB2 receptors. CB1 receptors are expressedprimarily in brain and peripheral nervous tissues (Pertwee,2006). CB2 receptors are intensely expressed in peripheral andimmune tissues but have also been reported in rat and mousebrain (Van Sickle et al., 2005; Gong et al., 2006; Onaivi et al., 2006).Both CB1 and CB2 receptors are coupled to G-proteins, and theactivation of CB1 receptors in many regions of the central ner-vous system (CNS) by endocannabinoids leads to inhibition ofneurotransmission (Alger, 2002, Kreitzer and Regehr, 2002).

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⁎ Corresponding author. Fax: +55 16 3633 0017.E-mail address: leaor@fmrp.usp.br (R.M. Leão).

0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.brainres.2008.01.011

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However, there is growing evidence for a distinct non-CB1 non-CB2 cannabinoid receptor in the CNS (Pertwee, 2005; Mackie andStella, 2006).

The nucleus tractus solitarius (NTS) in the brain stem,wheredigestory, cardiovascular, and respiratory afferents that parti-cipate in autonomic reflexes establish their first synapse (Alt-schuler et al., 1989; Andresen and Kunze, 1994) is an importantintegrative center and plays a critical role in modulating auto-nomic output. Both CB1 and CB2 receptors are expressed in thebrainstem but to date only CB1 receptors have been shown inthe NTS (Herkenham et al., 1991; Tsou et al., 1998; Van Sickleet al., 2005).

The endocannabinoid system in the NTS is associated withchanges in autonomic reflex function. For instance the AEAcontent in the NTS is increased by brief periods of hypertension

(Seagard et al., 2004) andmicroinjections into theNTSofAM404,an endocannabinoid uptake inhibitor prolong the baroreflex in-hibition associated with renal sympathetic nerve activity (Bro-zoski et al., 2005) and increase firing of baroreceptor neurons(Seagard et al., 2005). In addition, electrophysiological studiesshowed that approximately half of the NTS neurons respondedto cannabinoid receptor agonistswithachange indischarge rate(Himmi et al., 1996; 1998) and that synaptic transmission fromtheNTS to the dorsalmotor nucleus of the vagus is inhibited byapplication of the cannabinoid agonist WIN 55212-2 (Derbenevet al., 2004). These data point to an important role of canna-binoids in modulating the activity of the NTS neurons.

In this work we investigated how the activation of thecannabinoid systemaffects the physiological properties of fastneurotransmission in the NTS. We studied the effects of thecannabinoid agonist WIN 55212-2 (WIN) on spontaneous glu-tamatergic and GABAergic transmission in NTS slices in vitro,and the type of receptor involved. We observed that WIN in-hibits fast neurotransmission in the NTS by activation of apre-synaptic receptor pharmacologically different from clas-sical CB1 or CB2 receptor.

2. Results

In order to study effects of cannabinoids on neurotransmissionin the NTS, we applied the agonist WIN. Since vehicle for thisdrug (DMSO, 0.1%) has been shown to affect spontaneous re-lease at other synapse (Dr. Christopher Kushmerick, personalcommunication), we first tested DMSO 0.1% alone and foundthat it significantly inhibited the frequency of sPSPs (22±7%,n=21; p<0.05). As we show below, application of WIN causedadditional inhibition, and all of the key findings reported werereproduced by applyingWIN after first pre-applying the vehicleDMSO 0.1%.

2.1. WIN inhibits spontaneous synaptic transmission inthe NTS

We recorded spontaneous synaptic currents (sPSCs) from NTSneurons in brainstem slices and observed that acute applica-tion of WIN (5 μM) reduced the frequency of these events in 20out of 27 cells tested (Fig. 1). In these cells, WIN reduced themean frequency of sPSCs by 60% (from 1.1±0.2 Hz to 0.5±0.04 Hz; Fig. 1B), without altering amplitudes (control: 52±7 pA;WIN: 50±7 pA, Fig. 1C) or half-widths (control: 8.5±2 ms; WIN9.7±3 ms) of these events. The distribution of inter-event in-tervals and amplitudes of these events recorded from a repre-sentative neuron are shown in Figs. 1D and E). The inhibition ofthe sPSCs frequency by WIN was significantly different fromDMSO 0.1% alone (p<0.05, t-test) and in 4 cells the same con-centration ofWIN produced a significant decrease in the sPSCs

Fig. 2 – Dose-dependent effect of WIN on the frequency ofthe synaptic currents. (A): dose dependency of the effect ofWIN on the frequency of the total sPSCs. Bars are the mean±SEM of the doses applied sequentially in the same cells (n=3)(B): comparison of the effect of sequential application on thesame cell of 5 nM and 5 μM of WIN on the frequency of thetotal sPSCs (n=4) and the pharmacologically isolatedglutamatergic sEPScs (n=3) and GABAergic sIPSCs (n=4).

Fig. 1 – Inhibition of neurotransmission by WIN: (A): example of sPSCs recorded in a NTS neurons before (control) andafter application of WIN (5 μM). (B, C): Summary of the effect of WIN (5 μM) on the frequency (B) and mean amplitude (C) of thesPSCs recorded in 18 cells. Bars represent the mean and each point an individual cell. (D, E): cumulative frequency graphsof inter-time interval (D) and amplitude (E) of the sPSCS before and after application of WIN in a representative cell.(F, G): summary of the effect ofWIN (5μM) on the frequency of the pharmacologically glutamatergic (F) and GABAergic (G) sPSCs.* = significantly different from control p<0.05.

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frequency when applied after perfusion of DMSO 0.1% (from1.1±0.2 Hz to 0.9±0.1 Hz. p<0.05, paired t-test). Since sequen-tial application of 0.01%, 0.1% and 0.3% DMSO did not produceadditive effects (control: 2.2±0.7 Hz; 0.01% DMSO: 1.1±0.7 Hz;0.1% DMSO 1.2±0.3 Hz; 0.3% 1.3±0.3 Hz. p=0.06 repeated mea-sures ANOVA) this effect was not due to cumulative applica-tion of DMSO. We conclude that despite the effect of thevehicle, WIN inhibits the neurotransmission in the NTS.

Interestingly in the remaining 7 cells tested in these con-ditions,WIN had the opposite effect, increasing sPSC frequencyfrom 0.55±0.11 Hz to 0.72±0.1 Hz (p<0.05) without changing themean amplitude (control: 84.3±24.8 pA; WIN: 86.3±21.3 pA).

Spontaneous neurotransmission in NTS slices is composedof both excitatory and inhibitory (i.e. glutamatergic andGABAer-gic) components (Jin et al., 2004; Baptista et al., 2005; Accorsi-Mendonça et al., 2007).We therefore studied theeffect ofWINonboth pharmacologically isolated GABAergic and glutamatergicneurotransmissions, to identify which components are affected

by WIN. (Since the pharmacological antagonists were dissolvedinDMSOand the effects ofDMSOwerenot cumulative anyeffectof WIN on the pharmacologically isolated GABAergic and glu-tamatergic transmissionswerenotdue toDMSO).Glutamatergiccurrents (sEPSCs) were isolated by applying 20 μMof bicuculline,aGABAA receptor antagonist, in thebathperfusion. Fig. 1F showsthat in these conditions WIN (5 μM) decreased the frequency ofsEPSCs by 60% (from 0.7±0.2 Hz to 0.3±0.1 Hz; p<0.05, n=7)without changing the amplitude of excitatory events (control:18.9±1.1 pA, WIN: 17±1.0 pA) or their half-width (control: 1.6±0.2 ms; WIN: 1.35±0.1 ms). Similarly, WIN inhibited GABAergiccurrents (sIPSCs) (isolated using 10 μM DNQX, a non-NMDAreceptor antagonist, in the bath perfusion) by 60% (sIPSCs: 0.5±0.2 Hz; WIN: 0.2±0.1 Hz, p<0.05, n=7 (Fig. 1G) without changingtheirmeanamplitude (control 66±17pA;WIN: 58±14pA)orhalf-width (control: 7.1±1 ms; WIN: 5.7±1 ms).

The inhibitory effect of WIN on spontaneous neurotrans-missionwas dose-dependent. At a dose of 5 nM,WIN inhibitedthe sPSCs frequency by 43% (from 1.7±0.4 Hz to 0.87+0.2 Hz;n=9), after previous perfusion of the cells with 0.1% DMSO.Increasing the concentration of WIN resulted in increased in-hibition of the sPSCs frequency, reaching 80% inhibition at1 μM WIN (n=7). In Fig. 2A we present the dose dependence ofWIN applied in sequence on the same cell (n=3) A similarprofile of dose dependency was obtained when we comparedthe effects of sequential doses of 5 nM and 5 μMofWIN in cellsbathed with either bicuculline or DNQX (Fig. 2B), although theglutamatergic sEPSCs seemed to be less sensitive to a increasein the concentration of WIN to 1 μM, suggesting some dif-ferences in sensitivity to WIN between both transmissions.

Fig. 4 – Lack of effect of CB1 antagonism on WIN effect:(A): example of sPSCs recorded in a NTS neurons before(control), after application of WIN (5 μM) and afterco-application of WIN and the CB1 antagonist AM 251(10 μM). (B): summary of the effects of WIN andWIN plus AM251 on the frequency of the sPSCs (n=4).

Fig. 3 – Effect of WIN on the miniature IPSCs. Summary ofthe effect of WIN (5 μM) on the (A) frequency, (B) amplitudeand (C) half-width of the miniature GABAergic currentsrecorded in the presence of TTX (1 μM)(n=4).* = significantly different from control p<0.05.

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2.2. WIN decreases the frequency of miniature IPSCs

SinceWIN did not affect amplitudes or half-widths of sPSCs, theinhibitory effect ofWIN on the sPSCs frequency is suggestive of apre-synaptic effect on neurotransmitter release, for example areduction in synaptic vesicle release probability, but could alsoresult from changes in firing probability due to changes in post-synaptic excitability. To distinguish these possibilities, we testedthe effect of WIN in the presence of TTX to measure actionpotential-independent miniature synaptic currents. In these ex-periments, we choose to record the more abundant miniatureGABAergic synaptic currents (mIPSCs), in the presence of theNa+-channel blocker TTX (10 μM) and DNQX. We observed that5 μM of WIN decreased the mean frequency (TTX+DNQX: 0.53±0.09 Hz; TTX+DNQX+WIN: 0.35±0.07 Hz; n=4, p<0.05; Fig. 3A) ofmIPSCs but did not change their amplitude (TTX+DNQX: 37.3±5.9 pA; TTX+DNQX+WIN: 30.1±5.6 pA; Fig. 3B), or half-width(TTX+DNQX:8.8±0.7ms;TTX+DNQX+WIN: 8.0±1.0ms; Fig. 3C).These results suggest thatWINacts on thepre-synaptic terminalreducing the exocytosis of GABAergic vesicles, and in additiondoes not affect GABAA receptors kinetics.

2.3. Inhibition of sPSC frequency byWIN is notmediated byactivation of CB1, CB2 or putative central vanniloid receptors

Activation of CB1 receptors by WIN reduces GABA and gluta-mate release in several CNS regions (Hájos et al., 2000; Huanget al., 2001; Wilson and Nicoll, 2001; Kushmerick et al., 2004),

making this a likelymechanism for the effect ofWIN in theNTS.However, when we tested the role of CB1 in WIN-mediated in-hibition in the NTS using the specific CB1 receptor antagonistAM251 (10 μM), we found it did not revert the inhibition of thesPSCs caused by previous application of WIN (control: 1.3±0.2 Hz; WIN: 0.7±0.2 Hz; WIN+AM251: 0.7±0.2 Hz; n=6, Figs. 4Aand B). Pre-application of AM251 (10 μM) was similarly unableto prevent WIN effect (n=3, not shown). We also tested AM251on pharmacologically isolated excitatory and inhibitory neuro-transmission, and found that AM251 was unable to antago-nize the inhibitory effect of WIN on glutamatergic (n=4) andGABAergic (n=1) neurotransmission (data not shown). As acontrol for the efficacy of AM251, we tested its ability to revertinhibitory effects of WIN on GABAergic neurotransmission inhippocampal CA1 neurons. We found that AM251 completelyreverted the effect of WIN in this system within 5 min ap-plication to the bath (n=2) confirming the efficacy of this drug inour hands.

The inability of a CB1 antagonist to block the effect of WINled us to test other receptors that maymediateWIN inhibitionof sPSCs in the NTS. WIN is also an agonist for CB2 receptors,which have been described in the CNS (Van Sickle et al., 2005;Gong et al., 2006; Onaivi et al., 2006). In addition,WIN has beenreported to inhibit glutamate release in the hippocampus byactivating a putative central metabotropic vanniloid receptor

Fig. 6 – Effect of CB1 agonists on the frequency of the sPSCs:(A): summary of the effect of ACPA (3 μM) on the frequencyof the sPSCs. Note the increase in the sPSCs frequencyproduced in one cell by application of ACPA. Bars are themean±SEM. (B): summary of the effect of HU210 (1μM) on thefrequency of the sPSCs and the inhibition of the HU210effect by AM 251 in 3 cells. Note that in 4 cells HU 210increased frequency of the sPSCs. Bars are the mean±SEM ofonly the data that we apply HU210 and AM 251 sequentially.

Fig. 5 – Lack of effect of CB2 and TRPV1 antagonism on WINeffect. (A): summary of the effects of WIN and co-applicationof WIN with the CB2 antagonist AM 630 3 μM (n=5).(B): summary of the effects of WIN and co-application of WINwith the TRPV1 antagonist AMG 9810 2 μM (n=4).

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(Hájos and Freund, 2002). However neither the CB2 antagonistAM630 (3 μM) nor the vanilloid TVPR1 antagonist AMG9810(2 μM) was able to revert the reduction in frequency of sPSCsproduced byWIN (Fig. 5) These data strongly suggest thatWINinhibits GABA and glutamate release in the NTS via a non-CB1,non-CB2, non-putative central vanniloid receptor.

2.4. CB1 receptor agonists do not inhibit fastneurotransmission in the NTS

To further confirm that the effect of WIN on sPSCs is due thenon-CB1 non-CB2 receptor activationwe analyzed the effect oftwo other cannabinoid agonists on sPSCs in order to comparetheir effects: HU210 a CB1/CB2 agonist that has no reportedeffect on non-CB1, non-CB2 receptors (Breivogel et al., 2001;Monory et al., 2002) and ACPA, a specific CB1 agonist (Hillardet al., 1999). The results show that neither ACPA (after ap-plication of the vehicle Tocrissolve alone that produced a 26%inhibition of the sPSCs frequency) (control: 0.6±0.1 Hz; 3 μMACPA: 0.9±0.4 Hz; n=9, Fig. 6A) nor HU210 (control: 1.4±0.5 Hz;1 μM HU210: 1.9±0.7 Hz; n=9, Fig. 6B) decreased the meanfrequency of sPSCs. In fact in 5 neurons treated with HU210(Fig. 6B) and in one neuron treated with ACPA (Fig. 6A) weobserved an increase in the frequency of the sPSCs, suggestiveof a CB1 receptor dependent increase in sPSCs frequency.Application of AM251 in 3 neurons where HU210 increased thefrequency of the sPSCs reverted the effect of HU210 (control:1.4±0.5 Hz; HU210: 1.9±0.7; HU210+AM251: 1.2±0.5 Hz). Weconclude that since activation of CB1 receptors either had noeffect or increases neurotransmitter release in the NTS, theinhibitory effect of WIN cannot be mediated by CB1 receptors.

3. Discussion

Thecannabinoid ligandWIN is anaminoalkylindoleCBagonist(Comptonet al., 1992) and is used inmany studies on the role ofthe CB1 receptors in modulating the central neurotransmis-sion. Its main reported effect is the inhibition of the neuro-transmission by inhibition of pre-synaptic calcium channels(Kreitzer and Regehr, 2001; Kushmerick et al., 2004) and/oractivation of G-protein-gated inwardly rectifying potassiumchannels (GIRK) (Mackie et al., 1995).

We observed that WIN produced a reduction in the fre-quency of the sPSCs in the NTS of young rats. Doses as low as5 nM of WIN were effective in inhibit sPSCs frequency. This re-ductionwas seen onboth isolated glutamatergic andGABAergiccurrents. Interestingly the glutamatergic sEPSCs were less af-fected than GABAergic sIPSCs when WIN concentration wasincreased from 5 nM to 5 μM,whichmight suggest a differentialeffect on these synapses.

Since WIN reduced the frequency of the sPSCs withoutaffecting their mean amplitude or half-width, it is likely thatWIN acts on the pre-synaptic terminal reducing the release oftransmitterwithout affecting receptor properties. Reductions ofthe frequency of the appearance of the sPSCs could reflect adecrease in the release probability due to pre-synaptic calciumchannel inhibition. Since WIN also inhibited the frequency ofminiature IPSCs it is plausible that one likely mechanism ofaction of WIN in inhibiting GABA release is by an inhibition of

pre-synaptic calciumchannels. However, since the effect on theminiature IPSCswas smaller than the observedwith the sIPSCs,other effects affecting the action potential-driven release ofGABA cannot be discarded.

Contrary to our expectations, the CB1 antagonist AM251(10 μM), did not revert the inhibitory effect of WIN on thefrequency of the sPSCs, and the isolated sEPSCs and sIPSCs,although applied in the same conditions it did revert theinhibitory effect of WIN on hippocampal GABAergic trans-mission. Additionally, pre-treatment with AM251 did notprevent the inhibitory effect of WIN on sPSCs frequency.Therefore, the effects of WIN are not mediated by CB1 re-ceptors. WIN is both a CB1 and CB2 agonist, and functionalCB2 receptors have been identified in the brainstem (VanSickle et al., 2005) and WIN could be inhibiting neurotrans-mission in the NTS activating CB2 receptors. However,antagonists of CB2 receptors did not revert the effect of WINon the NTS, discarding a CB2 mediated action of WIN. Adifferent CB1/CB2 agonist, HU210, as well as the CB1 agonistACPA also did not inhibit neurotransmission in the NTS.Therefore, it seems that WIN is inhibiting neurotransmissionin the NTS by a receptor distinct from the classical CB1or CB2receptor.

Evidence shows that a different kind of receptor, a centralnon-CB1, non-CB2 receptor, can mediate some effects of WINand of endocannabinoids in the brain. Studies from CB1−/−

knock-out mice show that WIN and the endocannabinoid an-andamide promote [35S]GTPγS binding to CB1−/− mice brainmembranes (Breivogel et al., 2001; Di Marzo et al., 2000) andreduce hippocampal glutamatergic neurotransmission (Hájoset al., 2000; Hájos and Freund 2002; Köfalvi et al., 2003). How-ever more recent studies using CB1−/− knock-out mice havefailed to observe any effect of WIN on the glutamate release atthe hippocampus and cerebellum (Kawamura et al., 2006; Ta-kahashi and Castillo, 2006). Therefore, the existence of a cent-ral non-CB1, non-CB2 (or CB3) receptor is still controversial.

The molecular identity of this putative non-CB1, non-CB2receptor is still unknown. It has been reported that it differsfrom CB1 receptors in its central distribution pattern (Breivo-gel et al., 2001; Di Marzo et al., 2000; Monory et al., 2002) and,interestingly, the binding of [3H]R-(+)-WIN is intense in thebrainstem of CB1−/− mice (Breivogel et al., 2001), a region poorin CB1 labeling in wild-type mice (Herkenham et al., 1991).

Some differences have been observed between the pro-posed non-CB1, non-CB2 receptors enhancing [35S]GTPγS inbrain membranes and the receptors that inhibit glutamaterelease in the hippocampus. For instance the increase in [35S]GTPγS binding in brainmembranes prepared fromCB1−/−miceis sensitive to WIN and to anandamide but not to HU210, Δ9-THC or CP55940 (Breivogel et al., 2001; Di Marzo et al., 2000;Monory et al., 2002) but on the other hand in the hippocampusof CB1−/− mice not only WIN but CP55940 also inhibited glu-tamate release (Hájos et al., 2000; Hájos and Freund 2002;Köfalvi et al., 2003) andWIN did not enhance [35S]GTPγS bind-ing inmembranes from hippocampus of CB1−/− CD1mice (Mo-nory et al., 2002). The vanniloid agonist capsaicin also reducesthe EPSC amplitude in the hippocampus of thesemice and notonly the effect of capsaicin but also the inhibitory effects ofWIN and CP55940 are reverted by the TRPV1 antagonistcapsazepine (Hájos and Freund, 2002). Our data indicate that

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in NTS WIN is not acting on these putative vanilloid recep-tors observed in hippocampus, since the TRPV1 antagonistAMG9810 did not antagonize the effect ofWIN. This data alongwith the inability of ACPA and HU210 in inhibiting the sPSCsfrequency strongly suggest that the inhibitory effect ofWIN onthe neurotransmission is mediated by a receptor other thanthe CB1, CB2 or putative central vanniloid receptors.

Although a direct effect of WIN on pre-synaptic calciumchannels in higher micromolar doses, like observed in hip-pocampal neurons in culture (Shen and Thayer, 1998), cannotbe ruled out we find it unlikely since 10 μM WIN inhibition ofcalcium currents from freshly dissociated neurons from theNTS is reverted by AM251 (Endoh, 2006).

Our results suggest the presence of a cannabinoid non-CB1,non-CB2 receptor in the NTS neurons that inhibits GABA andglutamate release or that WIN is activating a different non-cannabinoid receptor. Interestingly a fraction of cells testedresponded to WIN, HU210 and ACPA increasing the sPSCsfrequency, and in 3 neurons that responded to HU210 its effectwas reverted by application of AM251, the CB1 antagonist.These data is suggestive of an unexpected excitatory effect ofactivation of CB1 receptors on the neurotransmission in theNTS. It is worth to note that it has been reported that acti-vation of CB1 receptors by WIN and CP55940 in the NTS alsohas a dual effect on the neuronal firing rate both increasingand decreasing it (Himmi et al., 1998). Thismight be suggestiveof a dual effect of these agonists on the NTS, although Seagardet al. (2005) have shown that the increase in the discharge rateof baroreceptive NTS neurons by application of anandamidewas blocked by GABAA receptors antagonists. Since the sti-mulatory effect of the CB agonists observed by us was onwhole sPSCs (GABAergic and glutamatergic) and was notobserved in isolated GABAergic and glutamatergic sPSCs, thisstimulatory effect could be due to a decrease in the inhibitorytone in the slice.

In conclusion, in NTS neurons of young rats WIN inhibitsneurotransmission by a mechanism distinct from activatingthe classical CB1 or CB2 receptors. However, since many re-ports show the presence of functional CB1 receptors in theNTS (Herkenham et al., 1991; Himmi et al., 1996; 1998; Tsouet al., 1998; Van Sickle et al., 2005) our data does not excludethe presence of CB1 receptors in the NTS.

4. Experimental procedure

All experimental protocols used in this work were reviewedand approved by the Institutional Ethical Committee for Ani-mal Experimentation of the School of Medicine of RibeirãoPreto-University of São Paulo.

4.1. Brain stem slices preparation

MaleWistar rats (22–30daysold)weredecapitatedand thebrainstem was rapidly removed and submerged in ice-cold (4°C)artificial cerebrospinal fluid (aCSF) containing (mM): 120 NaCl,2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 10 glucose and 2CaCl2, with osmolality of 300–310 mOsm/Kg H2O and pH 7.4when bubbled with 95% O2 and 5% CO2. Brain stem transversal

slices (200 μm thick) were cut using an oscillating slicer (Vib-ratome 1000 plus, Vibratome, USA) and kept in aCSF at 37 °C for45minand thereafter at roomtemperature (RT; 23–25 °C). Beforerecording, a single slice was placed into the recording cham-ber, held in place with a nylon thread, and continuously per-fusedwith oxygenated aCSF at a rate of approximately 3ml/minat RT.

4.2. Whole-cell patch-clamp recordings

Whole-cell recordings were made with patch pipettes pulledfrom thick-walled borosilicate glass capillaries (Sutter Instru-ments, Novato, CA, USA), using a P-97 puller (Sutter Instru-ments, USA). The patch pipettes were filled with an internalsolution containing (mM): 150 KCl, 5 ethylene glycol-bis (β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 10 N-2-hydroxy-ethylpiperazine-N'-2-ethnesulfonic acid (HEPES),osmolality of 310–20 mOsm/Kg H2O pH 7.3 adjusted withKOH. The final open tip resistance ranged from 4 to 8 MΩ.Neurons were visualized by infrared differential interferencecontrast (IR-DIC) microscopy (Olympus BX51WI, Olympus,Japan) through a 40× water immersion objective (LUMPlain F1-IR, Olympus, Japan) and a CCD camera (C7500-50, Hamamatsu,Japan). Signals were acquired using an Axopatch 200B ampli-fier (Axon Instruments, USA) connected to a microcomputervia a Digidata 1200 or 1440A board (Axon Instruments, USA).The Clampex 8 software was used to acquire the data. Datawas low-pass filtered at 2 kHz and acquired at 10 kHz. Seriesresistance (<30 MΩ) was checked regularly during the experi-ment and cells with large variations in series resistances werediscarded. The neuronswere held at −70mV and their identitywas confirmed by the presence of voltage-gated sodium cur-rents. Recordings were started 5 min after entering the whole-cell configuration.

4.3. Data Analysis

Spontaneous post-synaptic currents (sPSCs) were recorded for3 min and their frequency, amplitude and half-width wereanalyzed with the Minianalysis Program version 5.0 (Synapto-soft, USA). The data are expressed as mean±standard error(SEM) and statistical significance among values (p<0.05) wasdetermined by paired Student t-test using the GraphPad Prism4 software (GraphPad Prism, USA).

4.4. Drugs

The drugs WIN 55212-2, AMG9810, AM251, AM630, HU210, te-trodotoxin (TTX) and ACPA were obtained from Tocris (USA),6,7-Dinitroquinoxaline-2,3(1H,4H)-dione (DNQX) and bicucul-line were from Sigma (USA). ACPA stock solution (1000-fold)were dissolved in Tocrisolve® and the other stock solutions(1000 or 10,000-fold) were prepared in dimethylsulphoxide(DMSO), resulting in a final concentration of DMSO or Tocri-solve® in the bath <0.3%. Application of the vehicles at theconcentration of 0.1% had, on average, a significant effect oninhibiting spontaneous neurotransmission (see results), notaffecting the mean amplitudes. When the drug presented aneffect on the sPSCS frequency of at least 2-fold the effect ofDMSO and/or presented effect in control experiments done in

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the presence of previously added 0.1% DMSO, we consideredthe drug efficacious. All experiments with ACPA were done inthe presence of previously added Tocrisolve®. The concentra-tions of agonists and antagonists chosen were equivalent to100 times the reported IC50s obtained in the respective datasheets. The aCSF solutions for perfusion with the differentdrugs were selected using a 6-valve solenoid system (VC-6;Warner Instruments, USA), and applied to the slices by agravity-driven perfusion system (flow rate of 2–3 ml/min).Recordings started at least 5 min after initiating the drugperfusion. Alternatively the cannabinoid agonists and antago-nists were applied directly on the bath. In these cases the bathperfusion was stopped and the chamber was continuouslybubbled with carbogen. The bath volume in the chamber waskept at 1 ml and a 1 μl drug stock solution (or a 10-fold dilutionin water from a 1000-fold stock in DMSO) resulting in the finaldesired concentration was added directly in the bath. Effectsweremeasured starting 5min after drug application, when westarted recording. No difference on the effect of drugs wasobserved using both drug application methods.

Acknowledgements

We thank Dr. Christopher Kushmerick and Dr. Hitoshi Mori-kawa for reviewing themanuscript.Work supportedby FAPESP(04/03285-7 and 03/04319-0) and NIH Fogarty InternationalCenter (TW006955-01A1).

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