Role of CB2 Receptors and Cgmp Pathway on the Cannabinoid-Dependent Antiepileptic Effects in an in Vivo Model of Partial Epileps

Epilepsy Research (2014) 108, 1711—1718

jo urnal homepage: www.elsevier.com/locate/epilepsyres

Role of CB2 receptors and cGMP pathway on

the cannabinoid-dependent antiepileptic

effects in an in vivo model of partial epilepsy

a,b,∗,1 a,1 a

Valerio Rizzo , Fabio Carletti , Giuditta Gambino ,

a c a

Girolamo Schiera , Carla Cannizzaro , Giuseppe Ferraro , a

Pierangelo Sardo

Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (Bio.Ne.C.), Sezione di Fisiologia

umana ‘‘G. Pagano’’, Università degli Studi di Palermo, Corso Tukory, 129—90134 Palermo, Italy

Department of Neuroscience, The Scripps Research Institute, Scripps Florida 130 Scripps Way, Jupiter, FL 33458

Dipartimento di Scienze per la Promozione della salute, Università degli Studi di Palermo, Via del Vespro,

133, 90100 Palermo, Italy

Received 4 June 2014; received in revised form 12 September 2014; accepted 1 October 2014

Available online 19 October 2014

KEYWORDS Summary This study aimed at providing an insight on the possible role of cannabi-

Cannabinoid; noid (CB) type 2 receptors (CB2R) and cGMP pathway in the antiepileptic activity of

WIN 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-

Temporal lobe

epilepsy; benzoxazin-6-Yl]-1-naphthalenylmethanone, a non-selective CB agonist, in the maximal dentate

AM630; activation (MDA) model of partial epilepsy in adult male rats. We evaluated the activity

sGC; of a CB2 antagonist/inverse agonist AM630, [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-

Hippocampus; indol-3-yl](4-methoxyphenyl)methanone or 6-iodopravadoline, alone or in co-administration

Electrophysiology with WIN 55,212-2. Also, in the MDA model it was investigated the co-treatment of WIN

55,212-2 and 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a speciﬁc inhibitor of the

nitric oxide (NO)-activated soluble guanylyl cyclase (sGC), the cGMP producing enzyme. The

WIN 55,212-2-dependent (21 mg/kg) antiepileptic effects were signiﬁcantly increased by the

co-administration with AM630 and by the co-treatment with ODQ (10 mg/kg). Whereas, the

administration of AM630 (2 mg/kg), alone exerts no effects on hippocampal hyperexcitabi-

lity. Our data show that pharmacological blockade of CB2 receptors and of sGC seems to

∗

Corresponding author. Tel.: +39 091 655 58 06; fax: +39 091 655 58 16.

E-mail address: [email protected] (V. Rizzo).

These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.eplepsyres.2014.10.001

1712 V. Rizzo et al.

cooperate with WIN in its antiepileptic action. These ﬁndings shed light on CB signaling mecha-

nisms, hinting that the modulation of the effects of CB agonist in the hyperexcitability phenomena

may be exerted both by targeting CB receptors and their possible downstream effectors, such as

nitrergic-dependent cGMP pathway.

Introduction cortex (den Boon et al., 2012). Therefore, we explored if

the control by CB2R may be found on paroxysmal neuronal

activity in the MDA model.

Several evidences have outlined that in the brain neu-

Beyond this, irrespective of the involvement of CB recep-

ronal excitability and synaptic function may be modulated

tors, the pathway through which eCB exert their effects

by either endogenous cannabinoid (eCB) signaling or cGMP

on neuronal processes is not completely clear especially

pathway, for instance through the control on neurotrans-

concerning the interplay with other neuromodulators. As

mitter release and ion channels conductance (Ahern et al.,

regards the epileptic condition, we previously investigated

2002; Castillo et al., 2012; Robello et al., 1996). Besides this,

on the intervention of the cGMP pathway within the frame-

the eCB system and cGMP signaling are reported to be func-

work of nitrergic modulation of hippocampal seizures (Sardo

tionally related in certain neuronal paradigms (Azad et al.,

et al., 2006). In particular, we provided data that the block-

2001; Ghasemi et al., 2007; Howlett et al., 2004; Stefano

ade of the sGC exerted antiepileptic effects (Sardo et al.,

et al., 1998). Indeed, guanine nucleotides can inhibit CB

2006). In this light, in the present study, we exploited the

agonist binding (Devane et al., 1988), whereas cannabinoid

MDA model of partial epilepsy to deepen knowledge on the

agonists can stimulate both the production of cGMP and

underlying mechanisms of the antiepileptic effects of CB

the translocation of the nitric oxide (NO)-activated soluble

transmission and on its possible interaction with NO/cGMP.

guanylyl cyclase (sGC), the cGMP-producing enzyme (Jones

For the above described purposes, we separately targeted

et al., 2008). In the hippocampus, the distribution and co-

both CB2 receptors, via administration of the CB2 antago-

localization of sGC and CB receptors have been described

nist/inverse agonist AM630, and sGC, by administering the

in the pre-synaptic glutamatergic afferents (Burette et al.,

speciﬁc inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-

2002). In addition, anatomical evidences have proven that

one (hereafter named ODQ), to assess if signiﬁcant changes

CA1 inhibitory synapses equipped with cannabinoid receptor

in the WIN-induced antiepileptic effects occur.

type 1 (CB1R) receptors express both postsynaptic neu-

ronal NO synthase (nNOS) and presynaptic NO-activated sGC

Materials and methods

(Makara et al., 2007).

As for the hyperexcitability phenomena, a functional

Animals and surgical procedures

involvement of the G protein-coupled CB1R has been

observed in the cannabinoid-mediated antiepileptic effects

Male Wistar rats (weight 260—300 g, 2—3 months-old) were

(Hofmann and Frazier, 2013; Matsuda et al., 1990). In fact,

used in this study. Detailed surgical procedures have

systemic cannabinoid treatment suppresses seizures and

been described in our previous papers (Carletti et al.,

increases activation threshold in epileptic rats (Wallace

2013). Brieﬂy: rats were anaesthetized with urethane

et al., 2003), whereas CB1R antagonists cause seizure-like

(1.0—1.2 g/kg intraperitoneally, i.p.) (Maggi and Meli, 1986)

activity in hippocampal culture models of acquired epilepsy

and after craniotomy, a stimulating electrode (coaxial bipo-

(Deshpande et al., 2007) and may exacerbate paroxysmal

lar stainless steel electrode: external diameter 0.5 mm;

events in patients with temporal lobe epilepsy (Braakman

exposed tip 25—50 ␮m) was placed in the angular bundle

et al., 2009). Though, growing evidences suggest that the

(AB) on the right side according to the stereotaxic coor-

pathways involved in the cannabinoid antiepileptic effects

dinates of the Atlas of Paxinos and Watson (1986). A glass

could not be exclusively CB1-mediated (Hill et al., 2013;

recording electrode, ﬁlled with 1% fast Green in 2 M NaCl,

Jones et al., 2012). In this regard, in a previous paper

was stereotaxically placed in the ipsilateral dentate gyrus

we accounted for the CB1 antagonist AM251 ineffective-

(DG). The animal was grounded through a subcutaneous

ness when administered alone using the maximal dentate

Ag/AgCl wire in the scapular region. The DG bioelectric

activation (MDA) model of hippocampal epilepsy (Rizzo

activity was recorded through a low level DC pre-ampliﬁer

et al., 2009); on the other hand, we reported that AM251

(Grass 7B, West Warwick, RI, USA) and then processed by a

signiﬁcantly but incompletely antagonized the antiepileptic

software package provided by DataWave Technologies (Long-

effects of WIN 55,212-2 (CB non-selective agonist, hereafter

mont, CO, U.S.A.).

named WIN). The ﬁnding of a partial antagonism exerted by

The experiments were conducted in strict accordance

a selective CB1 antagonist on WIN-dependent antiepileptic

with the current Italian rules on animal experimentation

effect likely implies the functional involvement of a further

(D.L. 116/92) and European directive (2010/63/EU).

receptor underpinning CB antiepileptic effects. For this

reason, in this paper we ﬁrstly focused on the cannabinoid

Maximal dentate gyrus activation and ictal events

receptor type 2 (CB2R), a protein associated with a variety identiﬁcation

of brain processes (Cabral et al., 2008; Fernández-Ruiz

et al., 2007; Morgan et al., 2009; Van Sickle et al., 2005),

In order to obtain stable and reproducible MDA as well

such as the decrease of neuronal ﬁring in the prefrontal

as to avoid progressive changes in their duration due

Role of CB2 receptors and cGMP pathway on the cannabinoid-dependent antiepileptic effects 1713

Fig. 1 (A) Representative MDA trace. Measurements of time of onset, duration of maximal dentate gyrus activation (MDA) and

afterdischarge (AD) during and after a stimulus train (400 ␮A, 20 Hz) delivered for 10 s to the angular bundle (AB). The recordings

were ampliﬁed using a low level DC pre-ampliﬁer. (B) Time course of MDA parameters in controls and WIN treated animals. Each

value represents the mean of D% of each group (᭹ Control, WIN). ( ) indicates a signiﬁcant difference vs baseline values (P < 0.05).

to repetitive AB stimulations, we modiﬁed the technique which the evoked paroxysmal EEG events abruptly ceased);

originally described by Stringer and Lothman (1989) char- (iii) afterdischarge (AD) duration (from the end of AB stim-

acterized by variable stimulation train durations strictly ulation to the end of the epileptiform activity) (Fig. 1A).

related to the beginning of the MDA response. Our elec- Furthermore, we analyzed the % of responses to AB stim-

trophysiological technique has been extensively described ulation to evaluate the possible suppression of paroxysmal

in our previous papers (Carletti et al., 2013). Brieﬂy: 10 s events.

duration trains of 20 Hz stimuli were given through the

AB stimulating electrode. Individual stimuli consisted of

0.3 msec biphasic pulses. The stimulus intensity was ini- Drug treatment

tially below that necessary to elicit any response and it was

␮

increased in 100 A steps in the following stimulations until DMSO was purchased from Sigma Chemical Co. (Sigma, St.

MDA occurred (threshold intensity). The stimulus train was Louis,MO, USA), while WIN, AM630 and ODQ were purchased

administered every 2 min until a MDA appeared and then from Tocris Bioscience (Bristol, UK). The study took into

every 10 min for up to 3 h. MDA was recorded by the elec- consideration six groups of rats (n = 6 rats each). The 1st

trode placed in the DG and it was deﬁned by a shift of the (untreated controls) and 2nd groups (vehicle treated)

extracellular potential in DC-coupled recordings as well as were studied for a period of about 200 min in order to

by the presence of bursts of population spikes. Once the verify possible modiﬁcations of MDA parameters due to the

MDA was elicited, the following parameters were recorded: repetitive stimulations or to the vehicle administration. In

(i) Onset duration (time from the beginning of AB stimulation the remaining groups the animals received WIN (21 mg/kg,

to the midpoint of the DC potential shift); (ii) MDA duration i.p., 3rd group), AM630 (2 mg/kg, i.p., 4th group), a co-

(from the midpoint of the DC potential shift to the point at treatment with AM630 and WIN (2 mg/kg and 21 mg/kg, i.p,

1714 V. Rizzo et al.

respectively; 5th group) and a co-treatment with WIN and one-way repeated measures analysis of variance (ANOVA).

ODQ (21 mg/kg and 10 mg/kg, i.p, respectively; 6th group), In drug-treated animals, post-pharmacological treatment

at dosages previously described (García-Gutiérrez et al., parameters were statistically analyzed using an one-way

2012; Rizzo et al., 2009; Sardo et al., 2006). All drugs were multivariate ANOVA test (MANOVA) followed by Bonferroni

dissolved in the same ﬁnal vehicle volume for each animal post-hoc test. The factor studied in this analysis was the

␮

(300 l of DMSO). Each single pharmacological treatment time elapsed from drug administration, with 13 levels (the

was performed after ﬁve consecutive stable MDA responses time 0 control value plus the twelve post administration

(baseline period) and the subsequent observation period stimulations). The occurrence of null MDA response (and

lasted 120 min after the drug injection (2nd, 3rd and 4th consequently the lack of related response parameters) did

group). In the last two groups, receiving two treatments not allow the use of a repeated measures MANOVA. The same

each, due to different pharmacokinetic proﬁles of the analysis was used for a further between-treatments compar-

drugs administered, an interval was interposed between ison to assess the effects of the treatments of 4th, 5th and

administrations so as to allow coincident actions. In fact, 6th groups with respect to WIN group. The factor studied in

the 5th group was pretreated with AM630 30 min before this analysis was the treatment, with 4 levels (each treat-

WIN injection, and the 6th group was administered with ment group). In this study, the variance ratio and the degrees

ODQ 30 min after receiving pretreatment with WIN. For of freedom (DF) are indicated by F(DF among groups, DF within groups).

both co-treated groups the observational period after the Differences were considered statistically signiﬁcant when P

second drug administration lasted 120 min. was less than 0.05.

Histology Results

In our histological procedure, recording and stimulating Control, vehicle and WIN-treated groups

electrode positions were veriﬁed and marked through ion-

␮

tophoretic Fast Green injection (50 A for 10 min) and a In untreated controls and vehicle-treated groups, repet-

small electrolytic lesion (20 mA for 10 s), respectively. At itive AB stimulations always induced a MDA response

the end of each experiment, the animals were anaes- whose parameters were not altered along the experimen-

thetized by an overdose of pentobarbital i.p., then they tal observation period. The WIN treatment reduced the % of

were whole-body perfused with normal saline followed by responses and also signiﬁcantly inﬂuenced the MDA parame-

10% buffered formalin. The brains were removed, postﬁxed ters, increasing the onset time and shortening the duration

in the same ﬁxative overnight and then cryoprotected in 30% of both the MDA and AD, with respect to control group

␮

sucrose/PBS. Subsequently, brains were sliced in 30—50 m (Fig. 1B), as previously reported (Rizzo et al., 2009).

serial coronal sections and stained by using Nissl-cresyl vio-

let method (Sardo et al., 2008).

Modulation of WIN effects by AM630 and ODQ on

the number of MDA responses

Statistical analysis

A comprehensive bar graph showing the effects of each

The chi-square (X ) test was used to compare the % of treatment on MDA responses is reported in Fig. 2.

responses to AB electrical stimulation following each drug The co-treatment AM630-WIN enhanced WIN effect in

treatment within the same experimental group. A between- reducing the % of responses to AB stimulation, as described

treatments X test was used to compare the % of responses henceforth. In particular, a within-treatment analysis on

of 4th, 5th and 6th group (5th and 6th after second drug AM630-WIN group displayed signiﬁcant changes on the per-

administration) with WIN treated group. centage of responses to AB stimulation. Indeed, data showed

In order to normalize individual data, within a group, a marked decrease from 30th to 60th minutes and from 100th

for each studied parameter (the duration of onset, MDA to 110th minutes, with a maximal effect at 30th minute

or AD), data from each animal were expressed as % differ- when no animals exhibited any MDA response ( = 12.000,

ence (D%) versus the baseline values represented by the last DF = 1, P = 0.0005). A further analysis on the % of responses

MDA responses of the period preceding vehicle (in the 2nd in AM630-WIN group showed signiﬁcant differences with

group) or single drugs (in 3th and 4th groups) administra- respect to WIN alone: in fact, the co-treatment continuously

tion. As regards the 5th and 6th group, data collected after reduced the % of responses from 30th to 70th minutes, with

the second drug administration were compared to the last a signiﬁcant decrease at 3rd stimulus (maximal reduction

MDA response of either the baseline period or pretreatment. observed: 50%, = 4.000, DF = 1, P = 0.0455), with respect

Nevertheless, data and related graphs of 5th and 6th groups to WIN alone.

were referred to the comparison with the last MDA response The assessment of AM630 alone proved to be ineffec-

of baseline period. tive on MDA responses since AB stimulations were always

Then, in order to study the time course of effects, in followed by DG activation in the observation period.

each group D% data were averaged (mean S.D.) on the The co-treatment ODQ-WIN enhanced WIN effect in

basis of the time elapsed from the ﬁrst stimulation follow- reducing the % of responses to AB stimulation. The within-

ing a single or combined pharmacological treatment (time treatment test revealed that in ODQ-WIN group there was

of stimulus: 10 min for 1st stimulus, 20 min for 2nd stimulus, a clear reduction of the % of responses from 10th minutes to

etc.). The time course of response parameters in untreated 120th minutes, with a maximal effect of −83.3% ( = 8.571,

controls and vehicle-treated animals was analyzed using a DF = 1, P < 0.01) at all the stimulations where the number of

Role of CB2 receptors and cGMP pathway on the cannabinoid-dependent antiepileptic effects 1715

parameters, the data did not reach an adequate statisti-

cal outcome due to the massive ODQ-WIN-induced reduction

of the % of responses (n = 1 or 2 at most stimulation time points).

Fig. 3

Comparisons between treatments (Fig. 3)

One-way (treatment, four levels) MANOVA performed by

pooling the data of all time stimulation points for each

treatment revealed a signiﬁcant multivariate main effect

for treatment (Wilks’ = 0.399, F(9, 424) = 21.591, P < 0.0001).

Moreover, signiﬁcant univariate main effects for time

were obtained for all parameters (Onset, F(3, 176) = 20.139,

P < 0.0001, power = 100; MDA duration, F(3, 176) = 22.929,

P < 0.0001, power = 100; AD duration, F(3, 176) = 16.737,

P < 0.0001, power = 100). Post hoc analysis showed signif-

Fig. 2 The bar graph shows the percentage of responses to

icant differences between the treatments: in particular,

the AB stimulation recorded in the experimental groups, as

AM630-WIN co-treatment signiﬁcantly increased the mean

indicated in the legend: WIN, AM630-WIN, AM630 and ODQ-

onset time versus WIN treatment (from 13.49 ± 2.9%

WIN, during the twelve progressive stimuli. Within-treatment

to 56.61 ± 4.63% for AM630 + WIN, +319.64%, P < 0.0001),

statistically signiﬁcant differences were indicated for P < 0.05

◦

* ** *** whereas MDA and AD duration were not signiﬁcantly

( ), P < 0.01 ( ) or P < 0.001 ( ). ( ) Indicates a signiﬁcant dif-

affected. Remarkably, signiﬁcant differences were observed

ference between the effects of AM630-WIN treatment versus

for these two parameters between AM630 and all the other

WIN (P < 0.05). () Indicates a signiﬁcant difference between the

treatments, the latter inducing marked reduction in the

effects of ODQ-WIN treatment versus WIN (P < 0.05).

parameters duration.

In order to further explore the time course of the

responses was equal to 1. A further comparison on the % effects, a one-way (treatment, four levels) MANOVA was

of responses in ODQ-WIN group with respect to WIN alone performed by analyzing data for each stimulation time

showed signiﬁcant reductions of % of responses at 10th, point. This analysis revealed a signiﬁcant multivariate main

70th and 80th minutes (in all the stimulations the reduction effect for treatment for each stimulation with the excep-

observed was: 66.6%; = 6.000, DF = 1, P = 0.0143 at 10th tion of 3rd, 5th, 8th and 12th. Signiﬁcant univariate main

minute; = 5.33, DF = 1, P = 0.02 at 70th and 80th minutes). effects were observed at different time points, and were

further highlighted by post hoc test: in particular, AM630-

WIN co-treatment showed a signiﬁcant increase of the time

Modulation of WIN effects by AM630 and ODQ on

of onset versus WIN alone, from 60th to 110th minutes,

the MDA parameters

with the maximal effect at 110th minute of +80.56% (from

−

8.76% ± 15.2 to +71.8 ± 13.9%; P = 0.0003; F(1,6) = 59.236).

Comparisons within each treatment (Fig. 3)

The same analysis for MDA and AD durations did not reveal

In the group receiving pre-treatment with AM630 followed

signiﬁcant differences. Furthermore, the comparison of

by WIN administration, one-way MANOVA revealed a signiﬁ-

AM630- versus WIN-induced effects showed a signiﬁcant

cant multivariate main effect for stimulation time (Wilks’

reduction of the mean MDA duration in WIN group from 40th

= 0.275, F(33, 98) = 1.632, P = 0.034). Moreover, signiﬁcant

to 100th minutes, with a maximum effect at 60th minute

univariate main effects for stimulation time were obtained

of −54.79% (from −15.6% ± 23.82 to −70.39% ± 13.70;

for all parameters (Onset, F(11, 35) = 2.335, P = 0.028; MDA

P = 0.0114; F(1,9) = 10.033). Lastly, a signiﬁcant decrease

duration, F(11, 35) = 3.300, P = 0.00348; AD duration, F(11,

of AD duration was shown in WIN group from 40th to

35) = 3.272, P = 0.0036). Post hoc analysis revealed that

100th minutes, with a maximum effect at 90th minute

AM630-WIN induced an increase in the onset parameter

of −85.13% (from +17.65 ± 42.51% to −67.48 ± 10.86%;

from 60th to 120th minutes, with a maximum effect of

P = 0.007; F(16,51) = 13.662), compared to AM630. As for the

+86.05 ± 10.22% at 70th minute (P = 0.0003). Moreover, a

comparison of the effects of ODQ-WIN versus WIN alone on

reduction of MDA duration was evident from 60th to 120th

the MDA parameters, the data did not reach an adequate

minutes, with a maximum effect of −85.49 ± 3.92% at 70th

statistical outcome due to the massive ODQ-WIN-induced

minute (P = 0.0004). Similarly, the co-treatment induced a

reduction of the % of responses (n = 1 or 2 at most stimulation

reduction in the AD duration from 60th to 120th minutes,

time points).

with a maximum effect of −87.04 ± 3.20% at 70th minute

(P = 0.0011). In contrast, one-way MANOVA for stimulation

time did not reveal a signiﬁcant multivariate main effect Discussion

for stimulation time for the group treated with AM630 alone

(Wilks’ = 0.496, F(36, 187) = 1.391, P = 0.0830), when com- A functional involvement of CB transmission in the modula-

pared to baseline period. Moreover, signiﬁcant univariate tion of paroxysmal events has been widely investigated, but

main effects for stimulation time were not obtained for clear and deﬁnitive conclusions are not still available. Sev-

any parameter in AM630 group. As for the comparison of eral studies targeted the CB1 receptors in an attempt to clar-

the effects of ODQ-WIN versus baseline period on the MDA ify the mechanism underlying the possible CB modulation of

1716 V. Rizzo et al.

Fig. 3 Effects of AM630 and AM630-WIN on the time course of MDA parameters during the 12 progressive stimuli. Each value

represents the mean of D% of each treatment ( AM630 or AM630-WIN) per stimulus versus baseline values. The symbol NR

indicates no response to the stimulation (3rd stimulus of AM630-WIN group). Within-treatment statistically signiﬁcant D% is indicated

* ◦

for P < 0.05 ( ) vs baseline values. Between-treatment signiﬁcant differences in AM630-WIN group are indicated for P < 0.05 ( ) vs

WIN group. Between-treatment signiﬁcant differences in AM630 group are indicated for P < 0.05 ( ) vs WIN group.

hyperexcitability processes. As a matter of fact, CB1 recep- induced by WIN alone, the co-treatment with AM630 and

tors are linked to an inhibitory G-protein modulating, among WIN signiﬁcantly reduced the severity of ictal events and,

the others, A-type K channels and N and P/Q type voltage- even more, the percentage of responses to the stimulation,

gated Ca currents so as to stabilize the membrane poten- suggesting that AM630 improves WIN efﬁcacy. A possible

tials (Deadwyler et al., 1995; Pan et al., 1996). This action reduced proneness to the epileptogenic phenomena, as

on membrane conductance may determine the CB-mediated revealed by the increase of onset time, is not associated to

suppression of presynaptic neurotransmitter release, either signiﬁcant differences in MDA and AD parameters between

regarding Glutamate or GABA release, a neuronal process the AM630-WIN and WIN groups. Taken together, these data

known as depolarization-induced suppression of excitation might suggest that the efﬁcacy of the co-treatment is

(DSE) or inhibition (DSI), respectively (Kreitzer and Regehr, exerted mainly by augmenting seizure threshold in the DG,

2001; Wilson and Nicoll, 2001). In detail, CB1-mediated DSE rather than on the epileptic discharge, once elicited. On the

has been hypothesized to be involved in the reduction of basis of the enhancement of WIN-induced effects follow-

the seizure discharge in hippocampal cultures (Deshpande ing AM630 pretreatment, one group of animals was treated

et al., 2007). Thus, it is conceivable that the antiepilep- with the CB2 antagonist/inverse agonist to assess its efﬁ-

tic properties of CB agonists may consists of a modulation cacy when administered alone, but this treatment resulted

mainly directed towards the inhibition of the glutamatergic unexpectedly fruitless, suggesting that CB2 exerts no direct

neurotransmission than GABA release (Monory et al., 2006). effects on hippocampal hyperexcitability in MDA model.

Though, in the MDA experimental rat model of human partial From a pharmacological point of view, we hypothesize that

epilepsy, the lack of a complete blockade of WIN-induced WIN may have greater occupancy at CB1Rs when CB2Rs are

effect after pre-treatment with a selective CB1 antagonist, antagonized by AM630, hence eliciting a better response by

AM251, (Rizzo et al., 2009) raised the possibility of a further facilitating WIN selectivity on CB1-mediated pathway.

mechanism for CB-mediated modulation of hippocampal In order to analyze the downstream effectors of CB recep-

seizures. In this regard, multiple evidences report a wide tors activation in the phenomena examined, we focused on

distribution of CB2 in neuronal and glial cells in several NO/cGMP pathway by administering the sGC inhibitor, ODQ.

CNS areas such as cerebral cortex, hippocampus, thalamus, As previously reported, ODQ alone induced a signiﬁcant

brain stem and cerebellum (Gong et al., 2006), suggesting decrease of the severity of ictal events, but did not induce

a potential implication of CB2 receptors, besides CB1R role, any change in the number of responses, in any case sug-

in mediating CB signaling (García-Gutiérrez et al., 2012). gesting a functional involvement of the NO/sGC metabolic

With the aim of exploring the possible modulatory role of pathway in the DG paroxysmal activity (Sardo et al., 2006).

CB2 on the antiepileptic action exerted by WIN 55,212-2 In the present study, animals were co-treated with WIN and

on the MDA model, we administered the antagonist/inverse ODQ so as to assess if the inhibition of sGC may impact on

agonist, AM630 (Bolognini et al., 2012), known for its high WIN antiepileptic properties. Our data showed a signiﬁcant

potency and afﬁnity for rat CB2 receptors (Mukherjee et al., reduction in the percentage of responses to AB stimulation

2004). Our results showed that, when compared to the effect induced by WIN and ODQ co-treatment. Moreover, this fall

Role of CB2 receptors and cGMP pathway on the cannabinoid-dependent antiepileptic effects 1717

in the percentage of responses was signiﬁcantly enhanced, References

when compared to WIN alone. This impressive decrease of

MDA responses suggests an interplay between cGMP and CB

1 Ahern, G.P., Klyachko, V.A., Jackson, M.B., 2002. cGMP and S-

controlling the excitability of DG neurons in experimentally- nitrosylation: two routes for modulation of neuronal excitability

induced seizures. Providing evidences to the hypothesis of by NO. Trends Neurosci. 25, 510—517.

this co-role, physiological and anatomical studies strongly Arancio, O., Antonova, I., Gambaryan, S., Lohmann, S.M., Wood,

bridge NO and eCB inasmuch as regulating neuronal hyper- J.S., Lawrence, D.S., Hawkins, R.D., 2001. Presynaptic role of

cGMP-dependent protein kinase during long-lasting potentiat-

excitability in pathophysiological states such as epilepsy

ion. J. Neurosci. 21 (1), 143—149 (The Ofﬁcial Journal of the

(Bahremand et al., 2009; Jones et al., 2008; Makara et al.,

Society for Neuroscience).

2007; Stringer and Erden, 1995). The underlying mecha-

Azad, S.C., Marsicano, G., Eberlein, I., Putzke, J., Zieglgänsberger,

nism may regard the involvement of NO/cGMP pathway

W., Spanagel, R., Lutz, B., 2001. Differential role of the nitric

in CB-mediated DSE and DSI, in which a retrograde con-

oxide pathway on delta(9)-THC-induced central nervous system

trol of glutamate and GABA release is exerted in in CB

1 effects in the mouse. Eur. J. Neurosci. 13 (3), 561—568.

expressing axon terminals (Chevaleyre et al., 2006; Llano Bahremand, A., Nasrabady, S.E., Shafaroodi, H., Ghasemi, M.,

et al., 1991; Makara et al., 2007). Indeed, the inhibition Dehpour, A.R., 2009. Involvement of nitrergic system in the anti-

of NO/cGMP signaling at various levels diminished DSI in a convulsant effect of the cannabinoid CB(1) agonist ACEA in the

CB1R-dependent manner (Makara et al., 2007), hence mod- pentylenetetrazole-induced seizure in mice. Epilepsy Res. 84

(2—3), 110—119.

ulating interneuron GABA-inhibition. In agreement with the

Bolognini, D., Cascio, M.G., Parolaro, D., Pertwee, R.G., 2012.

aforementioned studies, our data convey the idea that, in

AM630 behaves as a protean ligand at the human cannabinoid

the MDA model, NO/cGMP pathway could be targeted as a

CB2 receptor. Br. J. Pharmacol. 165 (8), 2561—2574.

neuromodulator after previous CB1 activation (e.g. under

Braakman, H.M., van Oostenbrugge, R.J., van Kranen-Mastenbroek,

WIN pre-treatment). On this basis, providing that nNOS is

V.H., de Krom, M.C., 2009. Rimonabant induces partial seizures

a Ca-dependent enzyme (Mergia et al., 2009; Neitz et al.,

in a patient with a history of generalized epilepsy. Epilepsia 50

2011), it is conceivable that CB1 antiepileptic activity may (9), 2171—2172.

be exerted by reducing NMDA-dependent calcium signaling Burette, A., Zabel, U., Weinberg, R.J., Schmidt, H.H., Valtschanoff,

in glutamate synapses, thus lowering post-synaptic NO pro- J.G., 2002. Synaptic localization of nitric oxide synthase and sol-

duction. If so, according to previously reported evidences uble guanylyl cyclase in the hippocampus. J. Neurosci. 22 (20),

8961—8970.

(Arancio et al., 2001), the blockade of presynaptic sGC in

Cabral, G.A., Raborn, E.S., Grifﬁn, L., Dennis, J., Marciano-Cabral,

excitatory synapses enhances the WIN antiepileptic effects

F. , 2008. CB2 receptors in the brain: role in central immune

likely by further weakening glutamate transmission. As far as

function. Br. J. Pharmacol. 153 (2), 240—251.

these signaling systems might act on common synaptic func-

Carletti, F. , Ferraro, G., Rizzo, V. , Cannizzaro, C., Sardo, P. , 2013.

tions such as ion channels permeability, neurotransmitter

Antiepileptic effect of dimethyl sulfoxide in a rat model of tem-

release and synaptic plasticity, any change in cGMP synthe-

poral lobe epilepsy. Neurosci. Lett. 546, 31—35.

sis within CB1-expressing axon terminals, by modulating the Castillo, P.E., Younts, T.J., Chávez, A.E., Hashimotodani, Y. , 2012.

sGC activity, could have consequences on the CB1-induced Endocannabinoid signaling and synaptic function. Neuron 76 (1),

effects (Ahern et al., 2002; Castillo et al., 2012; Deadwyler 70—81.

et al., 1995; Feil and Kleppisch, 2008). Chevaleyre, V. , Takahashi, K.A., Castillo, P.E., 2006.

Endocannabinoid-mediated synaptic plasticity in the CNS.

In conclusion, our study showed that the manipulation

Annu. Rev. Neurosci. 29, 37—76.

of CB2 receptor is able to potentiate the antiepileptic

Deadwyler, S.A., Hampson, R.E., Mu, J., Whyte, A., Childers, S.,

action of the CB agonist, WIN, in a model of hippocam-

1995. Cannabinoids modulate voltage sensitive potassium A-

pal seizures; a similar effect is achieved by blocking the

current in hippocampal neurons via a cAMP-dependent process.

activity of sGC, a putative downstream effector of CB

J. Pharmacol. Exp. Ther. 273 (2), 734—743.

signaling. Taken together, these results shed light on the

den Boon, F.S., Chameau, P. , Schaafsma-Zhao, Q., van Aken, W.,

mechanisms underpinning CB antiepileptic effects and hint a Bari, M., Oddi, S., Kruse, C.G., Maccarrone, M., Wadman, W.J.,

possible novel pharmacological approach for the treatment Werkman, T.R., 2012. Excitability of prefrontal cortical pyrami-

of excitotoxicity-derived diseases. dal neurons is modulated by activation of intracellular type-2

cannabinoid receptors. Proc. Natl. Acad. Sci. U.S.A. 109 (9),

3534—3539.

Deshpande, L.S., Sombati, S., Blair, R.E., Carter, D.S., Martin, B.R.,

Acknowledgement DeLorenzo, R.J., 2007. Cannabinoid CB1 receptor antagonists

cause status epilepticus-like activity in the hippocampal neu-

ronal culture model of acquired epilepsy. Neurosci. Lett. 411

This work was supported by grants of Italian Ministry of

(1), 11—16.

the University and the Scientiﬁc Research (M.I.U.R.), MIUR-

Devane, W.A., Dysarz, F.A., Johnson, M.R., Melvin, L.S., Howlett,

UNIPA ORPA07BLYM Rome, Italy.

A.C., 1988. Determination and characterization of a cannabinoid

receptor in rat brain. Mol. Pharmacol. 34 (5), 605—613.

Feil, R., Kleppisch, T. , 2008. NO/cGMP-dependent modula-

tion of synaptic transmission. Handb. Exp. Pharmacol. 184,

Appendix A. Supplementary data 529—560.

Fernández-Ruiz, J., Romero, J., Velasco, G., Tolón, R.M., Ramos,

J.A., Guzmán, M., 2007. Cannabinoid CB2 receptor: a new target

Supplementary data associated with this arti-

for controlling neural cell survival? Trends Pharmacol. Sci. 28 (1),

cle can be found, in the online version, at http://dx.doi.org/10.1016/j.eplepsyres.2014.10.001. 39—45.

1718 V. Rizzo et al.

García-Gutiérrez, M.S., García-Bueno, B., Zoppi, S., Leza, J.C., Klugmann, M., Wölfel, B., Dodt, H.U., Zieglgänsberger, W., Wot-

Manzanares, J., 2012. Chronic blockade of cannabinoid CB2 jak, C.T., Mackie, K., Elphick, M.R., Marsicano, G., Lutz, B.,

receptors induces anxiolytic-like actions associated with alter- 2006. The endocannabinoid system controls key epileptogenic

ations in GABA(A) receptors. Br. J. Pharmacol. 165 (4), 951—964. circuits in the hippocampus. Neuron 51 (4), 455—466.

Ghasemi, M., Sadeghipour, H., Shafaroodi, H., Nezami, B.G., Morgan, N.H., Stanford, I.M., Woodhall, G.L., 2009. Functional

Gholipour, T. , Hajrasouliha, A.R., Tavakoli, S., Nobakht, M., CB2 type cannabinoid receptors at CNS synapses. Neuro-

Moore, K.P., Mani, A.R., Dehpour, A.R., 2007. Role of the nitric pharmacology 57 (4), 356—368.

oxide pathway and the endocannabinoid system in neurogenic Mukherjee, S., Adams, M., Whiteaker, K., Daza, A., Kage, K., Cassar,

relaxation of corpus cavernosum from biliary cirrhotic rats. Br. S., Meyer, M., Yao, B.B., 2004. Species comparison and phar-

J. Pharmacol. 151 (5), 591—601. macological characterization of rat and human CB2 cannabinoid

Gong, J.P., Onaivi, E.S., Ishiguro, H., Liu, Q.R., Tagliaferro, receptors. Eur. J. Pharmacol. 505 (1—3), 1—9.

P.A., Brusco, A., Uhl, G.R., 2006. Cannabinoid CB2 receptors: Neitz, A., Mergia, E., Eysel, U.T., Koesling, D., Mittmann, T. , 2011.

immunohistochemical localization in rat brain. Brain Res. 1071 Presynaptic nitric oxide/cGMP facilitates glutamate release via

(1), 10—23. hyperpolarization-activated cyclic nucleotide-gated channels in

Hill, T.D., Cascio, M.G., Romano, B., Duncan, M., Pertwee, R.G., the hippocampus. Eur. J. Neurosci. 33 (9), 1611—1621.

Williams, C.M., Whalley, B.J., Hil, A.J., 2013. Cannabidivarin- Pan, X., Ikeda, S.R., Lewis, D.L., 1996. Rat brain cannabinoid recep-

rich cannabis extracts are anticonvulsant in mouse and rat via tor modulates N-type Ca2+ channels in a neuronal expression

a CB1 receptor-independent mechanism. Br. J. Pharmacol. 170 system. Mol. Pharmacol. 49 (4), 707—714.

(3), 679—692. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordi-

Hofmann, M.E., Frazier, C.J., 2013. Marijuana, endocannabinoids, nates. Academic Press, San Diego, IL, USA.

and epilepsy: potential and challenges for improved therapeutic Rizzo, V. , Ferraro, G., Carletti, F. , Lonobile, G., Cannizzaro, C.,

intervention. Exp. Neurol. 244, 43—50. Sardo, P. , 2009. Evidences of cannabinoids-induced modula-

Howlett, A.C., Breivogel, C.S., Childers, S.R., Deadwyler, S.A., tion of paroxysmal events in an experimental model of partial

Hampson, R.E., Porrino, L.J., 2004. Cannabinoid physiology and epilepsy in the rat. Neurosc. Lett. 462 (2), 135—139.

pharmacology: 30 years of progress. Neuropharmacology 47 Robello, M., Amico, C., Bucossi, G., Cupello, A., Rapallino, M.V.,

(Suppl. 1), 345—358. Thellung, S., 1996. Nitric oxide and GABAA receptor function

Jones, J.D., Carney, S.T., Vrana, K.E., Norford, D.C., Howlett, in the rat cerebral cortex and cerebellar granule cells. Neuro-

A.C., 2008. Cannabinoid receptor-mediated translocation of science 74 (1), 99—105.

NO-sensitive guanylyl cyclase and production of cyclic GMP in Sardo, P. , Carletti, F. , D’Agostino, S., Rizzo, V. , Ferraro, G., 2006.

neuronal cells. Neuropharmacology 54 (1), 23—30. Involvement of nitric oxide-soluble guanylyl cyclase pathway in

Jones, N.A., Glyn, S.E., Akiyama, S., Hill, T.D.M., Hill, A.J., Weston, the control of maximal dentate gyrus activation in the rat. J.

S.E., Burnett, M.D.A., Yamasaki, Y. , Stephens, G.J., Whalley, Neural Transm. 113 (12), 1855—1861.

B.J., Williams, C.M., 2012. Cannabidiol exerts anti-convulsant Sardo, P. , D’Agostino, S., Carletti, F. , Rizzo, V. , La Grutta,

effects in animal models of temporal lobe and partial seizures. V. , Ferraro, G., 2008. Lamotrigine differently modulates 7-

Seizure 21 (5), 344—352 (The Journal of the Epilepsy Associa- nitroindazole and L-arginine inﬂuence on rat maximal dentate

tion). gyrus activation. J. Neural Transm. 115 (1), 27—34.

Kreitzer, A.C., Regehr, W.G., 2001. Retrograde inhibition of presyn- Stefano, G.B., Rialas, C.M., Deutsch, D.G., Salzet, M., 1998. Anan-

aptic calcium inﬂux by endogenous cannabinoids at excitatory damide amidase inhibition enhances anandamide-stimulated

synapses onto Purkinje cells. Neuron 29, 717—727. nitric oxide release in invertebrate neural tissues. Brain Res.

Llano, I., Leresche, N., Marty, A., 1991. Calcium entry increases 793 (1—2), 341—345.

the sensitivity of cerebellar Purkinje cells to applied GABA and Stringer, J.L., Erden, F. , 1995. In the hippocampus in vivo, nitric

decreases inhibitory synaptic currents. Neuron 6 (4), 565—574. oxide does not appear to function as an endogenous antiepileptic

Maggi, C.A., Meli, A., 1986. Suitability of urethane anesthesia for agent. Exp. Brain Res. 105, 391—401.

physiopharmacological investigations in various systems. Part 1: Stringer, J.L., Lothman, E.W., 1989. Maximal dentate gyrus activa-

General considerations. Experientia 42 (2), 109—114. tion: characteristics and alterations after repeated seizures. J.

Makara, J.K., Katona, I., Nyíri, G., Németh, B., Ledent, C., Watan- Neurophysiol. 62, 136—143.

abe, M., de Vente, J., Freund, F. T. , Hájos, N., 2007. Involvement Van Sickle, M.D., Duncan, M., Kingsley, P.J., Mouihate, A., Urbani,

of nitric oxide in depolarization-induced suppression of inhi- P. , Mackie, K., Stella, N., Makriyannis, A., Piomelli, D., Davi-

bition in hippocampal pyramidal cells during activation of son, J.S., Marnett, L.J., Di Marzo, V. , Pittman, Q.J., Patel,

cholinergic receptors. J. Neurosci. 27 (38), 10211—10222 (The K.D., Sharkey, K.A., 2005. Identiﬁcation and functional charac-

Ofﬁcial journal of the Society for Neuroscience). terization of brainstem cannabinoid CB2 receptors. Science 310

Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C., Bonner, (5746), 329—332.

T.I., 1990. Structure of a cannabinoid receptor and functional Wallace, M.J., Blair, R.E., Falenski, K.W., Martin, B.R., DeLorenzo,

expression of the cloned cDNA. Nature 346 (6284), 561—564. R.J., 2003. The endogenous cannabinoid system regulates

Mergia, E., Koesling, D., Friebe, A., 2009. Genetic mouse mod- seizure frequency and duration in a model of temporal lobe

els of the NO receptor ‘soluble’ guanylyl cyclases. Handb. Exp. epilepsy. J. Pharmacol. Exp. Ther. 307 (1), 129—137.

Pharmacol. 191, 33—46. Wilson, R.I., Nicoll, R.A., 2001. Endogenous cannabinoids medi-

Monory, K., Massa, F. , Egertová, M., Eder, M., Blaudzun, H., West- ate retrograde signalling at hippocampal synapses. Nature 410,

enbroek, R., Kelsch, W., Jacob, W., Marsch, R., Ekker, M., 588—659.

Long, J., Rubenstein, J.L., Goebbels, S., Nave, K.A., During, M.,