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Neuroscience Letters 602 (2015) 38–43

Contents lists available at ScienceDirect

Neuroscience Letters

journa l homepage: www.e lsev ier .com/ locate /neule t

esearch article

nilateral microinjection of carbenoxolone into the pontis caudalisucleus inhibits the pentylenetetrazole-induced epileptiform activity

n rats

avier Franco-Pérez a, Paola Ballesteros-Zebadúa b, Joaquín Manjarrez-Marmolejo a,∗

Laboratory of Physiology of Reticular Formation, National Institute of Neurology and Neurosurgery, M.V.S. Mexico, D.F., MexicoLaboratory of Medical Physics, National Institute of Neurology and Neurosurgery, M.V.S. Mexico, D.F., Mexico

i g h l i g h t s

The gap junction blocker carbenoxolone reduced the incidence of seizures.Microinjection of carbenoxolone inhibited the PTZ-induced epileptiform activity.The gap junction opener trimethylamine increased the duration of seizures.Trimethylamine exacerbated the PTZ-induced epileptiform activity.

r t i c l e i n f o

rticle history:eceived 8 April 2015eceived in revised form 28 May 2015ccepted 17 June 2015vailable online 30 June 2015

eywords:arbenoxolone

a b s t r a c t

Pontine reticular formation (PRF) is involved in the generation and maintenance of generalized epilepticseizures. Carbenoxolone (CBX) is a gap junction blocker with anticonvulsant properties. Therefore, thepresent study was designed to explore the effects of CBX microinjected into the pontis caudalis nucleus(PnC) on generalized tonic–clonic seizures (GTCS) and epileptiform activity induced by pentylenetetra-zole (PTZ).

All control rats presented GTCS after a single dose of PTZ. The microinjection of CBX into the PnCreduced the GTCS incidence induced by PTZ. Moreover, the CBX significantly increased the latency to

ap junctionsentylenetetrazoleontine reticular formationeizures

the first myoclonic jerk. Additionally, CBX significantly decreased the spectral power and the amplitudeof the epileptiform activity induced by PTZ. By contrast, the microinjection of a gap junction opener(trimethylamine) did not cause anticonvulsant effects and even increased the duration of the GTCS.

These findings suggest that the PnC is a particular nucleus where the CBX could exert its action mech-anisms and elicit anticonvulsant effects.

© 2015 Elsevier Ireland Ltd. All rights reserved.

. Introduction

Since several years ago, some authors have suggested that PRF is

nvolved in the generation and maintenance of generalized epilep-ic seizures [1–3]. Specifically, some studies have showed thatnC neurons participate in the generation of behaviors related to

Abbreviations: CBX, carbenoxolone; EEG, electroencephalographic recordings;TCS, generalized tonic–clonic seizures; i.p., intraperitoneal; PnC, pontis caudalisucleus; PRF, pontine reticular formation; PTZ, pentylenetetrazole; TMA, trimethy-

amine.∗ Corresponding author at: Laboratory of Physiology of Reticular Formationational Institute of Neurology and Neurosurgery, M.V.S. Insurgentes Sur 3877, Col.a Fama, C.P. 14269 Mexico D.F, Mexico. Fax: +52 55 5424 0808.

E-mail address: joaquinmanjarrez@hotmail.com (J. Manjarrez-Marmolejo).

ttp://dx.doi.org/10.1016/j.neulet.2015.06.037304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.

seizures events. It has been reported that microinjection of NMDAreceptor antagonists into the PnC decreased the incidence of GTCSand decreased the amplitude and frequency of the cortical epilep-tiform activity induced by PTZ [4].

Gap junctions are clusters of intercellular channels that providecytoplasmic continuity and permit direct communication betweencells. These intercellular channels contribute to the fast exchangeof ions and some small biological molecules allowing the elec-trical coupling and synchronized firing of neurons [5]. It is wellknown that some chemical agents have the ability to block gapjunctions [6]. Between the gap junction blockers, CBX is one of the

most widely used because reversibly block the electrical couplingin several neuronal circuits [7,8].

The hypothesis that electrical coupling mediated by gap junc-tions is a primary mechanism involved in the generation and

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aintenance of seizures has gained relevance. In fact, the neuronalypersynchronic activity is a common feature that describes con-ulsive events. For this reason, it has been described that systemicdministration of CBX reduces the clonic and tonic phases of theudiogenic seizures [9]. More recently, Sefil et al. [10] demonstratedhat intraperitoneal (i.p.) administration of CBX has anticonvulsantffects by shortening the generalized seizure duration and reducinghe total spike number in PTZ kindled rats.

CBX has showed anticonvulsant effects, and there is convincingvidence regarding the presence of gap junctions in pontine nuclei11,12]. For these reasons, we decided to evaluate the effects of the

icroinjection of CBX into the PnC on GTCS and the epileptiformctivity induced by PTZ in rats.

. Material and methods

.1. Animals

Male Wistar rats (270–300 g) were maintained under controlledonditions (24.4 ◦C; 7:00–19:00 light, 19:00–7:00 darkness; foodnd water ad libitum). All animals were treated according to reg-lations specified by the Bioethical Committee and the Mexicantandard for the production, care and use of laboratory animalsNOM-062-Z00-1999).

.2. Implantation of electrodes and guide cannula

All reagents were purchased from Sigma–Aldrich (St Louis,O., USA). Animals were anesthetized with ketamine (80 mg/kg,

.p.) and xylazine (10 mg/kg, i.p.). Two electrodes were deeplymplanted in the motor cortex (1.2 mm anterior to Bregma, 2.5 mmateral to the midline, 1.5 mm below the surface of the skull) forhe electroencephalographic (EEG) recordings. One more electrodemplanted above the cerebellum was used as a reference (11.9 mmosterior to Bregma, 3.0 mm lateral to the midline, 1.0 mm belowhe surface of the skull) [13]. Besides, a stainless-steel guide cannulaBecton Dickinson, Mexico) (25 gauge) was stereotaxically posi-ioned 4 mm above the right PnC (10.0 mm posterior to Bregma,.8 mm lateral to midline, 9.0 mm below the surface of the skull)13] for the microinjection of the vehicle, CBX or trimethylamineTMA).

.3. Groups

We used 78 rats; however, we only selected those 40 with therecise microinjection into the PnC. Rats were randomly divided

nto the following groups: control group (n = 8) administered withhe vehicle (saline solution). Three groups were microinjected withhe gap junction blocker: CBX 20 nmol (n = 8), CBX 50 nmol (n = 8)nd CBX 100 nmol (n = 8). Another group was microinjected withhe gap junction opener: TMA 50 nmol (n = 8).

.4. EEG recordings

The rats were connected to an amplifier (EBNeuro®, Firenze,taly) by means of flexible insulated cables. Before any manipu-ation, a basal recording was carried out for 10 min. Then, rats were

icroinjected with the vehicle, CBX or TMA through an injectioneedle (32-gauge) 4 mm longer than the guide cannula. Microin-

ections were carried out with freely moving animals using thenjection needle connected to a 10 �L Hamilton syringe (infusion

ate 0.2 �L/min). 15 min after the microinjection of the vehicle,BX or TMA the animals were administered with PTZ (70 mg/kg,

.p.). The video-EEG recordings were stored on the hard drive of aomputer for the off-line analysis.

e Letters 602 (2015) 38–43 39

2.5. Histological procedures

At the end of EEG recordings, methylene blue was microin-jected (0.2 �L), to localize the injection site and to rule out theanimals with microinjections outside of the PnC. Rats were sys-temically administered with sodium pentobarbital (100 mg/kg,i.p.) and were transcardially perfused with saline-heparin solu-tion (0.9%) followed by formalin (3.7%). Brains were then removedand postfixed in formalin (3.7%). Finally, the injection sites wereverified in coronal slices of 80 �m thickness stained with cresylviolet.

2.6. Power spectral analysis

EEG signals analysis was performed with Galileo NT software(EBNeuro®, Firenze, Italy). All EEG signals were filtered with alow-pass at 0.3 Hz and a high-pass at 70 Hz. During rat wakeful-ness, 60-s epochs of the EEG recordings were extracted. Afterward,the total spectral power (mV2) as well as the amplitude (mV)and frequency (Hz) of the EEG signal were calculated with afast Fourier transform method. It has been demonstrated thatPTZ induces epileptiform activity characterized by voltage fluc-tuations mainly at low frequencies (1–16 Hz) and with minimalfluctuations at high frequencies (>30 Hz) [14]. For this reason,the analysis was carried out limiting the frequency range until32 Hz.

2.7. Statistical analysis

Fisher’s exact probability tests (p < 0.05) were used to comparethe incidence of GTCS and percentage of survival after administra-tion of PTZ. Also, Kruskal–Wallis one-way analysis of variance onranks with Dunnett’s method were used to determine the statisticalsignificance (p < 0.05) of the others observed parameters.

3. Results

3.1. Incidence of GTCS and survival

The 100% of the animals administered with the vehicle intothe PnC and PTZ presented GTCS. The microinjection of CBX didnot induce an apparent dose-dependent effect since the same per-centage of animals with GTCS (62.5%) was observed in the groupspretreated with 20 and 100 nmol of CBX. On the other hand, themicroinjection of CBX 50 nmol significantly decreased (p < 0.05)75% the incidence of GTCS. By contrast, the gap junction openerTMA, did not protect against the GTCS induced by PTZ (Fig. 1A). Thepercentage of survival after PTZ administration also was analyzed.It was observed that no rat survived after the treatment with vehicleor TMA and PTZ. By contrast, the animals microinjected with CBX20, 50 and 100 nmol showed a survival of 50%, 75% (p < 0.05), and37.5%, respectively (Fig. 1B). Similarly, after comparing the durationof the GTCS induced by PTZ, we found that CBX (50 nmol) decreasedthis parameter. Interestingly, the microinjection of TMA into thePnC induced a significant increase of the GTCS duration (Fig. 1C).

3.2. Latency to the first myoclonic jerk and GTCS

The first seizure parameter observed after the PTZ administra-tion was the myoclonic jerk. This parameter was characterizedby a strong shaking of the whole body. In the vehicle group, thischaracteristic was observed 0.92 ± 0.03 min after PTZ administra-

tion. However, the CBX doses significantly (p < 0.05) delayed theappearance of this seizure parameter (Table 1).

Later, it was analyzed the latency to the GTCS. All of the con-trol rats presented only one GTCS with a latency of 1.55 ± 0.15 min.

40 J. Franco-Pérez et al. / Neuroscience Letters 602 (2015) 38–43

Fig. 1. Effects of CBX and TMA on seizures incidence, survival, and GTCS duration.The microinjection of CBX 50 nmol into the PnC: (A) significantly decreased the incidence of GTCS induced by PTZ and (B) significantly increased the percentage of survivalafter the administration of PTZ. *p < 0.05 as compared with the vehicle group (Fisher’s exact probability tests). (C) The GTCS duration was decreased by CBX (50 nmol) andi –Wall

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ncreased by TMA (50 nmol). *p < 0.05 as compared with the vehicle group (Kruskal

onetheless, the microinjection of CBX into the PnC significantlyp < 0.05) delayed the appearance of the GTCS (Table 1). Similarly,

e observed that the microinjection of CBX significantly modi-ed the latency to death when comparing with the control group

Table 1). Finally, the administration of the gap junction openerMA did not change the analyzed latencies (Table 1).

is one-way analysis of variance on ranks with Dunnett’s method).

3.3. Spectral power after PTZ administration

In the vehicle group, the PTZ administration triggered GTCScharacterized by a tonic phase described by hindlimb extensionand a clonic phase with myoclonus of the anterior and poste-rior limbs. In the EEG, these behaviors correlated with highly

J. Franco-Pérez et al. / Neuroscience Letters 602 (2015) 38–43 41

Table 1Analysis of seizure parameters induced by the administration of PTZ (70 mg/kg, i.p.) in rats.

Group Latency to first myoclonic jerk (min) Latency to GTCS (min) Latency to death (min)

Vehicle + PTZ 0.92 ± 0.03 1.55 ± 0.15 3.41 ± 0.33CBX 20 nmol + PTZ 2.70 ± 1.25* 47.01 ± 21.37* 71.84 ± 18.85*

CBX 50 nmol + PTZ 7.83 ± 4.12* 90.59 ± 19.25* 93.72 ± 17.20*

CBX 100 nmol + PTZ 5.32 ± 3.67* 46.67 ± 21.48* 51.21 ± 20.32*

TMA 50 nmol + PTZ 0.73 ± 0.06 1.97 ± 0.6 5.49 ± 1.28

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esults are expressed in minutes as mean ± SEM.* p < 0.05, differences statistically significant in relation to the vehicle group. Analyethod.

ynchronized bursting activity and with trains of voltage fluc-uations. As a consequence, these voltage fluctuations producedncrements of the spectral power (Fig. 2A and B). By contrast,he microinjection of CBX (50 nmol) into the PnC inhibited thexpression of GTCS. In the EEG, CBX also inhibited the presencef the EEG spikes typically observed in this epileptogenic model.

nstead of, we only found small synchronized bursting activity cor-elated with short facial myoclonus, but with no increments inhe spectral power (Fig. 3A and B). Interestingly, TMA induced

contrary effect to that observed with CBX. This gap junctionpener triggered GTCS characterized by a first tonic phase fol-

owed by a clonic phase and the occurrence of one or more tonichases. This phenomenon was correlated with highly synchro-ized activity and increments of the spectral power (Fig. 4A and).

.4. Localization of the guide cannula

The correct site of microinjection is showed in the supplemen-ary material. We found several animals microinjected with CBX in

egions as varied as intermediate reticular nucleus, fourth ventricle,enu of the facial nerve, superior paraolivary nucleus, tectospinalract, paraabducens nucleus. All the animals microinjected in these

ig. 2. Spectral power analysis after the treatment with vehicle and PTZ.A) 3D graphic showing high spectral power values at low frequencies (0–5 Hz)nduced by the administration of PTZ. (B) Representative EEG traces obtained in

60-s epoch during the post-administration of vehicle and PTZ. The epileptiformctivity correlated with the expression of a tonic phase and a clonic phase.

sed on Kruskal–Wallis one way analysis of variance on ranks followed by Dunnett’s

regions presented GTCS and did not show any protection againstmortality induced by PTZ (data not shown).

4. Discussion

There are several studies that have determined the presence ofgap junctions in neurons, astrocytes and even oligodendrocytes inthe pons and along the brainstem [11,12,15]. Our study provides thefirst experimental evidence that unilateral microinjection of a gapjunction blocker into the PnC prevents the incidence of GTCS as wellas the PTZ-induced epileptiform activity. The CBX is a widely usedgap junction blocker with favorable responses to epileptic activ-ity when is used in different animal models. Recent studies havedemonstrated that in vitro application of CBX to thalamocorticalslices, abolish the synchronous spontaneous seizure-like activitiesinduced by 4-aminopyridine [16,17]. Also, it has been reportedthat i.p. administration of CBX delays the onset of seizures andreduce the duration of GTCS induced by PTZ [10,18]. Some stud-ies have found specific nuclei in the brain where the CBX elicitsanticonvulsant effects. Thus, the microinjection of CBX into theinferior colliculus, substantia nigra, and the inferior olivary com-plex reduced the duration and severity of audiogenic seizures [9].

Our results suggest that CBX might be inducing a U-shapeddose–response curve related to the incidence of GTCS. It has beenproposed that opposite effects at low, or high doses be related to

Fig. 3. Spectral power analysis after the treatment with CBX (50 nmol) and PTZ.(A) 3D graphic showing no changes on the spectral power along the time after theadministration of PTZ. (B) Representative EEG traces obtained in a 60-s epoch duringthe post-administration of CBX and PTZ. CBX inhibited the presence of EEG spikesand induced small synchronized bursting activity correlated with the short facialmyoclonus (dashes under the EEG trace).

42 J. Franco-Pérez et al. / Neuroscienc

Fig. 4. Spectral power analysis after the treatment with TMA (50 nmol) and PTZ.(A) 3D graphic showing high spectral power values at different frequencies(0–20 Hz) induced by the administration of PTZ. (B) Representative EEG tracesoip

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btained in a 60-s epoch during the post-administration of TMA and PTZ. TMAnduced prolonged GTCS characterized by a first tonic phase followed by a clonichase and the occurrence of one or more tonic phases.

isruption of homeostasis and even cellular toxicity [19]. In accor-ance with the toxicity hypothesis, it has been showed that highoses of CBX can exacerbate the neurological deficits in mice with

ntracerebral hemorrhage [20]. Interestingly, it has been demon-trated that similar doses of CBX (50 nmol) inhibit the epileptiformctivity induced by 4-aminopyridine when administering into theippocampus and entorhinal cortex [21]. Consequently, we suggesthat 50 nmol of CBX is an optimal dose to inhibit seizures inducedy diverse experimental models.

Since several years ago it has been proposed that gap junc-ions play an important role in neuronal synchronization and as

consequence on the epileptogenesis and the maintenance ofeizure events [22,23]. Overall, it is assumed that CBX elicits annticonvulsant effect through blocking the electrotonic couplingnd synchronization between neurons. Interestingly, it has beenhowed that the gap junction opener, TMA, into the entorhinal cor-ex elicits continuous epileptiform activity when is co-injected with-aminopiridyne [24]. Similar to this study, our data also revealedhat TMA increased the duration of the GTCS induced by PTZ.

Some authors have proposed that CBX block neuronal networkctivity by means of mechanisms different from gap junctions.pecifically, it was reported that CBX significantly decreases theeuronal excitability reducing the AMPA and NMDA-mediatedxcitatory postsynaptic currents in hippocampal cultures [25,26].

study demonstrated that blocking the NMDA receptors into thenC provoked a substantial inhibition of the GTCS and the behav-

oral signs of the epileptic seizures induced by PTZ [4]. Probably,he similarity with our results is related to the ability of CBX to

educe NMDA-mediated excitatory currents [26]. Although ourata are clarifying, more studies are necessary to include or ruleut the effects of CBX on gap junction communication and chem-

cal synapses. Meanwhile, we only can suggest that CBX exert a

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e Letters 602 (2015) 38–43

series of complex mechanisms on neuronal networks, which actingtogether, could be eliciting a significant inhibition of the PTZ-induced epileptiform activity.

Classical studies proposed that PRF be involved in the genera-tion and maintenance of generalized epileptic seizures [1–3]. Morerecently, it was determined that PRF neurons expressed a tonic neu-ronal firing related to some behavioral signs of epileptic seizuressuch as wild running and generalized myoclonus [27]. Even, it hasbeen proposed that ultrastructural alterations in PRF nuclei corre-late with the continued expression of generalized seizures inducedby 4-aminopyridine [28]. Recently, it was suggested that, in humanswith Lennox–Gastaut syndrome, the epileptic activity arise in cor-tical areas and quickly spread to the PRF through a corticopontinenetwork [29]. In accordance with our results, these studies also sug-gest that PRF nuclei could be participating in the generation andmaintenance of seizures.

5. Conclusion

Current data show that microinjection of the gap junctionblocker (CBX) into the PnC, provokes remarkable anticonvulsanteffects. Although more studies are necessary to determine theinvolved mechanisms, the opposite effects observed after themicroinjection of a gap junction opener (TMA), give us some evi-dence for the gap junctions as an underlying mechanism.

Conflict of interest

The authors declare that they have no conflicts of interest withrespect to the work included here.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.neulet.2015.06.037

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