Temporally unstructured electrical stimulation to the amygdala
suppresses behavioral chronic seizures of the pilocarpine animal model
Jasiara Carla de Oliveira
a, Daniel de Castro Medeiros
b, Gustavo Henrique de Souza e Rezende
b,
Márcio Flávio Dutra Moraes
b, Vinícius Rosa Cota
a,⁎
aLaboratório Interdiciplinar de Neuroengenharia e Neurociências, Departamento de Engenharia de Biossistemas (DEPEB), Universidade Federal de São João Del-Rei, Pça. Dom Helvécio, 74, 36301-160 São João Del-Rei, MG, Brazil
bNúcleo de Neurociências, Instituto de Ciências Biológicas (ICB), Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, CEP 31270-901 Belo Horizonte, MG, Brazil
a b s t r a c t
a r t i c l e
i n f o
Article history:
Received 17 January 2014 Revised 30 April 2014 Accepted 6 May 2014 Available online 13 June 2014
Keywords:
Temporal lobe epilepsy Pilocarpine
Electrical stimulation Temporal coding Basolateral amygdala Desynchronization Chronic seizures
Electrical stimulation applied to the basolateral amygdala in the pentylenetetrazole animal model of seizures may result in either a proconvulsant or an anticonvulsant effect depending on the interpulse intervals used: pe-riodic or nonpepe-riodic, respectively. We tested the effect of this electrical stimulation temporal coding on the spontaneous and recurrent behavioral seizures produced in the chronic phase of the pilocarpine animal model of temporal lobe epilepsy, an experimental protocol that better mimics the human condition. After 45 days of the pilocarpine-inducedstatus epilepticus, male Wistar rats were submitted to a surgical procedure for the im-plantation of a bipolar electrical stimulation electrode in the right basolateral amygdala and were allowed to re-cover for seven days. The animals were then placed in a glass box, and their behaviors were recorded daily on DVD for 6 h for 4 consecutive days (control period). Spontaneous recurrent behavioral seizures when showed in animals were further recorded for an extra 4-day period (treatment period), under periodic or nonperiodic electrical stimulation. The number, duration, and severity of seizures (according to the modified Racine's scale) during treatment were compared with those during the control period. The nonperiodically stimulated group displayed a significantly reduced total number and duration of seizures. There was no difference between control and treatment periods for the periodically stimulated group. Results corroborate previousfindings from our group showing that nonperiodic electrical stimulation has a robust anticonvulsant property. In addition, results from the pilocarpine animal model further strengthen nonperiodic electrical stimulation as a valid therapeutic approach in current medical practice. Our working hypothesis is that temporally unstructured electrical stimula-tion may wield its effect by desynchronizing neural networks involved in the ictogenic process.
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
Epilepsy is a chronic neurological disorder characterized by recur-rent and spontaneous seizures caused by hypersynchronous and exces-sive neural activity[1]. It has high prevalence, affecting aboutfifty million people worldwide[2]. Temporal lobe epilepsy (TLE) is the most common type of partial epilepsy[3], which accounts for about 60% of all patients[4]. It has a focal onset, and it is the most common type of drug-resistant epilepsy[5].
Intracranial electrical stimulation has long been considered a poten-tially viable therapy for patients with drug-resistant epilepsy who are not eligible for ablative surgery[6]. Currently, electrical stimulation (i.e., current or voltage pulses) may be applied to the peripheral nervous system, in structures such as the vagus nerve (vagus nerve stimulation)
[7]and the trigeminal nerve (trigeminal nerve stimulation)[8], or di-rectly to the central nervous system, in substrates such as the anterior thalamic nucleus[9], subthalamic nuclei[10], and epileptogenic focus itself[11].
Although the literature reports an overwhelming amount of data showing the effect of electrical stimulation on seizure suppression (for a review, see[6]), its mechanisms of action on neural network modula-tion need further investigamodula-tion. While debatable, the most widely accepted framework for its therapeutic effectiveness posits that electri-cal stimulation would recruit substrates and/or neural networks capable of modulating seizure-like activity in areas involved in ictogenesis or rather by impairing the coupling of neural oscillators necessary to prop-agate and sustain aberrant activity[12,13]. Among others, in silico stud-ies have shown that neural circuits thatfire synchronously are coupled in a positive feedback fashion[14], that synaptic weights increase in proportion to the coincidence in neuronalfiring[15], corroborating
Hebbian postulates[16], and that this coincidence may be increased or decreased by a synchronizing or desynchronizing electrical stimulation, respectively[15]. Finally, Medeiros et al. have shown that electrical
⁎ Corresponding author at: DEPEB, Pça. Dom Helvécio, 74, B. Fábricas, 36301-160 São João Del-Rei, MG, Brazil. Tel.: +55 32 3379 2541 (office), +55 32 8861 8074 (mobile).
E-mail addresses:vrcota@ufsj.edu.br,vrcota@pq.cnpq.br,vrcota@gmail.com
(V.R. Cota).
http://dx.doi.org/10.1016/j.yebeh.2014.05.005
1525-5050/© 2014 Elsevier Inc. All rights reserved.
Contents lists available atScienceDirect
Epilepsy & Behavior
stimulation may drive the temporal occurrence of preictal oscillatory neural networks; that is, cortical preictal discharges gradually synchro-nize with electrical stimulation minutes before seizure onset[17].
In recent investigations, we approached this issue by testing the hy-pothesis that afixed four-stimuli-per-second electrical stimulation in the amygdaloid complex would modulate convulsive behavior of rats with acute pentylenetetrazole (PTZ)-induced seizures according to the temporal pattern used: structured (constant interpulse intervals or periodic — PS) or nonstructured (random interpulse intervals or
nonperiodic—NPS). It is important to highlight that the average fre-quency used (4 pulses per second) is substantially lower than what is usually considered to be anticonvulsant[6]. It was shown that PS is proconvulsant and NPS is anticonvulsant, suggesting that a putative synchronization/desynchronization effect of structured/nonstructured electrical stimulation may be an underlying mechanism of action[18]. In addition, these results corroborate the notion that reverberation of neural networks has an important role in ictogenesis.
Based on the promising results obtained with the use of a nonstruc-tured temporal pattern in the suppression of acutely induced seizures and on the urge to develop safe and efficient therapeutic alternatives to treatment-resistant epilepsy such as TLE, in this study, we set out to investigate the hypothesis that NPS may also have a positive effect on animal models that best mimic this clinical scenario. For this, we tested NPS applied to the amygdaloid complex of the pilocarpine animal model during chronic seizures. Pilocarpine is a cholinergic agonist that binds to muscarinic receptors to increase cholinergic excitatory neurotransmis-sion. When administered systemically in high doses, it induces limbic seizures that become generalized and are associated withstatus epilep-ticus(SE) in rodents[19–22]. Such a state of enduring seizures induces epileptogenesis due to large cellular reorganization that culminates in permanent late neural tissue hyperexcitability[23–35]. Due not only to the similarities between pathophysiological mechanisms but also to the behavioral manifestation of its spontaneous and recurrent chronic seizures, the pilocarpine animal model is considered an experimental protocol that mimics human TLE. The amygdala was used as a target for electrical stimulation because it has an important role in the cou-pling of neural oscillators within the limbic system during seizures [18,23,24,26,27]and also in the activation of modulatory circuits such as the nucleus accumbens–temporal lobe[36,37].
2. Materials and methods
2.1. Animals and groups
All experiments were done in accordance with the Ethical Commit-tee for Animal Experimentation (Comitê de Ética em Experimentação Animal — CETEA) of the Federal University of Minas Gerais
(Universidade Federal de Minas Gerais—UFMG) and with the Ethical Committee on Research Involving Animals (Comitê de Ética em Pesquisa Envolvendo Animais—CEPEA) of the Federal University of
São João del Rei (Universidade Federal de São João del Rei—UFSJ). The procedures for animal care were previously approved by these organizations under protocols 150/2006/UFMG and 01/2011/UFSJ. A total of 14 male Wistar rats, weighing 250–300 g, supplied by UFMG (n = 8) and UFSJ (n = 6) vivariums, were kept in a light–dark cycle of 12 h (lights on at 7 am and off at 7 pm) with free access to food and water.
The animals were randomly divided into two groups: NPS (n = 7) received nonperiodic stimulation and PS (n = 7) received periodic stimulation. After a recovery period of 5 to 7 days, the animals were placed in a glass box with free access to food and water, and their behav-iors were recorded on DVD for 6 h (10 am to 4 pm) daily for 4 consecu-tive days (control period—CRTL). Animals that showed spontaneous
recurrent seizures underwent electrical stimulation (pattern according to group) and video recording for an extra 4 days (treatment period).
2.2. Status epilepticus induction
Status epilepticuswas induced in the pilocarpine animal model by
first applying methylscopolamine (1 mg/kg; Sigma Aldrich, St. Louis, MO, USA) by means of an intraperitoneal (i.p.) injection, followed 30 min later by pilocarpine hydrochloride (320 mg/kg; Sigma Aldrich, St. Louis, MO, USA) i.p. injection. Methylscopolamine is a cholinergic antagonist that does not cross the blood–brain barrier and was used to avoid undesirable systemic effects of the cholinergic stimulation in-duced by pilocarpine (i.e., excessive defecation, urination, sweating, and bronchial secretion). Animals that did not develop SE during the
first 30 min after pilocarpine injection received an overdose of 40% of
the initial dose. Animals that did not develop SE even with the overdose were excluded from the study. At the 90-minute mark, SE was interrupted by i.p. injection of diazepam (20 mg/kg; Laboratório Teuto Brasileiro, Anápolis, GO, Brazil). The animals received intensive care, including rehydration with dextrose (2 mL) i.p., during 48 h after the SE induction to ensure survival.
2.3. Stereotaxic procedures
Between 45 and 50 days after the SE induction, the animals underwent a surgical procedure for implantation of a bipolar stimula-tion electrode in the right basolateral amygdala. Electrodes were made of a twisted pair of stainless-steel teflon-coated wires (model 791400, A-M Systems Inc., California, USA) and were surgically implanted at coordinates derived from the Paxinos and Watson's atlas for rats[38]: AP = 2.8 mm and ML = 5.0 mm referenced from the bregma suture and DV = 7.2 mm from dura mater.
Briefly, the animals were anesthetized by means of an i.p. injection
containing the mixture of ketamine (100 mg/kg—König do Brasil,
Santana do Paraíba, SP, Brazil), xylazine (5 mg/kg—Syntec do Brasil, Cotia, SP, Brazil), and fentanyl (0.025 mg/kg—Union Chemical do Brasil, Londrina, PR, Brazil). After hair shaving and proper asepsis proce-dures, the animals were positioned in a stereotaxic frame (Insight Equipamentos Ltda, Ribeirão Preto, SP, Brazil). The electrode wasfixed to the skull with zinc cement and soldered to a telephone jack (model RJ-11), which wasfixed onto the skull with dental acrylic. After surgery, the animals received a prophylactic pentabiotic (2.5 mg/kg) treatment and were allowed to recover for 5–7 days before the experimental
procedure.
2.4. Electrical stimulation
For stimulation delivery, we designed and built an electrical stimula-tor composed of a constant-voltage isolation unit driven by the output of an MP3 player (model NWZ-B152 26B, Sony). Control signals for both periodic electrical stimulation and nonperiodic electrical stimula-tion were digitally designed using Adobe Audistimula-tion 1.0 and transformed into a 44.1 KHz, 16-bit, mono-waveform, MP3 format compatible with the D/A hardware output. Pulses were always square, biphasic waves of 100μs duration. Current amplitudes, measured by a built-in shunt resistor, varied from 400 to 600μA due to differences in the impedance of electrode–brain sets among the animals. Yet, there was no difference in average currents between periodic and nonperiodic groups. Pulses were biphasic to prevent electrodeposition along the extended period of stimulation and subsequent tissue damage, as reported in the litera-ture[39,40].
2.5. Behavioral analysis
The number, duration, and severity–according to the modified Racine's scale[41]–of seizures, during control and treatment periods of both groups, were assessed by two separate and experienced researchers. Their independent results were compared, and reproduc-ibility of data was assessed in order to guarantee minimum subjectivity or bias. Between researchers, there was absolutely no difference in measures of the severity and number of seizures, while the duration of seizure measurements differed no more than 2 s. In this last case, we used the average between measures. Only animals displaying sei-zures with Racine's index greater than or equal to 3 during the control period were used to computefinal results.
2.6. Histology
After the end of stimulation, the animals were deeply anesthetized with ketamine (100 mg/kg) and xylazine (5.0 mg/kg) and were transcardially perfused with formaldehyde (4%) before brain removal. Coronal sections of 50μm thickness were cut on a vibratome (Leica), mounted on glass slides, and stained with cresyl violet. Animals with in-correct positioning of electrodes were not included in our analysis.Fig. 2 shows a representative location of the electrode tip.
2.7. Statistical analysis
The Kolmogorov–Smirnov normality test and Student t test were used to evaluate parametric data (number and duration of seizures)
and Wilcoxon test to evaluate nonparametric data (severity of seizures). Statistical significance was set at pb0.05. Values in the text are
displayed as means ± standard error.
3. Results
All animals had histological confirmation of the positioning of
elec-trode in the BLA. Only one animal was excluded from the study because it had no spontaneous and recurrent behavioral seizures during the control period (first 4 days of experimental protocol).
Seizures were observed during control and stimulation periods. The animals developed the typical behaviors of the pilocarpine animal model: myoclonic jerks, forelimbs and head clonus, rearing, and gener-alized tonic–clonic seizures (GTCS).
During treatment when compared to the control period, NPS was able to significantly reduce the total number (CTRL: 3.8 ± 1.3; NPS: 1.3 ± 0.6; pb0.05) (Fig. 3A) and duration (CTRL: 26.6 ± 2.1; NPS:
8.3 ± 3.9; pb0.05) (Fig. 3B) of seizures. Seizure severity in the
treat-ment period did not show significant difference (CTRL: 4.4 ± 0.3; NPS: 2.5 ± 0.9; p = 0.1077) (Fig. 3C).
In the animals of the periodically stimulated (PS) group, there were no statistically significant differences between control and treatment periods regarding any of the analyzed parameters. Averages and their respective standard errors were the following: total seizure number (CRTL: 1.7 ± 1.7; PS: 5.4 ± 6.1; p = 0.1359) (Fig. 4A), seizure duration (CTRL: 20.2 ± 21.4; PS: 33.4 ± 27.1; p = 0.2675) (Fig. 4B), and seizure severity (CTRL: 3.1 ± 2.2; PS: 3.5 ± 1.7; p = 0.7984) (Fig. 4C).
Fig. 1.Rats were stimulated with two different temporal patterns. Left column: interpulse interval (IPI) histograms for: (A) periodic (PS) and (C) nonperiodic (NPS) electrical stimulation. Right column: temporal distribution of pulses every second (vertical ticks) for PS (B) and NPS (D). Electrical stimulation for both patterns was bipolar pulses of 400 to 600μA amplitude,
100μs duration, and four-stimuli-per-second pulse count (see Cota et al.[18]for details).
4. Discussion
Our present results show that low pulse per second count, temporal-ly unstructured, electrical stimulation (nonperiodic or NPS) applied to the basolateral amygdala has a positive therapeutic effect on behavioral seizures of the pilocarpine animal model of temporal lobe epilepsy (TLE), being able to reduce their number and duration during the chron-ic phase. These results corroborate previousfindings from our group using the acute pentylenetetrazole (PTZ) animal model[18]and extend them to another important animal model used in epileptology (i.e., pilocarpine).
Previous studies reported the effect offixed frequency electrical stimulation on a pilocarpine or kainate animal model, but they were not accomplished in the chronic phase or did not show positive thera-peutic effects. Hamani et al. showed that bilateral stimulation (100 Hz) of the anterior thalamic nuclei was protective against SE in-duced by pilocarpine[42]. Jou et al. also found that anterior thalamic stimulation with high-frequency (200 Hz) and low-intensity currents may reduce the occurrence of seizure and SE[43]. Finally, Lado showed that high-frequency electrical stimulation (100 Hz) led to an increase in the frequency of seizures in animals with chronic epilepsy that received kainic acid[44]. To the best of our knowledge, there are no studies of electrical stimulation during the chronic phase of the pilocarpine animal
model targeting the amygdala or, most importantly, using unstructured temporal patterns with a low count of pulses per second.
The pathophysiology of the pilocarpine animal model is a complex, multivariate phenomenon, yet sharing many common mechanisms with human TLE[26]. Ultimately, molecular and cellular modifications lead to a state of highly excitable neural tissue, susceptible to developing spontaneous and recurrent seizures, that is similar to what is observed in humans affected with TLE[45]. Structures within the limbic system, prominently the hippocampus and the entorhinal cortex, are among the most affected neural substrates[23,46,47]. Aberrant epileptiform activity originating in a group of hyperexcitable neurons (epileptogenic focus) can propagate through aberrant pathological or, potentially, even apparently normal pathways in the central nervous system[48], cou-pling different brain areas for the full epileptic event by means of syn-chronization of activity[14,49,50]. In vitro studies in healthy neural tissue demonstrated that incoming activity from the entorhinal cortex enters the hippocampus through the perforant path passing the dentate gyrus to reach Ammon's horn via mossyfibers and Schaffer collaterals [51]. Suchflow of information seems to be powerfully modulated by
amygdala outputs to deep layers of the entorhinal cortex[26,27]. In pi-locarpine-treated animals, epileptiform activity may also propagate from the entorhinal cortex to the hippocampus laterally through the temporoammonic path to reach CA1[52,53]. Moreover, hippocampal outputs back to the entorhinal cortex form a reentrant circuit crucial for sustaining seizures in a reverberatory process[24,49,54–57]. Fig. 3.Number (A), duration (B), and severity (C) of seizures, according to the modified
Racine's scale, in the nonperiodically stimulated group during treatment period compared to its control period (CTRL). The number and duration of seizures was significantly decreased by NPS.
The present data do not allow elucidation of the mechanisms by which NPS attains its therapeutic effect. Yet, considering the importance of synchronization to ictogenesis and known correlations between be-havior and neural activation[58,59], we believe that NPS impairs the coupling of micro-oscillators within the entorhinal cortex to those in the hippocampus by imposing unstructured rhythms in the deep layers of the entorhinal cortex targeted by BLA outputs (connections in the limbic system of pilocarpine-treated animals are preserved[60]), and, thus, it suppresses seizures. Within this theoretical framework, the de-crease in the number and duration of seizures attained by NPS could be explained by a putative impairment of coupling and synchronization of micro-oscillators within the limbic system, crucial for limbic seizure initiation, propagation, and reverberation[61], by avoiding temporal co-incidence among activities of different substrates[14,49,62]. A real-time measurement of neural activity, such as recordings of localfield
poten-tial (or electroencephalogram), is necessary in order to clarify the role of each temporal pattern in the synchronization or desynchronization of these micro-oscillators and to more definitely state if such a mechanism is really underlying the observed effects.
Periodic stimulation (PS) did not show a clear proconvulsant effect, although there seemed to be an increasing trend in the number of sei-zures. It is plausible to believe that thefixed frequency used (4 Hz) is
not the proper stimulus design for precipitating seizures in the pilocar-pine animal model opposed to what has been demonstrated in PTZ-treated animals. On the other hand, the clear lack of an anticonvulsant effect of PS states that the therapeutic effect of NPS is not due to its low-frequency content.
NPS has both low frequency content and high frequency content as can be observed in its histogram inFig. 1. We ruled out the effect of high band in the anticonvulsant effect observed through experiments in a previous original work published in 2009[18]. There, we tested, in animals with PTZ-induced seizures, one-per-second 50-Hz bursts of electrical stimulation, with 4 pulses each, and they showed no anticon-vulsant effect. Finally, we also tested a variation (called LH) of the nonperiodic (temporally unstructured) pattern of electrical stimulation, obtained by a different computational algorithm, in addition to the one that is also used in the present work (called IH). We observed that LH variation results were no different from control, while IH had the anti-convulsant effect. It is important to stress that all these experiments used the very same pulse parameters (duration, amplitude, count per second, polarity, etc.) among groups and only the temporal coding was different. Particularly, this approach also rules out lesion-induced effects. If this were the case, all groups would display similar anticonvul-sant or even proconvulanticonvul-sant effects once the very same lesion-inducing charge (zero net charge in the present study once pulses are biphasic) was delivered in each case. It is also important to highlight that NPS had an anticonvulsant effect on dysfunctional neural tissue marked by aberrant exacerbated connectivity and excitability, including poor GABAergic inhibition[63]. This suggests that NPS does not act directly upon neurotransmitter systems; rather, it may have an effect on mech-anisms at the circuit level.
Taken together, these results led us to the conclusion that the temporal coding, rather than any other factor, is the responsible pa-rameter for the anticonvulsant effect. Moreover, these results and their plausible explanation are in agreement with previous results of our group, using noninvasive imaging, that synchronization and desynchronization of neural substrates may be determinant factors in ictogenesis and its suppression by NPS[37], respectively.
5. Conclusions
In this work, we showed that temporally unstructured (nonperiodic) electrical stimulation applied to the basolateral amygdala, even with a low count of pulses per second, is capable of decreasing the number and duration of spontaneous recurrent behavioral seizures of the rats during the chronic phase of the pilocarpine animal model, while periodic
stimulation has no clear effect. We believe these results to be of major importance because of three main reasons: 1) they corroborate previ-ousfindings of our group using the acute PTZ animal model[18], dem-onstrating that such temporal pattern has a robust effect in controlling seizures; 2) they add empirical evidence to the putative role of syn-chronization and desynsyn-chronization as underlying mechanisms of ictogenesis and its suppression, respectively; and 3) they clearly show that temporally unstructured electrical stimulation has a therapeutic effect even on dysfunctional hyperexcitable neural tissue susceptible to develop and sustain aberrant epileptiform activity.
Considering the common features of the pilocarpine animal model of epilepsy and human temporal lobe epilepsy (spontaneous recurrent sei-zures and limbic pathophysiology), these present results suggest that temporally unstructured electrical stimulation of the basolateral amyg-dala should be considered as a therapeutic approach in clinical trials.
Conflict of interest
The authors state that no other people or organization have inappro-priately influenced this work. Therefore, there is no pertinent claim of a conflict of interest.
Acknowledgments
We are grateful to Brazilian agencies CNPq (grants #484588/2011-7 and #484704/2012-5) and FAPEMIG (grant #APQ 01818-12) forfi nan-cial support. Márcio Flávio Dutra Moraes is a recipient of CNPq research fellowship.
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