Effect of low frequency repetitive transcranial magnetic stimulation on kindling-induced changes in electrophysiological properties of rat CA1 pyramidal neurons.

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Effect of low frequency repetitive transcranial magnetic stimulation on kindling-induced changes in electrophysiological properties of rat CA1 pyramidal neurons Q1

Homeira Moradi Chameha, Mahyar Janahmadib, Saeed Semnaniana, Amir Shojaeia, Javad Mirnajafi-Zadeha,n a

Department of Physiology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Neuroscience Research Centre and Department of Physiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

b

art i cle i nfo

ab st rac t

Article history:

In this study, the effect of repetitive transcranial magnetic stimulation (rTMS) on the

Accepted 13 February 2015

kindling induced changes in electrophysiological firing properties of hippocampal CA1 pyramidal neurons was investigated. Male Wistar rats were kindled by daily electrical

Keywords:

stimulation of the basolateral amygdala in a semi-rapid manner (12 stimulations/day) until

Electrophysiological properties

they achieved stage-5 seizure. One group (kindledþrTMS (KrTMS)) of animals received

Kindling

rTMS (240 pulses at 1 Hz) at 5 min after termination of daily kindling stimulations. Twenty-

Amygdala

four hours following the last kindling stimulation electrophysiological properties of

Repetitive transcranial magnetic

hippocampal CA1 pyramidal neurons were investigated using a whole-cell patch clamp

stimulation

technique, under current clamp condition. Amygdala kindling significantly decreased the Q2

Antiepileptogenic

adaptation index, post-afterhyperpolarization, rheobase current, utilization time, and delay to the first rebound spike. It also caused an increase in the voltage sag, number of rebound spikes and number of evoked action potential. Results of the present study revealed that application of rTMS following kindling stimulations had antiepileptogenic effects. In addition, application of rTMS prevented hyperexcitability of CA1 pyramidal neurons induced by kindling and conserved the normal neuronal firing. & 2015 Published by Elsevier B.V.

n Correspondence to: Department of Physiology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, 14117-13116 Tehran, Iran. Fax: þ98 21 82884528. E-mail address: [email protected] (J. Mirnajafi-Zadeh).

http://dx.doi.org/10.1016/j.brainres.2015.02.023 0006-8993/& 2015 Published by Elsevier B.V.

Please cite this article as: Chameh, H.M., et al., Effect of low frequency repetitive transcranial magnetic stimulation on kindling-induced changes in electrophysiological properties of rat CA1 pyramidal neurons. Brain Research (2015), http://dx.doi. org/10.1016/j.brainres.2015.02.023

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1.

Introduction

Temporal lobe epilepsy (TLE) is one of the most prevalent forms of epilepsy, which is a common chronic neurological disorder (Browne and Holmes, 2001). About one-third of patients with TLE are pharmacoresistant and do not respond to current treatments (Sasa, 2006).Therefore, much effort has been put towards finding new treatments for drug resistant epilepsy. Such efforts have led to development of the repetitive transcranial magnetic stimulation (rTMS) as an alternative therapy for pharmacoresistant epileptic patients (Kimiskidis, 2010). rTMS is a noninvasive brain stimulation method (Barker et al., 1985), where the coils put over the top of cortical and subcortical regions, resulting in long term changes in their neural activity and overall excitability (Fregni and PascualLeone, 2007; Mally and Stone, 2007; Post and Keck, 2001; Ridding and Rothwell, 2007; Walsh and Cowey, 2000). Previous studies on human showed that application of lowfrequency rTMS led to decrement in neuronal excitability and had anticonvulsant effect (Bae et al., 2007; Chen et al., 1997; Fregni et al., 2005; Hsu et al., 2011; Joo et al., 2007; Sun et al., 2011, 2012; Tergau et al., 1999). The anticonvulsant action of rTMS has been also observed in laboratory models of epilepsy, including amygdala kindling (Akamatsu et al., 2001; Anschel et al., 2003; Fleischmann et al., 1999; Lin et al., 2014; Mongabadi et al., 2013; Rotenberg et al., 2008; Shojaei et al., 2014; Yadollahpour et al., 2014). The mechanisms of these effects may be similar to those involved in induction of longterm depression or depotentiation (Chen et al., 1997; Hallett, 2000; Hoffman and Cavus, 2002; Kobayashi and PascualLeone, 2003; Tokay et al., 2009). Amygdala kindling results in synaptic potentiation (Teskey et al., 2002). This potentiation induces changes in the electrophysiological properties of neurons and synaptic transmission in epileptic focus and areas which are important in seizure development and progression (Kemppainen and Pitkanen, 2004), including the hippocampus. There are anatomical and functional connections between hippocampus and amygdala (Lopes da Silva et al., 1990; Witter et al., 1989; Witter, 1993). It has been suggested that hippocampus has a role in initiating, maintaining and probably terminating the amygdaloid kindled seizure (Namba et al., 1991), and several alterations in the structure or biochemistry seen following amygdala kindling have been observed in hippocampus (Morimoto et al., 1997).It has been shown that amygdala kindling is suppressed when degenerations are observed in bilateral hippocampus (Dasheiff and McNamara, 1982; Tanaka et al., 1991; Yoshida, 1984). In vivo studies have shown that induction of long-term potentiation in hippocampus affects long-term potentiation in the amygdala and vice versa (Ikegaya et al., 1994; Maren and Fanselow, 1995). Therefore, the anticonvulsant effects of rTMS in amygdala kindling may be mediated through preventing the kindlinginduced changes in electrophysiological properties of hippocampal CA1 neurons. rTMS may also induce new changes in the electrophysiological properties of these neurons to reduce their hyperexcitability. In the present study we examined the effect of rTMS applied during amygdala kindling procedure

on changes in electrophysiological properties of hippocampal CA1 pyramidal neurons.

2.

Results

The afterdischarge threshold (123.3723.9 mA in kindled and 111.7722.42 mA in KrTMS groups) and afterdischarge duration after the first kindling stimulation (10.1970.72 s in kindled and 9.8970.31 s in KrTMS groups) had no significant difference in kindled and KrTMS groups. Therefore, the seizure susceptibility was similar in these two groups at the beginning of experimental procedure. Because the animals of kindled group reached stage 5 seizure after 670.44 days of kindling procedure, kindling stimulations and/or rTMS were applied in KrTMS and rTMS groups for 6 days. Seizure stage and afterdischarge duration were decreased in KrTMS compared to kindled group. Whole cell patch clamp recordings were obtained to investigate the effect of rTMS on kindling induced changes in the electrophysiological properties of hippocampal CA1 pyramidal neurons using depolarizing, hyperpolarizing and ramp current injections. In hippocampal slices of kindled rats (n ¼15), adaptation index in response to injection of depolarizing currents (200–500 pA, 650 ms) significantly decreased compared to control group (n¼ 15) (po0.001). Application of rTMS following kindling stimulations in KrTMS group (n ¼14) prevented the decrease in adaptation index, so that there was no significant difference between KrTMS and control group (Fig. 1A and C). The amplitude of post-AHP in response to depolarizing current steps (100–500 pA, 650 ms) was also measured. In kindled rats (n ¼7), it was significantly decreased in response to 400 and 500 pA depolarizing currents (po0.05 and po0.01 respectively), but not in response to 100–300 pA, when compared to control group (n¼ 7). However, when rTMS was applied following kindling stimulations (KrTMS group, n ¼12) no decrement was observed in the amplitude of postAHP in response to 500 pA depolarizing pulse (po0.01) and there was no significant difference between KrTMS and control groups (Fig. 1B and D). As Fig. 1A shows, there was a significant difference between instantaneous frequency of the first four spikes in kindled (46.3976.73 Hz) and control (20.371.88 Hz) groups following a 200 pA depolarizing current. However, application of rTMS during kindling procedure could not prevent the kindling-induced increase in this parameter (42.4275.11 Hz in KrTMS group). The same results were also observed after injection of higher depolarizing currents (300–500 pA; data not shown). To evaluate the effect of rTMS on the kindling induced changes in neuronal excitability, a ramp current pulse was applied under current clamp condition. The rheobase current was significantly decreased in kindled (n ¼23) compared to control group (n ¼11) (po0.00 1). rTMS administration following kindling stimulations(KrTMS group) (n¼ 15) had an inhibitory effect on kindling-induced changes in rheobase; so that there was no significant difference between rheobase current in KrTMS and control groups (Fig. 2A and B). Similarly, application of rTMS prevented the kindling-induced decrease


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Fig. 1 – Effects of kindling stimulation and low frequency rTMS on adaptation index and post-AHP. (A) Sample of voltage traces from CA1 pyramidal neurons in response to 500 pA depolarizing current steps in different groups. (B) The same records have been shown in a new scale to show better differences in post-AHP amplitude in difference groups. The tip of spikes has been cut. (C) The adaptation index and (D) post-AHP reduced in kindled rats (n ¼15) compared to control group (n ¼15) but, application of rTMS in KrTMS group (n ¼ 14) prevented the decrement of these parameters. rTMS alone (n ¼10) had no significant effect on these parameters. *po0.05, **po0.01 and ***po0.001 compared with control group.þpo0.05, þþþpo0.001 compared with KrTMS group.

of utilization time (the time needed to generate action potential). There was a significant difference between utilizing time in kindled (n ¼23) and KrTMS groups (n¼ 15) (po0.001), but no difference observed between control and KrTMS groups (Fig. 2A and C). In addition, in slices obtained from kindled rats (n¼23) the number of evoked action potentials in response to depolarizing ramp current injection was significantly increased compared to control group (n¼ 11) (po0.001) and application of rTMS significantly decreased it in KrTMS rats (n¼ 15) (po0.001) )Fig. 2A and D). We also measured the number of rebound spikes following hyperpolarizing current steps (100–500, 650 ms). As shown in Fig. 3A

and B, in slices from kindled rats (n¼ 18) the number of rebound spikes was significantly increased in comparison to control group (n ¼13) (po0.01). In addition, the latency to generation of rebound spikes was shorter in kindled compared to control group (po0.01) (Fig. 3A and C). However, rTMS application in KrTMS group prevented the shortening effect of kindling on the first spike latency (n ¼15). In hippocampal CA1 pyramidal neurons, a voltage sag can be observed following hyperpolarization of the membrane. The amplitude of voltage sag was increased in kindled rats (n¼ 18) compared to control (n¼ 13), but rTMS prevented the kindling induced-changes in voltage sag when administered


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Fig. 2 – Effects of kindling stimulation and low frequency rTMS on number of action potential, rheobase current and utilization time following depolarizing ramp protocol. (A) Sample of voltage traces from CA1 pyramidal neurons in response to depolarizing ramp current in different groups. (B) Changes in rheobase, (C) in utilization time and (D) number of action potentials in different experimental groups. Kindled group (n¼ 23) required less rheobase current and utilization time but higher number of action potentials compared to control group (n¼ 11). rTMS application (KrTMS group; n ¼15) prevented kindling-induced changes in these parameters. Interestingly, effects of rTMS alone (n ¼10) were similar to kindling-induced changes. *po0.05, **po0.01 and ***po0.001.

following kindling stimulations in KrTMS group (n¼15) (Poo0.00) (Fig. 3A and D).

3.

Discussion

Results of the present study revealed that application of rTMS following kindling stimulations had antiepileptogenic effects. In addition, rTMS prevented kindling induced hyperexcitability in the hippocampal CA1 region and conserved the normal firing properties of CA1 pyramidal cells. Several studies have shown that after low frequency rTMS treatment in epileptic patients the number of interictal spikes on EEG decreases significantly (Brodbeck et al., 2010; Fregni et al., 2005; Joo et al., 2007; Menkes and Gruenthal, 2000; Santiago-Rodriguez et al., 2008) and this effect lasts approximately for two months (Misawa et al., 2005). The anticonvulsive action of rTMS has also been shown in laboratory animals

(Akamatsu et al., 2001; Mongabadi et al., 2013; Tokay et al., 2009; Yadollahpour et al., 2014); however, a few studies have been done to determine the effect of rTMS on neuronal excitability. Previous study in our lab showed that application of rTMS following kindling stimulations prevents the kindlinginduced potentiation in the synaptic plasticity (Yadollahpour et al., 2014). In addition, it decreases the seizure-induced changes in some electrophysiological properties of hippocampal CA1 pyramidal neurons (Shojaei et al., 2014). To complete and confirm our previous studies other electrophysiological parameters were addressed in this study. In the present experiment, following amygdala kindling, the neuronal excitability of hippocampal CA1 pyramidal neurons was increased. The minimum current to evoke action potential (rheobase), utilization time, adaptation index, amplitude of post-AHP and the delay to the first rebound spike decreased, but the number of action potentials in response to a depolarizing current ramp, the number of rebound spikes


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Fig. 3 – Effects of kindling stimulation and low frequency rTMS on amplitude of voltage sag, delay of first rebound spike, and number of rebound spikes following hyperpolarizing protocol. (A) Sample of voltage traces from CA1 pyramidal neurons in response to hyperpolarizing current in different groups. (B) Changes in number of rebound spikes in response to 400 pA (left) and 500 pA (right), (C) changes in delay of first rebound spike and (D) in amplitude of voltage sag in different experimental groups. In kindled group (n ¼18) the number of rebound spikes and amplitude of voltage sag increased, but delay of first rebound spikes decreased compared to control (n ¼13) significantly; all of these changes returned to normal situation in KrTMS group (n ¼15). rTMS alone (n ¼ 11) had no significant effect on the above parameters. *po0.05, **po0.01 and ***po0.001 compared with control group. þþpo0.01 and þþþpo0.001 compared with KrTMS group.

and the amplitude of voltage sag increased. Application of rTMS almost preserved the firing properties of CA1 pyramidal neurons. In kindled animals, decrease in the rheobase current, utilization time and increase in the number of action potentials during ramp current injection may reflect the possible changes in sodium channel kinetic or density. Voltage-gated sodium channels are crucial in the generation of action potential and excitability (Offord and Catterall, 1989; Waxman et al., 2000). Previous studies have revealed that mutations in voltage gated sodium channels can cause genetic forms of epilepsy (Berkovic Q3 et al., 2006; Mulley et al., 2005). The expression of four voltage gated sodium channel subtypes (NaV1.1, NaV1.2, NaV1.3 and

NaV1.6) and persistent sodium current increase in the hippocampus and temporal lobe cortex following spontaneous epileptic discharges. These changes may be involved in the generation of epileptiform activity and participate in neuronal excitability(Berkovic et al., 2006; Blumenfeld et al., 2009; Mulley et al., 2005; Waxman et al., 2000; Xu et al., 2013). In addition, it has been shown that the number of sodium channels available for activation will increase due to enhancement of sodium current inactivation and it can result in increasing excitability of pyramidal cells after kindling (Vreugdenhil et al., 1998). Therefore, the increases in the rheobase current and utilization time and a decrease in the number of action potentials after application of rTMS in


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kindled rats could be due to a decrease in the availability of voltage-gated sodium channels (Gu et al., 2007; Shruti et al., 2008). The amplitude of post-AHP following the depolarizing current pulse was also measured as an index of Ca2þ-activated Kþ channels function. The AHP repolarizes neurons following burst firing activity. It has been reported that blockade of AHP makes neurons more responsive to excitatory stimuli (Madison and Nicoll, 1984, 1986).The decrease of post-AHP amplitude in kindled animals may contribute to the enhanced excitability of the CA1 neurons and may act to promote synchronous neuronal firing within these neurons (Asprodini et al., 1992). Application of rTMS following kindling stimulation prevented the decrement of post-AHP amplitude so that there was no significant difference between KrTMS and control groups. However, the mechanism of this effect needs to be investigated further using voltage clamp. The other parameter which may be affected by Ca2þactivated and/or M (muscarine-sensitive) Kþ-currents is adaptation index. Adaptation is the ability of a neuron to decrease action potential firing in response to a long depolarizing current injection. Drugs that reduce these currents have been shown to decrease adaptation and AHP (Figenschou et al., 1996; Storm, 1990). Application of rTMS following kindling could completely prevent the kindling-induced changes in the adaptation index. According to changes observed in post-AHP amplitude and adaptation index it is possible to consider a possible role for Kþ channels in mediating the preventing effect of rTMS on neuronal excitability. However, application of rTMS in KrTMS group had no significant effect on instantaneous frequency of the first four spikes. This phenomenon is mediated by different Kþ currents including SK (Pedarzani et al., 2005), BK and M-type (Gu et al., 2007), A-type (Lien et al., 2002) and D-type (Storm, 1990). Therefore, more experiments need to shed light on the role of Kþ channels in rTMS antiepileptogenic effects. Kindling stimulation significantly increased the number and decreased the latency of rebound spikes which were rarely observed in control slices, as reported previously (Lien et al., 2002). These spikes have a crucial role in persistent activity and oscillatory dynamics (Bottjer, 2005) and in most neurons are due to the activity of transient calcium and sodium currents (McCormick and Huguenard, 1992; Zhang et al., 2004). In addition, these currents can increase the activity of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels which has a significant effect on the frequency of rebound spike Q4 and first rebound latency per se (Biel et al., 2009). It seems that rebound spikes in CA1 pyramidal neurons may be generated under pathological conditions as it has been described in febrile models of seizures (Chen et al., 2001) or after perisomatic GABAergic inhibition (Buhl et al., 1994). In KrTMS group, the number of rebound spikes decreased and their latency increased toward control values, so that there was no significant difference between these parameters in KrTMS and control group. However, rTMS application could prevent these changes. The other aspect of ameliorative effect of rTMS was its preventing effect on kindling induced increase of voltage sag. The size of the voltage sag is an index of the degree of activation of HCN channels. These channels are activated by hyperpolarization of membrane potential and are permeable to Naþ and Kþ. The cationic current flowing through HCN

channels (known as Ih) causes membrane to be depolarized toward threshold for action potential generation, and reduces membrane resistance. Therefore, Ih currents have a main role in regulating neuronal excitability, integration of synaptic potentials in dendrites, synaptic transmission (Kase and Imoto, 2012), and rhythmic oscillatory activity in individual neurons and neuronal networks (Maccaferri and McBain, 1996; McCormick and Pape, 1990). Experimental evidence indicates that abnormal regulation of HCN expression or function has been implicated in epilepsy (Noam et al., 2011). Altered HCN1 or HCN2 expression has been demonstrated in hippocampus of patients with severe temporal lobe epilepsy (Bender et al., 2003). However, the role of HCN disorders in epilepsy is complex. Studies in experimental models indicate that both up-regulation (Dyhrfjeld-Johnsen et al., 2008; Gill et al., 2006) and down-regulation (Powell et al., 2008; Richichi et al., 2008; Santoro et al., 2010; Shah et al., 2004; Strauss et al., 2004) of HCN channels can be associated with seizures. Our data were Q5 clearly inconsistent with an increase in Ih current during the neuronal hyperexcitability in CA1 region of the hippocampus. Therefore, preventing the increase in voltage sag by rTMS may be partly accounted for its possible antiepileptogenic effect. Interestingly, application of rTMS alone exerted some alterations in some electrophysiological properties of CA1 pyramidal neurons. On the other hand, rTMS alone could increase the Q6 neuronal excitability per se, which is consistent with a recent study in which daily rTMS of 1 Hz remarkably increases neuronal excitability (Silvanto and Pascual-Leone, 2008). To explain the observed controversial effects of rTMS in the present study (i.e. the inhibitory action when applied in kindled group and the excitatory effect when applied alone), it has been shown that the effect of 1 Hz rTMS is dependent on the baseline neuronal excitability (Siebner et al., 2004; Silvanto and Pascual-Leone, 2008). Therefore, the observed opposite effects of rTMS may be related to the different levels of neuronal activity of these two groups. It has to be considered that although we tried to apply the magnetic field onto the hippocampus by using a figure-8 shaped coil, however, other structures around the hippocampus, especially the amygdala in which we implanted a metal electrode, could also be affected by the magnetic field. Thus, because of anatomical connections between the hippocampus and amygdala (Witter and Amaral, 2004), the possible rTMS inducedchanges in the activity of this region may also affect the hippocampal excitability. In summary, our data confirmed the inhibitory effect of rTMS on amygdala kindling induced hyperexcitability in hippocampal CA1 region. These data show that rTMS can exert its antiepileptogenic effects not only on seizure focus (i.e. amygdala), but also on the regions involved in seizure propagation to other brain areas. However, it is necessary to study the changes in the activity and/or expression of some probable specific ion channels that may be involved in future studies.

4.

Experimental procedure

4.1.

Animals

Adult male Wistar rats (4–6 weeks old at the time of surgery) were obtained from the Pasteur Institute of Iran (Tehran, Iran).


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Animals were kept in a colony room with a constant temperature (257721 C), humidity (50775%), and artificial 12:12-h light–dark cycle. The lights were turned on at 7:00 a.m. Animals were kept in individual cages with wood-chip bedding and had free access to standard food and water. All protocols were approved by the “EtZZf Medical Sciences, Tarbiat Modares University,” which completely coincides with “National Institutes of Health Guide for the Care and Use of Laboratory Animals”.

4.2.

Surgical procedure

Surgical procedure was performed using a stereotaxic instrument as described previously (Paxinos and Watson, 2013). Under ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia, animals underwent stereotaxic implantation with a tripolar electrode (consists of a bipolar stimulating and a monopolar recording electrode), in the right basolateral nucleus of amygdala (2.5 posterior, 4.8 lateral and 7.5 ventral Q7 with respect to bregma; Paxinos and Watson, 2013). Electrodes were stainless steel, Teflon-coated, 127 μm in diameter, and insulated except at their tips (A-M Systems, Inc., Carlsborg, WA, USA). Two other electrodes connected to stainless steel screws were positioned in the skull above the frontal and occipital cortices as reference and ground electrodes. The incisor bar was set 3.3 mm below the interaural line. All electrodes were connected to pins of a lightweight multichannel miniature socket as a head-stage and fixed to the skull with dental acrylic. Electrophysiological experiments began after a 10-day recovery period.

4.3.

Semi-rapid kindling procedure

Following a 10-day recovery period, rats were transferred from the home cage to a recording box. The head-stage of the rat was connected to a flexible, shielded cable. Afterdischarge (AD) threshold was determined by 1 ms monophasic square Q8 wave of 50 Hz with 3 s train duration as described previously (Mohammad-Zadeh et al., 2007; Sadegh et al., 2007). The stimulating currents were initially delivered at 10 mA and then its intensity was increased in increments of 10 mA at 5 min intervals until ADs of at least 8 s were recorded. In each animal, the minimum intensity sufficient to induce the ADs for at least 8 s was selected as the AD threshold of that animal and used for kindling stimulation. The AD was defined as spikes with a frequency of at least 1 Hz and amplitude of at least twice the baseline activity originating immediately post stimulation. In this study, the AD threshold intensity of different animals ranged from 20 to 150 μA. Rats were electrically stimulated at the AD threshold 12 times a day with an interval of 5 min. Epileptiform ADs were continuously recorded from the amygdala following each kindling stimulation. The AD duration (ADD) and the behavioral progression of kindling (stages 1–5), according to Racine (1972)scores were monitored during kindling stimulations. The kindling stimulations were applied to the focus until the development of stage 5 seizure.

4.4.

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rTMS application

rTMS (1 Hz for 4 min) was applied at 5 min after the end of kindling stimulation as described previously (Shojaei et al., 2014; Yadollahpour et al., 2014). Briefly, the animals were exposed to CO2 during the application of rTMS. rTMS was delivered by a magstim rapid stimulator (Magstim Ltd., Whitland, Wales, UK) and 25 mm figure-of-eight shaped coil (figure8 coil; inner diameter (ID)), 14 mm, outer diameter (OD) 43772 and turns 14771). The intensity of rTMS was determined according to the percentage of motor threshold (MT). The MT was assessed by using single pulse stimulation upon the hindlimb representation of the motor cortex to evoke motor response in the hindlimb muscles. The motor response was revealed by both visual observation and EMG recording.

4.5.

Hippocampal slice preparation

In all experimental groups, the hippocampal slices were prepared 24 h after the last kindling stimulation. Animals were anesthetized with ether, and their brain was removed rapidly and immersed in ice-cold cutting solution containing (in mM): 2.5KCl, 0.5CaCl2, 2MgCl2, 1NaH2PO4, 26.2NaHCO3, 238sucrose and 11D-glucose bubbled with 95% O2 and 5% CO2. Then, 400 mm thick horizontal slices were cut by the vibrotome (Vibratum, Series 1000, Technical Products International Inc., USA). The slices were incubated at 32–35 1C temperature in aCSF containing (in mM): 125NaCl, 3KCl, 1.25NaH2PO4, 25NaHCO3, 10D-Glucose, 2CaCl2, 1.3MgCl2 continuously bubbled with 95% O2 and 5% CO2, for at least 1 h before the whole-cell recording at room temperature (23– 25 1C). The pH of both solutions was adjusted to 7.3–7.4 using NaOH 1 M and their osmolarity was 290–300 mOsm/L.

4.6.

Electrophysiological recordings

Slices were transferred to the recording chamber and superfused with aCSF at the rate of 2 ml/min. Borosilicate glass microelectrodes with an inner filament (1.5 mm o.d., 0.86 mm i.d.; Sutter Instrument Company, USA) were pulled on a horizontal puller (P-97, Sutter, USA) using a four step program giving an electrode resistance of 4–6 MΩ. The microelectrodes were filled with internal solution containing (in mM): 115Kgluconate, 20KCl, 10HEPES, 2EGTA, 10 disodium–phosphocreatine, 2MgATP and 0.3NaGTP. pH was adjusted to 7.25– 7.30 and osmolality was in the range of 285–290 mOsm. Whole-cell patch-clamp recordings were obtained from CA1 pyramidal neurons at room temperature under visual guidance by using infrared differential interference contrast microscopy (Axioskop 2 FS MOT model, Carl Zeiss, Germany). Recordings with series resistance 427 MΩ or over 15% changes during the experiment were discarded. Recordings were done by using a Multiclamp 700 A amplifier (Axon Instruments), filtered at 3 kHz and digitized at 10 kHz. Digidata 1440 (Axon, Molecular Devices, Sunnyvale, CA, USA) was used for data acquisition to computer. Data were acquired to computer by using a PClamp 9.2 (Axon instrument). Intrinsic membrane parameters were obtained in current clamp mode. Depolarizing currents were injected at different intensities (100–500 pA in increments of 100 pA, 650 ms)


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to estimate the firing properties and spike accommodation including adaptation index and amplitude of post-afterhyperpolarization (AHP). Hyperpolarizing currents (100–500 pA, 650 ms) were also applied to measure voltage sag, numbers of rebound spikes and delay to the first rebound spikes. In addition, a depolarizing ramp protocol (from 0 to 200 pA during 1000 ms; i.e. 0.16 pA/ms) was injected to measure the rheobase, utilization time and the number of spike. The adaptation index was calculated by dividing the mean of the three last inter-spike intervals to the mean of the three first inter-spike intervals following positive current step. The post-AHP (which is produced following termination of depolarizing current) was assessed from the baseline (before the stimulus) to the peak of hyperpolarization. The rheobase (the minimum current is needed to generate an action potential) was calculated from the onset of ramp current to first spike threshold. The utilization time is the time needed to reach the threshold of an action potential. Voltage sag amplitude in response to hyperpolarizing current pulses was determined as the voltage difference between the peak hyperpolarization and the steady-state membrane potential.

4.7.

Experimental design

Animals were randomly divided into control, sham, kindled and kindledþrTMS (KrTMS) groups. In the kindled group, animals were stimulated according to the semi-rapid kindling protocol (12 stimulations/day) to show at least one stage 5 seizure. The kindling parameters were also recorded following each kindling stimulation. In the KrTMS group, animals received the daily kindling stimulations (12 stimulations/day) and underwent the CO2-stunning process, fixed by the restrainer and received rTMS (1 Hz, at the intensity of 40% of MT, for 4 min) at 5 min following the last (12th) kindling stimulation. Animals of the sham group were handled similar to the KrTMS group except that they were stimulated by placebo coil which made a click sound (similar to the real coil), but did not produce the magnetic field. The number of kindling stimulation days in KrTMS and sham groups was equal to the mean number of days needed for the animal of kindled group to show a stage 5 seizure (5 or 6 days). Animals of the control group were handled similar to kindled rats, but did not receive kindling stimulations.

4.8.

Statistical analysis

The values were expressed as mean77SEM. Statistical analysis was performed by Graph Pad prism (version 5) software and a value of po0.05 was considered statistically significant. The electrophysiological data obtained were subjected to one-way or two-way ANOVA followed by Bonferroni's test. In the case of non-parametric data, Kruskal–Wallis and Mann–Whitney U-test were used.

Acknowledgments This study was supported by grants from Tarbiat Modares University and Iran National Science Foundation (INSF) (Grant #92040251).

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