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Effect of transient hippocampal inhibition on amygdaloid kindled seizures and amygdaloid kindling rate
Brain Research 954 (2002) 220–226 www.elsevier.com / locate / brainres
Research report
Effect of transient hippocampal inhibition on amygdaloid kindled seizures and amygdaloid kindling rate Javad Mirnajafi-Zadeh*, Mostafa Mortazavi, Yaghoub Fathollahi, Masoud Alasvand Zarasvand, Mohammad Reza Palizvan Department of Physiology, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran Accepted 16 April 2002
Abstract In this study the effect of transient inhibition of the CA1 region of the dorsal hippocampus by lidocaine on amygdala kindling rate and amygdaloid kindled seizures was investigated. In experiment 1, rats were divided into four groups. In group 1, animals were implanted only with a tripolar electrode into the amygdala but in groups 2–4, two guide cannulae were also implanted into the CA1 regions of the dorsal hippocampi. Animals were stimulated daily to be kindled. In groups 3 and 4, saline or 2% lidocaine (1 ml / 2 min) was also injected respectively into the hippocampus, 5 min before each stimulation. Results obtained showed that amygdala kindling rate and the number of stimulations to receive from stage 4 to stage 5 seizure were significantly increased in group 4. In experiment 2, lidocaine (1% and 2%) was infused (1 ml / 2 min) into the hippocampus of amygdala kindled rats bilaterally and animals were stimulated at 5, 15 and 30 min after drug injection. Twenty four h before lidocaine injection, saline was also infused (1 ml / 2 min) into the hippocampus as control. Obtained results showed that afterdischarge duration was reduced 5 min after lidocaine (1% and 2%) injection. Stage 5 seizure duration was also decreased 5 and 15 min after 2% lidocaine. Thus, it may be suggested that in amygdala kindling, activation of the hippocampal CA1 region has a role in seizure acquisition and seizure severity so that inhibition of this region results in decreasing of seizure severity and retards amygdala kindling rate. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animal models Keywords: Epilepsy; Seizure; Kindling; Hippocampus; Amygdala; Lidocaine
1. Introduction Temporal lobe epilepsy (TLE) is a common epileptic syndrome [15]. Identifying the neural circuits critical to the amplification and propagation of TLE into their convulsive form, could provide new insights into the treatment of TLE [11]. The typical seizure of temporal origin is the complex partial seizure and kindling has been considered as an animal model of complex partial and secondary generalized seizures [5]. Kindling is the progressive development of behavioral convulsions and epileptic activity following the repeated administration of an initially subconvulsant low intensity, electrical stimulation of the brain [5]. *Corresponding author. Fax: 198-21-800-6544. E-mail address: [email protected] (J. Mirnajafi-Zadeh).
Among different brain regions, amygdala and hippocampus are recognized as two of the important structures involved in the development and control of kindled seizures [1,12] and amygdalohippocampectomy is a surgery treatment for drug resistance TLE in some patients [36]. Thus, determining the precise interconnection between these two regions in the kindling model of epilepsy can provide important insights into the anatomy of complex partial seizures. Although there are many reports showing changes in the neural properties of hippocampus following amygdala kindling [6,8,16,27,30,35,39] results from studies on influence of hippocampal lesions upon amygdala kindling are controversial [3,23,33,38]. In addition, our previous study showed that intrahippocampal injection of 2-chloroadenosine has no effect on amygdala kindled seizures [20].
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03292-4
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Thus, the precise role of the hippocampus in amygdala kindling is still to be resolved. On the other hand, lidocaine has a reversible inhibitory effect on neural activity [26], and it can produce a transient inhibition after microinjection into different brain areas [26]. So, in this study we have tried to investigate the effect of transient hippocampal inhibition by lidocaine on amygdala kindling development as well as amygdaloid kindled seizures.
2. Materials and methods
2.1. Experiment 1 In this experiment, the effect of transient hippocampal inhibition on amygdala kindling rate was investigated. Four groups of animals were used. Male Sprague–Dawley rats (Razi Institute, Tehran) weighing 300–330 g, under sodium pentobarbital anesthesia (50 mg / kg; i.p.) were stereotaxically implanted with bipolar stimulation and monopolar recording electrodes (twisted into a tripolar configuration) terminating in the basolateral amygdala of the right hemisphere (coordinates: A, 22.5 mm; L, 4.8 mm from Bregma and 7.5 mm below dura) and except for group 1, two 23 gauge guide cannulae also implanted in the CA1 regions of the dorsal hippocampi (coordinates: A, 23.6 mm; L, 2.3 mm from Bregma and 2.2 mm below dura) of the right and left hemispheres [19]. Electrodes (stainless steel, teflon coated, A.M. Systems, Inc., USA) were insulated except at the tips. The incisor bar was set 3 mm below intraural line. In all groups, two other electrodes were connected to skull screws, placed above the left cortical surface as earth and differential electrodes.
2.1.1. Kindling procedure One week after surgery, using stimulator (Grass S88, USA) stimulus isolated unit (Grass SIU5, USA) and a constant current unit (Grass CCU 1A, USA), afterdischarge (AD) threshold was determined in the amygdala by a 2 s, 60 Hz monophasic square wave stimulus of 1 ms per wave. The stimulations were initially delivered at 10 mA and then at 5 min intervals increasing stimulus intensity in increments of 10 mA until at least 5 s of AD was recorded as previously described [19]. Then, different groups of animals were stimulated daily at AD threshold until the first stage 5 seizure was elicited. In groups 1 (control, n59) and 2 (sham operated, n57), animals did not receive any drug, but in groups 3 (saline injected, n56) and 4 (lidocaine injected, n56), animals were daily received saline and 2% lidocaine respectively. The solutions were bilaterally injected into the CA1 region of the hippocampus (1 ml / 2 min; as described in experiment 1) 5 min before each stimulation. So during each injection, the total volume of lidocaine delivered to each of the hemispheres was 1 ml.
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2.1.2. Kindling parameters During the course of the study, following kindling parameters were recorded: (a) number of stimulations required before a stage 5 (amygdala kindling rate) or other seizure stages were triggered, (b) number of stimulations from one seizure stage to the later stage, (c) amygdala AD threshold and (d) AD duration after the first stimulation. 2.2. Experiment 2 In this experiment, the effect of transient hippocampal inhibition on amygdaloid kindled seizures was investigated. Animals were implanted in the basolateral amygdala with tripolar electrodes and two guide cannulae were also implanted in the CA1 regions of the right and left hippocampi. In all groups, two other electrodes were connected to the skull screws as earth and differential electrodes.
2.2.1. Kindling procedure One week after surgery, AD threshold was determined as explained in experiment 1. Then, animals were stimulated daily at the AD threshold intensity until 5 consecutive stage 5 seizures (according to the Racine [22]) were elicited. When all rats reached this criterion, AD threshold was determined again, by delivery stimulation at an initial intensity of 25 mA followed by incrementing stimulations in 25 mA steps separated by at least 30 min. The threshold was the lowest current required to initiate stage 5 seizure. 2.2.2. Kindling parameters In the kindled animals, the recorded parameters were: Seizure stage, amygdala AD duration, the latency to the onset of stage 4 seizure and the duration of stage 5. 2.2.3. Drug administration For intrahippocampal injection, 1% and 2% lidocaine hydrochloride (Bayer) were used. The solutions were sterilized through microfilters (0.2 mm, Minisart, NML, Sartorius, Germany). Drugs were infused (1 ml / 2 min) via two 30-gauge cannulae as injectors, which extended 1 mm below the tip of the guide cannulae. Each of the injector was connected to PE-10 tubing that attatched to a 25 ml Hamilton syringe. A microsyringe pump (EICOM EP-60, Japan) controlled flow rate. The total volume of the lidocaine delivered to each of the hemispheres was 1 ml. Lidocaine (1 and 2%) was infused in situ and 5, 15 and 60 min later, animals were stimulated at an intensity of 25 mA over the AD threshold. To show that the procedures were not producing lasting damage or some other long term effects, the animals were also stimulated 24 h post lidocaine infusion. In each case, 24 h prior to the experiment, animals received saline, were stimulated by the same way (5, 15 and 30 min after saline injection) and the results recorded as control values. Six to nine rats were
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used in each group. A different group of animals was used for each of the times and concentrations employed.
2.3. Histology At the end of procedure, methylene blue was injected bilaterally through guide cannulae into the CA1 regions of the right and left hippocampi. Fifteen min after methylene blue injection, the rats used in experiment 1 were perfused with formalin 10% under deep anesthesia. The animals used in experiment 2 were randomly perfused with formalin 10% at 5, 15 and 30 min after methylene blue injection. After formalin perfusion, the rats brain were removed and sectioned. The track of the electrode and each of the cannulae and the location of the dye were examined under a light microscope. In the case of any abnormality, the data from that particular animal were not included in the results.
2.4. Statistical analysis Obtained results are expressed as the mean6S.E.M. and accompanied by the numbers of observations. In experiment 1, data were compared with one-way ANOVA and a post-hoc test of Tukey. In experiment 2, a two-way ANOVA and Tukey’s post test was done to compare different groups of animals at different times post different doses of drug injections. In the case of seizure stage, the Kruskal–Wallis test was used to compare different groups of animals. Data expressed as percentage of control were statistically compared using Wilcoxon test. A P value less than 0.05 was considered to represent a significant difference.
3. Results Histological analysis confirmed that the tripolar electrodes and cannulae were located in or near the basolateral amygdala and CA1 region of dorsal hippocampus respectively (Fig. 1a). Determination the extent of spread of injection showed that the drug is spread within 0.5 mm diameter at 5 min, 1.2 mm diameter at 15 min and about 1.6 mm at 30 min, thus is restricted to the site of injection (CA1 region of the hippocampus) (Fig. 1b). In experiment 2, the comparison between seizure parameters after saline infusion and 24 h post drug injection showed no significant difference. At the concentrations employed, lidocaine had no noticeable effect on behavioral or locomotor activity.
3.1. Experiment 1 No difference was observed in AD threshold [F(3,34)5 0.63, P50.59] or AD duration after the first stimulation [F(3,34)52.2, P51.55] in different groups of animals used in this experiment.
Fig. 1. (a) A typical photomicrograph of a coronal section through the injection site in the CA1 regions of right and left hippocampi, Scale bar5400 mm. (b) Schematic drawing of coronal plane through the CA1 region of the hippocampus has been adapted from the atlas of Paxinos and Watson [19]. Solid dots reveal the location of the cannula tips.
One way ANOVA revealed a significant difference between amygdala kindling rate in four groups [F(3,3)5 4.95, P,0.01] which showed a significant increase in the number of stimulations required before a stage 5 seizure in lidocaine injected group with respect to control and sham operated groups (Fig. 2). However, there were no significant differences between the number of stimulations to induce stage 1 [F(3,34)50.55, P50.65], stage 2 [F(3,34)50.32, P50.80], stage 3 [F(3,34)50.77, P50.51] or stage 4 [F(3,34)51.03, P50.39] seizures (Fig. 2).
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In addition, one-way ANOVA showed a significant difference between the number of stimulations from stage 4 to stage 5 seizure in four groups [F(3,34)57.3, P,0.01] and there was a significant increase in the number of stimulations in lidocaine injected group with respect to other groups (Table 1). However, there was no significant difference between the number of stimulations from stage 1 to stage 2 [F(3,34)50.32, P50.80], from stage 2 to stage 3 [F(3,34)50.44), P50.72] or from stage 3 to stage 4 [F(3,34)51.52, P50.23] (Table 1).
3.2. Experiment 2 Lidocaine administration into the CA1 region of the hippocampus led to significant suppression of AD duration generated within the amygdala after electrical stimulation. Although a two-way ANOVA of resultant data revealed no significant effects of concentration [F(1,34)54.03, P5 0.33], time [F(2,34)51.52, P50.23] or interaction between these variables [concentration3time; F(2,34)51.99, P50.15], there was a significant reduction in AD duration 5 min after injection of 2% lidocaine according to Wilcoxon test (Fig. 3a). Likewise, intrahippocampal infusion of lidocaine resulted in prolongation of latency to stage 4 (shown as a reduction in 1 / stage 4 latency in Fig. 3b). The two-way ANOVA showed only a significant effect of concentration [F(1,34)59.78, P,0.01] but no significant effect of time [F(2,34)53.09, P50.06] or concentration3time [F(2,34)50.93, P50.41]. In addition, 2% lidocaine reduced stage 5 duration after intrahippocampal administration (Fig. 3c) and there was a significant effect of concentration3time [F(2,34)52.94, P,0.05]. Also, there was a significant reduction in stage 5 duration at 5 and 15 min after 2% lidocaine administration according to Wilcoxon test. Lidocaine had no significant effect on seizure stage at different time points post infusion, although after 2% lidocaine administration, 4 / 8 rats did not show stage 4 or 5 seizures (Table 2). However, no significant difference was observed in seizure parameters of the animals which stimulated 24 h post lidocaine injection with compared to the control groups.
4. Discussion
Fig. 2. Number of stimulation trials to stage 1 (S1), Stage 2 (S2), Stage 3 (S3), Stage 4 (S4) and Stage 5 (S5) in 4 groups of rats (explained in text). Values are mean6S.E.M. of groups of 9 (control), 7 (sham operated), 6 (saline injected), and 6 (lidocaine injected) rats. *P,0.05 by the one-way ANOVA and Tukey’s test with respect to control and sham operated groups.
Results obtained in the present study show that bilateral inhibition of hippocampal CA1 region has a partial anticonvulsant effect on amygdala kindled seizures and moderately increased amygdala kindling rate. There exists some association changes in the hippocampus with the amygdala kindling [18,34,37] and many of the alterations in structure or biochemistry seen following amygdala kindling have been observed in the hippocampus [1,16,30,35]. Using the 2-deoxyglucose technique, it has been shown that amygdala kindling seizures induce post
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Table 1 Effect of intrahippocampal lidocaine on difference between the number of stimulations from one seizure stage to the later stage Groups
Number of stimulations
Control (n56) Sham operated (n57) Saline injected (n56) Lidocaine injected (n56)
From S a 1 to S2
From S2 to S3
From S3 to S4
From S4 to S5
3.060.8 2.761.0 2.562.2 4.061.9
3.961.1 2.960.6 4.361.3 3.260.7
0.960.4 2.460.9 1.860.4 2.360.5
1.860.5 2.460.6 1.760.3 5.360.9**
All rats were stimulated 5 min post injection values are mean6S.E.M. **P,0.01 when compared to other groups by one-way ANOVA and Tukey’s test. a Seizure stage.
Fig. 3. Effect of intrahippocampal lidocaine (1% and 2%) on afterdischarge duration (a), 1 / Stage 4 Latency (b) and Stage 5 duration (c) in amygdaloid kindled rats. Values are mean6S.E.M. (n56–9). *P,0.05 when compared with related control by Wilcoxon test.
ictal enhanced glucose metabolism in the hippocampus [2,9,18]. Amygdala kindling has also been found to induce pronounced hippocampal mossy fiber sprouting [25]. Alteration in gene expression and extracellular neurotransmitter release in the hippocampus of amygdala kindled rats has also been reported [1,8,13,16,17,27,39]. It is important to note that many of these changes are related to an increase in excitability of the hippocampal neurons. Table 2 Effect of intrahippocampal lidocaine on seizure stage at different time points post infusion Drugs
1% Lidocaine 2% Lidocaine
Seizure stage 5 min
15 min
30 min
3.3361.0 2.3761.1
5.0060.0 5.0060.0
5.0060.0 4.6760.3
Animals were stimulated at 5, 15 and 30 min post lidocaine injection. Values are mean6S.E.M. (n56–9). The Kruskal–Wallis test showed no significant difference between groups.
On the other hand, results from studies of influence of the hippocampal lesions upon amygdala kindling are controversial. While it has been reported that large dorsal hippocampal lesion dose not affect amygdala kindling [23], it has been shown that amygdala kindling is significantly suppressed when degenerations are observed in bilateral hippocampus [11,33,38]. In addition, we previously showed that unilateral injection of 2-chloroadenosine, an adenosine receptors agonist, into the CA1 region of the ipsilateral hippocampus, has no significant effect on amygdala kindled seizures [20]; although, unilateral injection of this drug into the amygdala [21] and perirhinal cortex [14] has anticonvulsant effect on amygdala kindled seizures. The results of this study confirmed that hippocampal activation has a role in kindling acquisition [3,33] and showed that daily intrahippocampal lidocaine retards kindling acquisition. This retardation is reflected by increasing the kindling rate in lidocaine injected group. However, it
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should be noted that there was no significant difference in kindling rate between lidocaine injected and saline injected groups. So, it may be suggested that the injection procedure may have an inhibitory effect on kindling rate by itself. But, increasing the number of stimulations from stage 4 to stage 5 seizure in lidocaine injected but not saline injected group, shows that retardation in acquisition of stage 5 motor convulsion is related to inhibitory effect of lidocaine on the hippocampal neuronal activity, and the CA1 region of the hippocampus has a role in generalization of amygdala kindled seizures. On the other hand, the hippocampal CA1 region plays a role in late but not early stages of amygdala kindling. In this experiment, there was no significant difference between AD threshold and AD duration after the first stimulation in different groups of animals. It reveals that there was no difference between the animals at the beginning, and the observed difference in the number of stimulations is related to lidocaine injection and inhibition of the hippocampal activity. On the other hand, in fully amygdala kindled animals, transient hippocampal inhibition reduced the duration of ADs recorded from the amygdala. As the AD duration is an index of neural excitability in the epileptic focus, it may be concluded that the hippocampal neural activity enhances the signal emitted from stimulation of the amygdala [31,32]. The latency to stage 4 was also increased after intrahippocampal injection of lidocaine. This parameter is an index of generalized seizure starting and its increment reveals that the hippocampus may play a role in spreading of epileptic spikes from amygdala to other brain region(s). Decrease of stage 5 duration is also another sign of lidocaine anticonvulsant action. However, there was no significant difference in seizure stage at different time points post lidocaine, which means that the hippocampal activation is not crucial for kindling state. There are also another reports, which show that other brain regions except the hippocampus are more crucial for amygdala kindling [20,23]. Here, we used lidocaine for transient inhibition of neuronal activity and this kind of inhibition has been used in many studies [7,29]. However, lidocaine has a concentration dependent effect on seizures. At lower concentrations, it has anticonvulsant properties, whereas concentrations above 15 mg / ml frequently result in seizures in laboratory animals [4]. The highest concentration of lidocaine in our experiments was 2% (which is equal to 0.02 mg / ml), so it was too low to result in seizure. The anticonvulsant effect of lidocaine is due to Na 1 channel inhibition [26,28] and this inhibition takes 30 min [24,28]. This is why in our experiments the anticonvulsant effect of lidocaine was observed only at 5 and 15 min after its intrahippocampal injection. The spatial extent of 0.5 ml of 2% lidocaine blockade [10,28] suggests that inactivation
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is effective within 1 mm diameter and thus is restricted to the injection site. Therefore, obtained results from the experiments 1 and 2 are due to inhibition of neural activity of the CA1 region of the hippocampus. It means that activation of this region has a role in amygdala kindled seizures [24,28].
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Research report
Effect of transient hippocampal inhibition on amygdaloid kindled seizures and amygdaloid kindling rate Javad Mirnajafi-Zadeh*, Mostafa Mortazavi, Yaghoub Fathollahi, Masoud Alasvand Zarasvand, Mohammad Reza Palizvan Department of Physiology, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran Accepted 16 April 2002
Abstract In this study the effect of transient inhibition of the CA1 region of the dorsal hippocampus by lidocaine on amygdala kindling rate and amygdaloid kindled seizures was investigated. In experiment 1, rats were divided into four groups. In group 1, animals were implanted only with a tripolar electrode into the amygdala but in groups 2–4, two guide cannulae were also implanted into the CA1 regions of the dorsal hippocampi. Animals were stimulated daily to be kindled. In groups 3 and 4, saline or 2% lidocaine (1 ml / 2 min) was also injected respectively into the hippocampus, 5 min before each stimulation. Results obtained showed that amygdala kindling rate and the number of stimulations to receive from stage 4 to stage 5 seizure were significantly increased in group 4. In experiment 2, lidocaine (1% and 2%) was infused (1 ml / 2 min) into the hippocampus of amygdala kindled rats bilaterally and animals were stimulated at 5, 15 and 30 min after drug injection. Twenty four h before lidocaine injection, saline was also infused (1 ml / 2 min) into the hippocampus as control. Obtained results showed that afterdischarge duration was reduced 5 min after lidocaine (1% and 2%) injection. Stage 5 seizure duration was also decreased 5 and 15 min after 2% lidocaine. Thus, it may be suggested that in amygdala kindling, activation of the hippocampal CA1 region has a role in seizure acquisition and seizure severity so that inhibition of this region results in decreasing of seizure severity and retards amygdala kindling rate. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animal models Keywords: Epilepsy; Seizure; Kindling; Hippocampus; Amygdala; Lidocaine
1. Introduction Temporal lobe epilepsy (TLE) is a common epileptic syndrome [15]. Identifying the neural circuits critical to the amplification and propagation of TLE into their convulsive form, could provide new insights into the treatment of TLE [11]. The typical seizure of temporal origin is the complex partial seizure and kindling has been considered as an animal model of complex partial and secondary generalized seizures [5]. Kindling is the progressive development of behavioral convulsions and epileptic activity following the repeated administration of an initially subconvulsant low intensity, electrical stimulation of the brain [5]. *Corresponding author. Fax: 198-21-800-6544. E-mail address: [email protected] (J. Mirnajafi-Zadeh).
Among different brain regions, amygdala and hippocampus are recognized as two of the important structures involved in the development and control of kindled seizures [1,12] and amygdalohippocampectomy is a surgery treatment for drug resistance TLE in some patients [36]. Thus, determining the precise interconnection between these two regions in the kindling model of epilepsy can provide important insights into the anatomy of complex partial seizures. Although there are many reports showing changes in the neural properties of hippocampus following amygdala kindling [6,8,16,27,30,35,39] results from studies on influence of hippocampal lesions upon amygdala kindling are controversial [3,23,33,38]. In addition, our previous study showed that intrahippocampal injection of 2-chloroadenosine has no effect on amygdala kindled seizures [20].
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03292-4
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Thus, the precise role of the hippocampus in amygdala kindling is still to be resolved. On the other hand, lidocaine has a reversible inhibitory effect on neural activity [26], and it can produce a transient inhibition after microinjection into different brain areas [26]. So, in this study we have tried to investigate the effect of transient hippocampal inhibition by lidocaine on amygdala kindling development as well as amygdaloid kindled seizures.
2. Materials and methods
2.1. Experiment 1 In this experiment, the effect of transient hippocampal inhibition on amygdala kindling rate was investigated. Four groups of animals were used. Male Sprague–Dawley rats (Razi Institute, Tehran) weighing 300–330 g, under sodium pentobarbital anesthesia (50 mg / kg; i.p.) were stereotaxically implanted with bipolar stimulation and monopolar recording electrodes (twisted into a tripolar configuration) terminating in the basolateral amygdala of the right hemisphere (coordinates: A, 22.5 mm; L, 4.8 mm from Bregma and 7.5 mm below dura) and except for group 1, two 23 gauge guide cannulae also implanted in the CA1 regions of the dorsal hippocampi (coordinates: A, 23.6 mm; L, 2.3 mm from Bregma and 2.2 mm below dura) of the right and left hemispheres [19]. Electrodes (stainless steel, teflon coated, A.M. Systems, Inc., USA) were insulated except at the tips. The incisor bar was set 3 mm below intraural line. In all groups, two other electrodes were connected to skull screws, placed above the left cortical surface as earth and differential electrodes.
2.1.1. Kindling procedure One week after surgery, using stimulator (Grass S88, USA) stimulus isolated unit (Grass SIU5, USA) and a constant current unit (Grass CCU 1A, USA), afterdischarge (AD) threshold was determined in the amygdala by a 2 s, 60 Hz monophasic square wave stimulus of 1 ms per wave. The stimulations were initially delivered at 10 mA and then at 5 min intervals increasing stimulus intensity in increments of 10 mA until at least 5 s of AD was recorded as previously described [19]. Then, different groups of animals were stimulated daily at AD threshold until the first stage 5 seizure was elicited. In groups 1 (control, n59) and 2 (sham operated, n57), animals did not receive any drug, but in groups 3 (saline injected, n56) and 4 (lidocaine injected, n56), animals were daily received saline and 2% lidocaine respectively. The solutions were bilaterally injected into the CA1 region of the hippocampus (1 ml / 2 min; as described in experiment 1) 5 min before each stimulation. So during each injection, the total volume of lidocaine delivered to each of the hemispheres was 1 ml.
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2.1.2. Kindling parameters During the course of the study, following kindling parameters were recorded: (a) number of stimulations required before a stage 5 (amygdala kindling rate) or other seizure stages were triggered, (b) number of stimulations from one seizure stage to the later stage, (c) amygdala AD threshold and (d) AD duration after the first stimulation. 2.2. Experiment 2 In this experiment, the effect of transient hippocampal inhibition on amygdaloid kindled seizures was investigated. Animals were implanted in the basolateral amygdala with tripolar electrodes and two guide cannulae were also implanted in the CA1 regions of the right and left hippocampi. In all groups, two other electrodes were connected to the skull screws as earth and differential electrodes.
2.2.1. Kindling procedure One week after surgery, AD threshold was determined as explained in experiment 1. Then, animals were stimulated daily at the AD threshold intensity until 5 consecutive stage 5 seizures (according to the Racine [22]) were elicited. When all rats reached this criterion, AD threshold was determined again, by delivery stimulation at an initial intensity of 25 mA followed by incrementing stimulations in 25 mA steps separated by at least 30 min. The threshold was the lowest current required to initiate stage 5 seizure. 2.2.2. Kindling parameters In the kindled animals, the recorded parameters were: Seizure stage, amygdala AD duration, the latency to the onset of stage 4 seizure and the duration of stage 5. 2.2.3. Drug administration For intrahippocampal injection, 1% and 2% lidocaine hydrochloride (Bayer) were used. The solutions were sterilized through microfilters (0.2 mm, Minisart, NML, Sartorius, Germany). Drugs were infused (1 ml / 2 min) via two 30-gauge cannulae as injectors, which extended 1 mm below the tip of the guide cannulae. Each of the injector was connected to PE-10 tubing that attatched to a 25 ml Hamilton syringe. A microsyringe pump (EICOM EP-60, Japan) controlled flow rate. The total volume of the lidocaine delivered to each of the hemispheres was 1 ml. Lidocaine (1 and 2%) was infused in situ and 5, 15 and 60 min later, animals were stimulated at an intensity of 25 mA over the AD threshold. To show that the procedures were not producing lasting damage or some other long term effects, the animals were also stimulated 24 h post lidocaine infusion. In each case, 24 h prior to the experiment, animals received saline, were stimulated by the same way (5, 15 and 30 min after saline injection) and the results recorded as control values. Six to nine rats were
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used in each group. A different group of animals was used for each of the times and concentrations employed.
2.3. Histology At the end of procedure, methylene blue was injected bilaterally through guide cannulae into the CA1 regions of the right and left hippocampi. Fifteen min after methylene blue injection, the rats used in experiment 1 were perfused with formalin 10% under deep anesthesia. The animals used in experiment 2 were randomly perfused with formalin 10% at 5, 15 and 30 min after methylene blue injection. After formalin perfusion, the rats brain were removed and sectioned. The track of the electrode and each of the cannulae and the location of the dye were examined under a light microscope. In the case of any abnormality, the data from that particular animal were not included in the results.
2.4. Statistical analysis Obtained results are expressed as the mean6S.E.M. and accompanied by the numbers of observations. In experiment 1, data were compared with one-way ANOVA and a post-hoc test of Tukey. In experiment 2, a two-way ANOVA and Tukey’s post test was done to compare different groups of animals at different times post different doses of drug injections. In the case of seizure stage, the Kruskal–Wallis test was used to compare different groups of animals. Data expressed as percentage of control were statistically compared using Wilcoxon test. A P value less than 0.05 was considered to represent a significant difference.
3. Results Histological analysis confirmed that the tripolar electrodes and cannulae were located in or near the basolateral amygdala and CA1 region of dorsal hippocampus respectively (Fig. 1a). Determination the extent of spread of injection showed that the drug is spread within 0.5 mm diameter at 5 min, 1.2 mm diameter at 15 min and about 1.6 mm at 30 min, thus is restricted to the site of injection (CA1 region of the hippocampus) (Fig. 1b). In experiment 2, the comparison between seizure parameters after saline infusion and 24 h post drug injection showed no significant difference. At the concentrations employed, lidocaine had no noticeable effect on behavioral or locomotor activity.
3.1. Experiment 1 No difference was observed in AD threshold [F(3,34)5 0.63, P50.59] or AD duration after the first stimulation [F(3,34)52.2, P51.55] in different groups of animals used in this experiment.
Fig. 1. (a) A typical photomicrograph of a coronal section through the injection site in the CA1 regions of right and left hippocampi, Scale bar5400 mm. (b) Schematic drawing of coronal plane through the CA1 region of the hippocampus has been adapted from the atlas of Paxinos and Watson [19]. Solid dots reveal the location of the cannula tips.
One way ANOVA revealed a significant difference between amygdala kindling rate in four groups [F(3,3)5 4.95, P,0.01] which showed a significant increase in the number of stimulations required before a stage 5 seizure in lidocaine injected group with respect to control and sham operated groups (Fig. 2). However, there were no significant differences between the number of stimulations to induce stage 1 [F(3,34)50.55, P50.65], stage 2 [F(3,34)50.32, P50.80], stage 3 [F(3,34)50.77, P50.51] or stage 4 [F(3,34)51.03, P50.39] seizures (Fig. 2).
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In addition, one-way ANOVA showed a significant difference between the number of stimulations from stage 4 to stage 5 seizure in four groups [F(3,34)57.3, P,0.01] and there was a significant increase in the number of stimulations in lidocaine injected group with respect to other groups (Table 1). However, there was no significant difference between the number of stimulations from stage 1 to stage 2 [F(3,34)50.32, P50.80], from stage 2 to stage 3 [F(3,34)50.44), P50.72] or from stage 3 to stage 4 [F(3,34)51.52, P50.23] (Table 1).
3.2. Experiment 2 Lidocaine administration into the CA1 region of the hippocampus led to significant suppression of AD duration generated within the amygdala after electrical stimulation. Although a two-way ANOVA of resultant data revealed no significant effects of concentration [F(1,34)54.03, P5 0.33], time [F(2,34)51.52, P50.23] or interaction between these variables [concentration3time; F(2,34)51.99, P50.15], there was a significant reduction in AD duration 5 min after injection of 2% lidocaine according to Wilcoxon test (Fig. 3a). Likewise, intrahippocampal infusion of lidocaine resulted in prolongation of latency to stage 4 (shown as a reduction in 1 / stage 4 latency in Fig. 3b). The two-way ANOVA showed only a significant effect of concentration [F(1,34)59.78, P,0.01] but no significant effect of time [F(2,34)53.09, P50.06] or concentration3time [F(2,34)50.93, P50.41]. In addition, 2% lidocaine reduced stage 5 duration after intrahippocampal administration (Fig. 3c) and there was a significant effect of concentration3time [F(2,34)52.94, P,0.05]. Also, there was a significant reduction in stage 5 duration at 5 and 15 min after 2% lidocaine administration according to Wilcoxon test. Lidocaine had no significant effect on seizure stage at different time points post infusion, although after 2% lidocaine administration, 4 / 8 rats did not show stage 4 or 5 seizures (Table 2). However, no significant difference was observed in seizure parameters of the animals which stimulated 24 h post lidocaine injection with compared to the control groups.
4. Discussion
Fig. 2. Number of stimulation trials to stage 1 (S1), Stage 2 (S2), Stage 3 (S3), Stage 4 (S4) and Stage 5 (S5) in 4 groups of rats (explained in text). Values are mean6S.E.M. of groups of 9 (control), 7 (sham operated), 6 (saline injected), and 6 (lidocaine injected) rats. *P,0.05 by the one-way ANOVA and Tukey’s test with respect to control and sham operated groups.
Results obtained in the present study show that bilateral inhibition of hippocampal CA1 region has a partial anticonvulsant effect on amygdala kindled seizures and moderately increased amygdala kindling rate. There exists some association changes in the hippocampus with the amygdala kindling [18,34,37] and many of the alterations in structure or biochemistry seen following amygdala kindling have been observed in the hippocampus [1,16,30,35]. Using the 2-deoxyglucose technique, it has been shown that amygdala kindling seizures induce post
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Table 1 Effect of intrahippocampal lidocaine on difference between the number of stimulations from one seizure stage to the later stage Groups
Number of stimulations
Control (n56) Sham operated (n57) Saline injected (n56) Lidocaine injected (n56)
From S a 1 to S2
From S2 to S3
From S3 to S4
From S4 to S5
3.060.8 2.761.0 2.562.2 4.061.9
3.961.1 2.960.6 4.361.3 3.260.7
0.960.4 2.460.9 1.860.4 2.360.5
1.860.5 2.460.6 1.760.3 5.360.9**
All rats were stimulated 5 min post injection values are mean6S.E.M. **P,0.01 when compared to other groups by one-way ANOVA and Tukey’s test. a Seizure stage.
Fig. 3. Effect of intrahippocampal lidocaine (1% and 2%) on afterdischarge duration (a), 1 / Stage 4 Latency (b) and Stage 5 duration (c) in amygdaloid kindled rats. Values are mean6S.E.M. (n56–9). *P,0.05 when compared with related control by Wilcoxon test.
ictal enhanced glucose metabolism in the hippocampus [2,9,18]. Amygdala kindling has also been found to induce pronounced hippocampal mossy fiber sprouting [25]. Alteration in gene expression and extracellular neurotransmitter release in the hippocampus of amygdala kindled rats has also been reported [1,8,13,16,17,27,39]. It is important to note that many of these changes are related to an increase in excitability of the hippocampal neurons. Table 2 Effect of intrahippocampal lidocaine on seizure stage at different time points post infusion Drugs
1% Lidocaine 2% Lidocaine
Seizure stage 5 min
15 min
30 min
3.3361.0 2.3761.1
5.0060.0 5.0060.0
5.0060.0 4.6760.3
Animals were stimulated at 5, 15 and 30 min post lidocaine injection. Values are mean6S.E.M. (n56–9). The Kruskal–Wallis test showed no significant difference between groups.
On the other hand, results from studies of influence of the hippocampal lesions upon amygdala kindling are controversial. While it has been reported that large dorsal hippocampal lesion dose not affect amygdala kindling [23], it has been shown that amygdala kindling is significantly suppressed when degenerations are observed in bilateral hippocampus [11,33,38]. In addition, we previously showed that unilateral injection of 2-chloroadenosine, an adenosine receptors agonist, into the CA1 region of the ipsilateral hippocampus, has no significant effect on amygdala kindled seizures [20]; although, unilateral injection of this drug into the amygdala [21] and perirhinal cortex [14] has anticonvulsant effect on amygdala kindled seizures. The results of this study confirmed that hippocampal activation has a role in kindling acquisition [3,33] and showed that daily intrahippocampal lidocaine retards kindling acquisition. This retardation is reflected by increasing the kindling rate in lidocaine injected group. However, it
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should be noted that there was no significant difference in kindling rate between lidocaine injected and saline injected groups. So, it may be suggested that the injection procedure may have an inhibitory effect on kindling rate by itself. But, increasing the number of stimulations from stage 4 to stage 5 seizure in lidocaine injected but not saline injected group, shows that retardation in acquisition of stage 5 motor convulsion is related to inhibitory effect of lidocaine on the hippocampal neuronal activity, and the CA1 region of the hippocampus has a role in generalization of amygdala kindled seizures. On the other hand, the hippocampal CA1 region plays a role in late but not early stages of amygdala kindling. In this experiment, there was no significant difference between AD threshold and AD duration after the first stimulation in different groups of animals. It reveals that there was no difference between the animals at the beginning, and the observed difference in the number of stimulations is related to lidocaine injection and inhibition of the hippocampal activity. On the other hand, in fully amygdala kindled animals, transient hippocampal inhibition reduced the duration of ADs recorded from the amygdala. As the AD duration is an index of neural excitability in the epileptic focus, it may be concluded that the hippocampal neural activity enhances the signal emitted from stimulation of the amygdala [31,32]. The latency to stage 4 was also increased after intrahippocampal injection of lidocaine. This parameter is an index of generalized seizure starting and its increment reveals that the hippocampus may play a role in spreading of epileptic spikes from amygdala to other brain region(s). Decrease of stage 5 duration is also another sign of lidocaine anticonvulsant action. However, there was no significant difference in seizure stage at different time points post lidocaine, which means that the hippocampal activation is not crucial for kindling state. There are also another reports, which show that other brain regions except the hippocampus are more crucial for amygdala kindling [20,23]. Here, we used lidocaine for transient inhibition of neuronal activity and this kind of inhibition has been used in many studies [7,29]. However, lidocaine has a concentration dependent effect on seizures. At lower concentrations, it has anticonvulsant properties, whereas concentrations above 15 mg / ml frequently result in seizures in laboratory animals [4]. The highest concentration of lidocaine in our experiments was 2% (which is equal to 0.02 mg / ml), so it was too low to result in seizure. The anticonvulsant effect of lidocaine is due to Na 1 channel inhibition [26,28] and this inhibition takes 30 min [24,28]. This is why in our experiments the anticonvulsant effect of lidocaine was observed only at 5 and 15 min after its intrahippocampal injection. The spatial extent of 0.5 ml of 2% lidocaine blockade [10,28] suggests that inactivation
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is effective within 1 mm diameter and thus is restricted to the injection site. Therefore, obtained results from the experiments 1 and 2 are due to inhibition of neural activity of the CA1 region of the hippocampus. It means that activation of this region has a role in amygdala kindled seizures [24,28].
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