Original Article
Increased Expression of GAP43 in Interneurons in a Rat Model of Experimental Polymicrogyria
Journal of Child Neurology 2015, Vol. 30(6) 716-728 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0883073814541476 jcn.sagepub.com
Tomoyuki Takano, MD, PhD1, and Katsuyuki Matsui, MD, PhD1
Abstract To investigate seizure susceptibility in polymicrogyria, the seizure threshold and growth-associated protein GAP43 expression were analyzed in a rat experimental model of polymicrogyria induced by intracerebral injection of ibotenate. A total of 72 neonates from 9 pregnant rats were used. Intraperitoneal pentylenetetrazole injection did not induce any seizure activity in the control rats, although it elicited seizures of variable severity in the polymicrogyria rats. Fluoro-Jade B–positive degenerating interneurons were found in the polymicrogyria brains; however, no such neurons were detected in the control brains. In the polymicrogyria rats, the GAP43 expression was significantly and widely distributed in the brain, and the percentage of parvalbumin-positive interneurons in the GAP43-positive cells was significantly higher than that observed in the nonphosphorylated neurofilament-positive pyramidal cells. We conclude that the relatively selective vulnerability of inhibitory interneurons constitutes the basis for the decreased seizure threshold observed in this model of polymicrogyria. Keywords polymicrogyria, GAP43, interneuron, seizure susceptibility Received May 05, 2014. Received revised May 20, 2014. Accepted for publication June 01, 2014.
Polymicrogyria involves the malformation of cortical development characterized by an excessively folded cortical ribbon of miniature, individually thin convolutions that may be fused together or piled on top of one another.1 Such malformations result in highly epileptogenic lesions, with approximately 80% of patients eventually developing seizures, the majority within the first 5 years.2 However, the epileptogenic mechanisms of this disease remain largely unknown. Polymicrogyria can be modeled in rats using transcortical freezing or the formation of excitotoxic lesions in the developing brain that mimic the histologic characteristics of human 4-layered polymicrogyria. These experimental models do not exhibit spontaneous epileptiform activity in vivo; however, several investigations have been presented concerning seizure susceptibility, as follows: (1) alterations in glutamate receptor distribution,3-5 (2) abnormalities in ion channels,6-11 (3) the formation of new excitatory or inhibitory connections,12-17 and (4) the downregulation of GABAA receptor subunits.18 One of the most characteristic findings of this brain malformation is that the anomalous cortex associated with polymicrogyria includes less excitable neural tissue, such as a cell sparse zone, while also involving a larger epileptogenic network extending to adjacent and distant cortical areas.19,20 The growth-associated protein, GAP43, is a presynaptic phosphoprotein of growth cones that is expressed during
development and other events of axonal growth, such as the regeneration of injured axons.21,22 Developing neurons express high levels of GAP43, which is transported to the growth cones of immature synapses. Its expression in neurons correlates closely with axonal elongation, synaptogenesis and nerve sprouting during development. Following the establishment of mature synaptic connections, the protein levels decline abruptly. Although the GAP43 levels in the brain decrease after birth, the basal levels persist in certain nerve ends throughout life.23 In the mature nervous system, the correlation between GAP43 phosphorylation by protein kinase C and neurotransmitter release,24 along with the establishment and persistence of long-term potentiation,25,26 indicates that this molecule plays a role in synaptic plasticity.27 In the adult hippocampus, a well-described pattern of axon growth from the granule cell mossy fiber system into the inner molecular layer of the dentate gyrus has been described in several models of epilepsy.28,29
1
Department of Pediatrics, Shiga University of Medical Science, Seta-Tsukinowa, Otsu, Japan
Corresponding Author: Tomoyuki Takano, MD, PhD, Department of Pediatrics, Shiga University of Medical Science, Seta-Tsukinowa, Otsu 520-2192, Japan. Email: [email protected]
Takano and Matsui Because of its nature and localization in the hippocampus, several studies of epilepsy have suggested the GAP43 messenger RNA30,31 and protein expression32,33 present in the dentate inner molecular layer to be linked with this sprouting mechanism. This protein also plays a role in the plasticity of adult neurons after brain damage, such as that involving cerebral infarction or trauma.34-36 Therefore, GAP43 is considered to be a sensitive neuritic marker of axonal damage and regenerative responses.37 Pentylenetetrazole is a well-described proconvulsant agent used in rat kindling models and the generation of acute shortlived seizures in animals.38 Pentylenetetrazole-induced kindling was first described by Mason and Cooper in rats,39 in which the phenomenon is characterized by an increased susceptibility to seizures following repeated injections. Pentylenetetrazole is used in small doses as a model of absence seizures, whereas higher doses produce convulsive seizures.40 Although pentylenetetrazole is widely used in several animal models, the exact mechanisms underlying the pathogenesis of pentylenetetrazole-induced convulsions remain unclear.41 The most accepted theory highlights pentylenetetrazole’s action as an antagonist to the picrotoxinin-sensitive site of the g-aminobutyric acid (GABA) receptor complex.42,43 The threshold for the development of acute seizures in normal adult rats is between 40 and 50 mg/kg.44 In the present study, in order to evaluate seizure susceptibility in the setting of polymicrogyria, we used pentylenetetrazole to test the hypothesis that the polymicrogyria brain has a lower seizure threshold and suffers more extended neuronal damage following repeated injections of subconvulsive doses of pentylenetetrazole. In addition, the relationship between seizure susceptibility and the GAP43 expression was investigated by examining the immunohistochemical alterations of GAP43 in excitatory and inhibitory neurons in the cerebral cortex of rats with polymicrogyria.
Methods Animals Pregnant Wistar rats (CLEA Japan, Inc, Tokyo, Japan) and their litters were used in this study. The delivery day was designated as postnatal day 0 (P0). The rat pups were weaned on P21. All animals were maintained on a 12-hour light-dark cycle with food and water available ad libitum in plastic cages. This study was approved by the Committee on Animal Experimentation and Animal Care at Shiga University of Medical Science (Otsu, Japan).
Excitotoxic Brain Lesions Newborn rats (P0) were anesthetized using hypothermia (placed on ice for 5-10 minutes) and positioned in a stereotaxic apparatus adapted for neonatal surgery. The developmental level of the cerebral cortex in neonatal rats corresponds to around 16 weeks of gestation in human brains.45 Ibotenate is an agonist of the N-methyl-D-aspartate (NMDA) complex receptor. Intracerebral injections of ibotenate (Sigma, St Louis, MO) diluted in 0.1 M phosphate-buffered saline were administered using a 27-gauge needle in a 10-mL Hamilton syringe mounted
717
Figure 1. The 3 experimental groups are shown. The PMG group received an intracerebral injection of ibotenate (3 mg, dissolved in 3 mL of 0.1 M phosphate-buffered saline) on P0 (the day of birth) (filled short arrow) and intraperitoneal injections of pentylenetetrazole (30 mg/kg) once daily from P15 to P20 (filled long arrows). The Control group received an intracerebral injection of 0.1 M phosphate-buffered saline (3 mL) on P0 (open short arrow) and intraperitoneal injections of pentylenetetrazole (30 mg/kg) once daily from P15 to P20 (filled long arrows). The Normal group received intraperitoneal injections of saline once daily from P15 to P20 (open long arrows). IC, intracerebral injection; IP, intraperitoneal injection; PTZ, pentylenetetrazole. on a calibrated microdispenser. The needle was inserted 2 mm under the external surface of scalp skin in the frontoparietal area of the right hemisphere 3 mm from the midline (on the lateral-medial plane) and 3 mm from the junction between the sagittal and lambdoid sutures (on the rostrocaudal plane). A 3-mL bolus (3 mg of ibotenate) was injected, and the needle was left in place for an additional 30 seconds. The ibotenate injections were performed within 12 hours after birth. The pups were allowed to recover on an electric warming pad before being returned to the cages of their mothers, where they remained until weaning on P21. A total of 24 neonates from 3 pregnant rats were subjected to intracerebral injections of ibotenate as the polymicrogyria group (PMG). The same number of animals injected in the same manner with the same amount of 0.1 M phosphate-buffered saline served as the control group (Control).
Pentylenetetrazole Kindling Pentylenetetrazole (Sigma) was freshly prepared throughout the study dissolved in 0.9% saline (solution: 20 mg of pentylenetetrazole dissolved in 1 mL of saline). The rats were injected intraperitoneally with a subconvulsive dose of pentylenetetrazole (30 mg/kg) once daily from P15 to P20 in both the PMG and Control groups. Age-matched normal animals received the same number of saline injections, grouped as the normal rats (Normal). The 3 groups (PMG, Control, and Normal) of rats were observed for 30 minutes after each pentylenetetrazole or saline injection for convulsive behavior via video recordings. The 3 experimental groups of rats and the injection protocols are summarized in Figure 1. The seizure activity was evaluated according to the modified Racine Scale46 as follows: stage 0, no response; stage 1, mouth and facial movements; stage 2, head nodding; stage 3, forelimb clonus; stage 4, rearing with forelimb clonus; stage 5, rearing and falling with loss of postural control. The animals were considered kindled if they exhibited stage 5 seizures on
718 2 consecutive trials. Two observers who were required to reach full agreement regarding the scoring of the behavior analyzed the video recordings. The Kruskal-Wallis test was used to compare the seizure scale scores among the 3 groups. A P value of
Increased Expression of GAP43 in Interneurons in a Rat Model of Experimental Polymicrogyria
Journal of Child Neurology 2015, Vol. 30(6) 716-728 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0883073814541476 jcn.sagepub.com
Tomoyuki Takano, MD, PhD1, and Katsuyuki Matsui, MD, PhD1
Abstract To investigate seizure susceptibility in polymicrogyria, the seizure threshold and growth-associated protein GAP43 expression were analyzed in a rat experimental model of polymicrogyria induced by intracerebral injection of ibotenate. A total of 72 neonates from 9 pregnant rats were used. Intraperitoneal pentylenetetrazole injection did not induce any seizure activity in the control rats, although it elicited seizures of variable severity in the polymicrogyria rats. Fluoro-Jade B–positive degenerating interneurons were found in the polymicrogyria brains; however, no such neurons were detected in the control brains. In the polymicrogyria rats, the GAP43 expression was significantly and widely distributed in the brain, and the percentage of parvalbumin-positive interneurons in the GAP43-positive cells was significantly higher than that observed in the nonphosphorylated neurofilament-positive pyramidal cells. We conclude that the relatively selective vulnerability of inhibitory interneurons constitutes the basis for the decreased seizure threshold observed in this model of polymicrogyria. Keywords polymicrogyria, GAP43, interneuron, seizure susceptibility Received May 05, 2014. Received revised May 20, 2014. Accepted for publication June 01, 2014.
Polymicrogyria involves the malformation of cortical development characterized by an excessively folded cortical ribbon of miniature, individually thin convolutions that may be fused together or piled on top of one another.1 Such malformations result in highly epileptogenic lesions, with approximately 80% of patients eventually developing seizures, the majority within the first 5 years.2 However, the epileptogenic mechanisms of this disease remain largely unknown. Polymicrogyria can be modeled in rats using transcortical freezing or the formation of excitotoxic lesions in the developing brain that mimic the histologic characteristics of human 4-layered polymicrogyria. These experimental models do not exhibit spontaneous epileptiform activity in vivo; however, several investigations have been presented concerning seizure susceptibility, as follows: (1) alterations in glutamate receptor distribution,3-5 (2) abnormalities in ion channels,6-11 (3) the formation of new excitatory or inhibitory connections,12-17 and (4) the downregulation of GABAA receptor subunits.18 One of the most characteristic findings of this brain malformation is that the anomalous cortex associated with polymicrogyria includes less excitable neural tissue, such as a cell sparse zone, while also involving a larger epileptogenic network extending to adjacent and distant cortical areas.19,20 The growth-associated protein, GAP43, is a presynaptic phosphoprotein of growth cones that is expressed during
development and other events of axonal growth, such as the regeneration of injured axons.21,22 Developing neurons express high levels of GAP43, which is transported to the growth cones of immature synapses. Its expression in neurons correlates closely with axonal elongation, synaptogenesis and nerve sprouting during development. Following the establishment of mature synaptic connections, the protein levels decline abruptly. Although the GAP43 levels in the brain decrease after birth, the basal levels persist in certain nerve ends throughout life.23 In the mature nervous system, the correlation between GAP43 phosphorylation by protein kinase C and neurotransmitter release,24 along with the establishment and persistence of long-term potentiation,25,26 indicates that this molecule plays a role in synaptic plasticity.27 In the adult hippocampus, a well-described pattern of axon growth from the granule cell mossy fiber system into the inner molecular layer of the dentate gyrus has been described in several models of epilepsy.28,29
1
Department of Pediatrics, Shiga University of Medical Science, Seta-Tsukinowa, Otsu, Japan
Corresponding Author: Tomoyuki Takano, MD, PhD, Department of Pediatrics, Shiga University of Medical Science, Seta-Tsukinowa, Otsu 520-2192, Japan. Email: [email protected]
Takano and Matsui Because of its nature and localization in the hippocampus, several studies of epilepsy have suggested the GAP43 messenger RNA30,31 and protein expression32,33 present in the dentate inner molecular layer to be linked with this sprouting mechanism. This protein also plays a role in the plasticity of adult neurons after brain damage, such as that involving cerebral infarction or trauma.34-36 Therefore, GAP43 is considered to be a sensitive neuritic marker of axonal damage and regenerative responses.37 Pentylenetetrazole is a well-described proconvulsant agent used in rat kindling models and the generation of acute shortlived seizures in animals.38 Pentylenetetrazole-induced kindling was first described by Mason and Cooper in rats,39 in which the phenomenon is characterized by an increased susceptibility to seizures following repeated injections. Pentylenetetrazole is used in small doses as a model of absence seizures, whereas higher doses produce convulsive seizures.40 Although pentylenetetrazole is widely used in several animal models, the exact mechanisms underlying the pathogenesis of pentylenetetrazole-induced convulsions remain unclear.41 The most accepted theory highlights pentylenetetrazole’s action as an antagonist to the picrotoxinin-sensitive site of the g-aminobutyric acid (GABA) receptor complex.42,43 The threshold for the development of acute seizures in normal adult rats is between 40 and 50 mg/kg.44 In the present study, in order to evaluate seizure susceptibility in the setting of polymicrogyria, we used pentylenetetrazole to test the hypothesis that the polymicrogyria brain has a lower seizure threshold and suffers more extended neuronal damage following repeated injections of subconvulsive doses of pentylenetetrazole. In addition, the relationship between seizure susceptibility and the GAP43 expression was investigated by examining the immunohistochemical alterations of GAP43 in excitatory and inhibitory neurons in the cerebral cortex of rats with polymicrogyria.
Methods Animals Pregnant Wistar rats (CLEA Japan, Inc, Tokyo, Japan) and their litters were used in this study. The delivery day was designated as postnatal day 0 (P0). The rat pups were weaned on P21. All animals were maintained on a 12-hour light-dark cycle with food and water available ad libitum in plastic cages. This study was approved by the Committee on Animal Experimentation and Animal Care at Shiga University of Medical Science (Otsu, Japan).
Excitotoxic Brain Lesions Newborn rats (P0) were anesthetized using hypothermia (placed on ice for 5-10 minutes) and positioned in a stereotaxic apparatus adapted for neonatal surgery. The developmental level of the cerebral cortex in neonatal rats corresponds to around 16 weeks of gestation in human brains.45 Ibotenate is an agonist of the N-methyl-D-aspartate (NMDA) complex receptor. Intracerebral injections of ibotenate (Sigma, St Louis, MO) diluted in 0.1 M phosphate-buffered saline were administered using a 27-gauge needle in a 10-mL Hamilton syringe mounted
717
Figure 1. The 3 experimental groups are shown. The PMG group received an intracerebral injection of ibotenate (3 mg, dissolved in 3 mL of 0.1 M phosphate-buffered saline) on P0 (the day of birth) (filled short arrow) and intraperitoneal injections of pentylenetetrazole (30 mg/kg) once daily from P15 to P20 (filled long arrows). The Control group received an intracerebral injection of 0.1 M phosphate-buffered saline (3 mL) on P0 (open short arrow) and intraperitoneal injections of pentylenetetrazole (30 mg/kg) once daily from P15 to P20 (filled long arrows). The Normal group received intraperitoneal injections of saline once daily from P15 to P20 (open long arrows). IC, intracerebral injection; IP, intraperitoneal injection; PTZ, pentylenetetrazole. on a calibrated microdispenser. The needle was inserted 2 mm under the external surface of scalp skin in the frontoparietal area of the right hemisphere 3 mm from the midline (on the lateral-medial plane) and 3 mm from the junction between the sagittal and lambdoid sutures (on the rostrocaudal plane). A 3-mL bolus (3 mg of ibotenate) was injected, and the needle was left in place for an additional 30 seconds. The ibotenate injections were performed within 12 hours after birth. The pups were allowed to recover on an electric warming pad before being returned to the cages of their mothers, where they remained until weaning on P21. A total of 24 neonates from 3 pregnant rats were subjected to intracerebral injections of ibotenate as the polymicrogyria group (PMG). The same number of animals injected in the same manner with the same amount of 0.1 M phosphate-buffered saline served as the control group (Control).
Pentylenetetrazole Kindling Pentylenetetrazole (Sigma) was freshly prepared throughout the study dissolved in 0.9% saline (solution: 20 mg of pentylenetetrazole dissolved in 1 mL of saline). The rats were injected intraperitoneally with a subconvulsive dose of pentylenetetrazole (30 mg/kg) once daily from P15 to P20 in both the PMG and Control groups. Age-matched normal animals received the same number of saline injections, grouped as the normal rats (Normal). The 3 groups (PMG, Control, and Normal) of rats were observed for 30 minutes after each pentylenetetrazole or saline injection for convulsive behavior via video recordings. The 3 experimental groups of rats and the injection protocols are summarized in Figure 1. The seizure activity was evaluated according to the modified Racine Scale46 as follows: stage 0, no response; stage 1, mouth and facial movements; stage 2, head nodding; stage 3, forelimb clonus; stage 4, rearing with forelimb clonus; stage 5, rearing and falling with loss of postural control. The animals were considered kindled if they exhibited stage 5 seizures on
718 2 consecutive trials. Two observers who were required to reach full agreement regarding the scoring of the behavior analyzed the video recordings. The Kruskal-Wallis test was used to compare the seizure scale scores among the 3 groups. A P value of
