- Email: [email protected]
In vitro formation of neuromuscular junction with NG108-15 cells and C2C12 myotubes
Abstracts / Neuroscience Research 71S (2011) e108–e415
Reference Tanaka, Y., et al., 2011. Multiple EMG activity and intracortical inhibition and facilitation during a fine finger movement under pressure. J. Mot. Behav. 43, 73–81.
e243
Research fund: This study was supported by the Initiative for the Promotion of Young Scientists’ Independent Research program (MEXT), KAKENHI (No. 20500365) and the Brain Science Foundation. doi:10.1016/j.neures.2011.07.1062
doi:10.1016/j.neures.2011.07.1060
P3-h03 In vitro formation of neuromuscular junction with NG108-15 cells and C2C12 myotubes Ayumi Ishihara 1,3 Miyazawa 1,2,3
, Junichi Saito 1 , Yuko Fukunaga 1,2,3 , Atsuo
1
Div. of Cell Biochem., Grad. Sch. of Life Sci., Univ. of Hyogo, Hyogo, Japan 2 RIKEN, SPring-8 Center, Bio-multisome Research Team, Hyogo, Japan 3 JSTCREST, Japan In mature neuromuscular junction (NMJ), the postsynaptic membrane has specialized folds, designated as junctional folds. As NMJ mature, nicotinic acetylcholine receptors (nAChRs) and voltage dependent sodium channels are concentrated to the crests and the troughs of them, respectively. It is well known that they are critical for proper neuronal transmission. The in vitro NMJ model is advantageous to elucidate the mechanisms underlying forming the junctional folds and the precise configurations of these proteins at NMJ by using bio-imaging analysis, genetic engineering and/or electrophysiological experiment. Here, we report the establishment of the in vitro model of NMJ replicating the structure of the in vivo NMJ, using NG108-15 (neuroblastoma and glioma hybrid clone) cells and C2C12 myotubes. Many aspects of differentiated NG108-15 cells are similar to motor neurons. For example, they are cholinergic and express the primary nAChR-aggregating factor, agrin. For the long term culture of C2C12 myotubes, we examined the surface modification for cell attachment. C2C12 myotubes cultured on collagen gel was maintained for more than 5 weeks. Therefore, we seeded differentiated NG108-15 cells on C2C12 myotubes, which are cultured on collagen gel. Colocalization between nAChR and synaptophysin, a presynapse marker, was observed most abundantly in 5 weeks. Scanning electron microscopy showed that the presynaptic varicosities of NG108-15 cells were invaginated into the C2C12 myotubes, resembling primary postsynaptic cleft at the in vivo NMJ. The basal lamina-like density and the junctional fold-like structure between varicosities and C2C12 myotubes were identified by transmission electron microscopy. These results indicate that prolonged (longer than 5 weeks) co-culture and/or other factors are required for maturation of the in vitro NMJ. Research fund: JST-CREST. doi:10.1016/j.neures.2011.07.1061
P3-h04 Characterization of ipsilaterally projecting inhibitory neurons in the ventral spinal cord in mouse neonates Hiroshi Nishimaru 1 Yuchio Yanagawa 3,4
, Hiroyuki
Sakagami 2 , Miyo
Kakizaki 1 ,
1
Grad. Sch. Comprehensive Human Sci. Univ. Tsukuba, Tsukuba, Japan 2 Dept. Anat. Kitazato Univ. Sch. Med., Sagamihara 3 Dept. Genet. Behav. Neurosci., Gunma Univ. Grad. Sch. Med., Maebashi 4 JST, CREST, Japan
Neuronal elements essential for the spinal locomotor circuit are localized in the ventral region of the spinal cord in mammals. However, the physiological and morphological characteristics of many of the inhibitory interneurons involved in this circuit are yet to be revealed. In this study, we examined the morphology and the firing pattern during locomotion of ipsilaterally projecting GABAergic inhibitory interneurons located ventral to the lumbar motoneuron pool. Utilizing the isolated spinal cord preparation taken from glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mouse neonates, we performed whole-cell recording of visually identified GFP-positive neurons. The recorded neurons were labeled with neurobiotin and their anatomical characteristics were examined using the ABC-DAB staining protocol. Among these neurons, those that were excited by electrical stimulation of the ventral root (VR-S) were identified as Renshaw cells. More than 90% of these neurons extended their axons along the rostrocaudal axis out of the segment in which the soma is located. By contrast, more than 90% of the neurons that did not respond to VR-S had their axon localized within the same segment. More than two-thirds of these segmental neurons were rhythmically active during the locomotor-like rhythmic activity indicating that they are involved in information processing in the local locomotor circuit.
P3-h05 Ghrelin protects spinal cord motoneurons against chronic glutamate-induced excitotoxicity via ERK1/2 and phosphatidyl-3-kinase/Akt/glycogen synthase kinase-3beta pathways Yumi Kim , Eunjin Lim, Sungyoub Lee, Endan Li, Seungjoon Park KyungHee Univ Excitotoxic degeneration of spinal cord motoneurons has been proposed as a pathogenic mechanism in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R) 1a, functions as a neuroprotective factor in various animal models of neurodegenerative diseases. In this study, the potential neuroprotective effects of ghrelin against chronic glutamate-induced cell death were studied by exposing organotypic spinal cord cultures (OSCC) to threohydroxyaspartate (THA), as a model of excitotoxic motoneruon degeneration. Ghrelin receptor was expressed on spinal cord motoneurons. Exposure of OSCC to THA for 3 weeks resulted in a significant loss of motoneurons. However, THA-induced loss of motoneurons was significantly reduced by treatment of ghrelin. Exposure of OSCC to the receptor-specific antagonist D-Lys-3-GHRP-6 abolished the protective effect of ghrelin against THA. Treatment of spinal cord cultures with ghrelin caused rapid phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and glycogen synthase kinase3beta (GSK-3beta). The effect of ghrelin on motoneuron survival was blocked by the MAPK inhibitor PD98059 and the phosphatidylinositol-3-kinase (PI3 K) inhibitor LY294002. Taken together, these findings indicate that ghrelin has neuroprotective effects against chronic glutamate toxicity by activating the MAPK and PI3 K/Akt signaling pathways and suggest that administration of ghrelin may have the potential therapeutic value for the prevention of motoneuron degeneration in human ALS. Our data also suggest that PI3 K/Akt-mediated inactivation of GSK-3beta in motoneurons contribute to the protective effect of ghrelin. Research fund: This study was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 2010-0028329 and 2010-0016284). doi:10.1016/j.neures.2011.07.1063
P3-h06 Comparison of neuronal activity in primary motor cortex with ongoing muscle activity during locomotor movements of an unrestrained Japanese monkey Katsumi Nakajima 1 , Futoshi Mori 2 , Akira Murata 1 , Masahiko Inase 1 1
Dept. of Physiol., Facult. of Med., Kinki Univ., Osaka-Sayama, Japan 2 Dept. of Vet. Neurophysiol., Facult. of Agr., Yamaguchi Univ., Yamaguchi, Japan To further understand cortical mechanisms underlying bipedal gait in humans, we recorded neuronal activity from M1 together with EMGs of a monkey exerting quadrupedal and bipedal locomotion (QL and BL) on a treadmill. We examined to what extent neuronal activity correlates with EMG activity during QL and BL. During QL, EMG activity of antagonistic pairs of trunk and hindlimb muscles displayed an alternate burst per step. For Bp, the burst activity was drastically enhanced; its amplitude and duration were larger and longer compared to QL. Notably, antagonistic muscle pairs in the trunk and thigh were co-activated, and all but ankle flexor muscles peaked during mid stance (ST) phase. To date, 92 neurons were recorded from trunk/hindlimb regions and 57 analyzed. During QL, most neurons discharged phasically or phasically and tonically per step. The peak activity for different cells occurred at around the swing-ST transition and around mid-late ST phase over the step cycle. For BL, the cells showed primarily an increase in their peak discharge frequency with preserving phasic pattern of modulation. The peak activity formed bimodal distribution over the step cycle. Compared to QL, however, the cells peaking during mid ST phase were strikingly smaller in number. The burst duration was unchanged or rather shortened. Spike-triggered averaging revealed that 5 cells showed post-spike facilitation in the hindlimb muscles. Interestingly, the peak activity of these cells was always preceded by that of the projecting muscles. Dissociation of activity patterns between neurons and EMGs indicates that monkey M1 does not control all aspects of locomotor movements. Our results suggest that M1 output modulate on-going locomotor activities of spinal interneurons and
Reference Tanaka, Y., et al., 2011. Multiple EMG activity and intracortical inhibition and facilitation during a fine finger movement under pressure. J. Mot. Behav. 43, 73–81.
e243
Research fund: This study was supported by the Initiative for the Promotion of Young Scientists’ Independent Research program (MEXT), KAKENHI (No. 20500365) and the Brain Science Foundation. doi:10.1016/j.neures.2011.07.1062
doi:10.1016/j.neures.2011.07.1060
P3-h03 In vitro formation of neuromuscular junction with NG108-15 cells and C2C12 myotubes Ayumi Ishihara 1,3 Miyazawa 1,2,3
, Junichi Saito 1 , Yuko Fukunaga 1,2,3 , Atsuo
1
Div. of Cell Biochem., Grad. Sch. of Life Sci., Univ. of Hyogo, Hyogo, Japan 2 RIKEN, SPring-8 Center, Bio-multisome Research Team, Hyogo, Japan 3 JSTCREST, Japan In mature neuromuscular junction (NMJ), the postsynaptic membrane has specialized folds, designated as junctional folds. As NMJ mature, nicotinic acetylcholine receptors (nAChRs) and voltage dependent sodium channels are concentrated to the crests and the troughs of them, respectively. It is well known that they are critical for proper neuronal transmission. The in vitro NMJ model is advantageous to elucidate the mechanisms underlying forming the junctional folds and the precise configurations of these proteins at NMJ by using bio-imaging analysis, genetic engineering and/or electrophysiological experiment. Here, we report the establishment of the in vitro model of NMJ replicating the structure of the in vivo NMJ, using NG108-15 (neuroblastoma and glioma hybrid clone) cells and C2C12 myotubes. Many aspects of differentiated NG108-15 cells are similar to motor neurons. For example, they are cholinergic and express the primary nAChR-aggregating factor, agrin. For the long term culture of C2C12 myotubes, we examined the surface modification for cell attachment. C2C12 myotubes cultured on collagen gel was maintained for more than 5 weeks. Therefore, we seeded differentiated NG108-15 cells on C2C12 myotubes, which are cultured on collagen gel. Colocalization between nAChR and synaptophysin, a presynapse marker, was observed most abundantly in 5 weeks. Scanning electron microscopy showed that the presynaptic varicosities of NG108-15 cells were invaginated into the C2C12 myotubes, resembling primary postsynaptic cleft at the in vivo NMJ. The basal lamina-like density and the junctional fold-like structure between varicosities and C2C12 myotubes were identified by transmission electron microscopy. These results indicate that prolonged (longer than 5 weeks) co-culture and/or other factors are required for maturation of the in vitro NMJ. Research fund: JST-CREST. doi:10.1016/j.neures.2011.07.1061
P3-h04 Characterization of ipsilaterally projecting inhibitory neurons in the ventral spinal cord in mouse neonates Hiroshi Nishimaru 1 Yuchio Yanagawa 3,4
, Hiroyuki
Sakagami 2 , Miyo
Kakizaki 1 ,
1
Grad. Sch. Comprehensive Human Sci. Univ. Tsukuba, Tsukuba, Japan 2 Dept. Anat. Kitazato Univ. Sch. Med., Sagamihara 3 Dept. Genet. Behav. Neurosci., Gunma Univ. Grad. Sch. Med., Maebashi 4 JST, CREST, Japan
Neuronal elements essential for the spinal locomotor circuit are localized in the ventral region of the spinal cord in mammals. However, the physiological and morphological characteristics of many of the inhibitory interneurons involved in this circuit are yet to be revealed. In this study, we examined the morphology and the firing pattern during locomotion of ipsilaterally projecting GABAergic inhibitory interneurons located ventral to the lumbar motoneuron pool. Utilizing the isolated spinal cord preparation taken from glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mouse neonates, we performed whole-cell recording of visually identified GFP-positive neurons. The recorded neurons were labeled with neurobiotin and their anatomical characteristics were examined using the ABC-DAB staining protocol. Among these neurons, those that were excited by electrical stimulation of the ventral root (VR-S) were identified as Renshaw cells. More than 90% of these neurons extended their axons along the rostrocaudal axis out of the segment in which the soma is located. By contrast, more than 90% of the neurons that did not respond to VR-S had their axon localized within the same segment. More than two-thirds of these segmental neurons were rhythmically active during the locomotor-like rhythmic activity indicating that they are involved in information processing in the local locomotor circuit.
P3-h05 Ghrelin protects spinal cord motoneurons against chronic glutamate-induced excitotoxicity via ERK1/2 and phosphatidyl-3-kinase/Akt/glycogen synthase kinase-3beta pathways Yumi Kim , Eunjin Lim, Sungyoub Lee, Endan Li, Seungjoon Park KyungHee Univ Excitotoxic degeneration of spinal cord motoneurons has been proposed as a pathogenic mechanism in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R) 1a, functions as a neuroprotective factor in various animal models of neurodegenerative diseases. In this study, the potential neuroprotective effects of ghrelin against chronic glutamate-induced cell death were studied by exposing organotypic spinal cord cultures (OSCC) to threohydroxyaspartate (THA), as a model of excitotoxic motoneruon degeneration. Ghrelin receptor was expressed on spinal cord motoneurons. Exposure of OSCC to THA for 3 weeks resulted in a significant loss of motoneurons. However, THA-induced loss of motoneurons was significantly reduced by treatment of ghrelin. Exposure of OSCC to the receptor-specific antagonist D-Lys-3-GHRP-6 abolished the protective effect of ghrelin against THA. Treatment of spinal cord cultures with ghrelin caused rapid phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and glycogen synthase kinase3beta (GSK-3beta). The effect of ghrelin on motoneuron survival was blocked by the MAPK inhibitor PD98059 and the phosphatidylinositol-3-kinase (PI3 K) inhibitor LY294002. Taken together, these findings indicate that ghrelin has neuroprotective effects against chronic glutamate toxicity by activating the MAPK and PI3 K/Akt signaling pathways and suggest that administration of ghrelin may have the potential therapeutic value for the prevention of motoneuron degeneration in human ALS. Our data also suggest that PI3 K/Akt-mediated inactivation of GSK-3beta in motoneurons contribute to the protective effect of ghrelin. Research fund: This study was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 2010-0028329 and 2010-0016284). doi:10.1016/j.neures.2011.07.1063
P3-h06 Comparison of neuronal activity in primary motor cortex with ongoing muscle activity during locomotor movements of an unrestrained Japanese monkey Katsumi Nakajima 1 , Futoshi Mori 2 , Akira Murata 1 , Masahiko Inase 1 1
Dept. of Physiol., Facult. of Med., Kinki Univ., Osaka-Sayama, Japan 2 Dept. of Vet. Neurophysiol., Facult. of Agr., Yamaguchi Univ., Yamaguchi, Japan To further understand cortical mechanisms underlying bipedal gait in humans, we recorded neuronal activity from M1 together with EMGs of a monkey exerting quadrupedal and bipedal locomotion (QL and BL) on a treadmill. We examined to what extent neuronal activity correlates with EMG activity during QL and BL. During QL, EMG activity of antagonistic pairs of trunk and hindlimb muscles displayed an alternate burst per step. For Bp, the burst activity was drastically enhanced; its amplitude and duration were larger and longer compared to QL. Notably, antagonistic muscle pairs in the trunk and thigh were co-activated, and all but ankle flexor muscles peaked during mid stance (ST) phase. To date, 92 neurons were recorded from trunk/hindlimb regions and 57 analyzed. During QL, most neurons discharged phasically or phasically and tonically per step. The peak activity for different cells occurred at around the swing-ST transition and around mid-late ST phase over the step cycle. For BL, the cells showed primarily an increase in their peak discharge frequency with preserving phasic pattern of modulation. The peak activity formed bimodal distribution over the step cycle. Compared to QL, however, the cells peaking during mid ST phase were strikingly smaller in number. The burst duration was unchanged or rather shortened. Spike-triggered averaging revealed that 5 cells showed post-spike facilitation in the hindlimb muscles. Interestingly, the peak activity of these cells was always preceded by that of the projecting muscles. Dissociation of activity patterns between neurons and EMGs indicates that monkey M1 does not control all aspects of locomotor movements. Our results suggest that M1 output modulate on-going locomotor activities of spinal interneurons and