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Characterization of a transneuronal cytokine family Cbln
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Abstracts / Neuroscience Research 58S (2007) S1–S244
P1-aØ8 Characterization of a transneuronal cytokine family Cbln
Iijima 1 ,
Miura 2 ,
Matsuda 1 ,
P1-a11 Cbln1 directly induces excitatory presynaptic assembly in cerebellar Purkinje cells
Takatoshi Eriko Keiko Yuichi Kamekawa 1 , Masahiko Watanabe 2 , Michisuke Yuzaki 1 1 Department of Physiology, School of Medicine, Keio University, Tokyo, Japan; 2 Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan
Aya Ishida 1 , Keiko Matsuda 1 , Tetsuro Kondo 1,2 , Takatoshi Iijima 1 , Michisuke Yuzaki 1 1 Department of Physiology, Keio University, Tokyo, Japan; 2 National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan
Cbln1, a member of the C1q and tumor necrosis factor superfamily, plays crucial roles as a cerebellar granule cell-derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although other Cbln family members Cbln2–Cbln4 have distinct spatial and temporal patterns of expression throughout the central nervous system, their biochemical and biological properties have remained largely uncharacterized. Here, we demonstrated that Cbln2 and Cbln4 were secreted glycoproteins like Cbln1. In contrast, Cbln3 was not secreted when expressed alone, and retained in the endoplasmic reticulum or cis-Golgi. In wild-type granule cells expressing Cbln1 and Cbln3, Cbln3 proteins were secreted and reached postsynaptic sites on Purkinje cell dendrites, while Cbln3 was dramatically degraded in cbln1-null granule cells. These results indicate that Cbln2–Cbln4 may also serve as a trans-neuronal regulator of synaptic functions in various brain regions.
Cbln1 is a hexameric glycoprotein secreted from cerebellar granule cells. Although Cbln1−/− mice show impaired synapse formation and plasticity at parallel fiber-Purkinje cell synapses, its direct function has not been assessed. Here, we examined the effect of exogenous Cbln1 on Purkinje cell synapses. We found that Cbln1 in the medium increased synaptophysin-positive puncta that colocalized with dendrites of cultured Purkinje cells. Whole-cell patch-clamp recordings revealed that incubation of cerebellar slices with Cbln1 for as short as 8 h could restore the reduced frequency, but not the amplitude, of miniature excitatory postsynaptic currents in Cbln1−/− Purkinje cells. Interestingly, mutant Cbln1, in which two cysteine residues were replaced with alanine to prevent hexamerization, could not rescue the structural and functional properties of synapses in Cbln1−/− Purkinje cells. These results indicate that Cbln1 forms a hexamer and directly induces excitatory presynaptic assembly.
Research funds: KAKENHI (18700324)
P1-aØ9 Change in superfusion rate alters excitatory synaptic transmission in rat hippocampal slice Shingo Kimura, Satoshi Kawasaki, Shuji Watanabe, Reiko Fujita, Kazuhiko Sasaki Department of Physiology, School of Medicine, Iwate Medical University, Morioka, Japan
P1-a12 Ca2+ clearance mechanisms in presynaptic terminals: Ca2+ uptake to mitochondria started when [Ca2+ ]i increased to a critical level Naoya Suzuki, Yuji Motou Department of Physics, School of Science, Nagoya University, Nagoya, Japan
In CA1 pyramidal neurons of hippocampal slice, increase in rate of superfusion of the medium (fast superfusion) markedly augments the amplitude of EPSC evoked by stimulation of Schaffer collateral. However, the fast superfusion did not affect both AMPA-induced current response and IPSC evoked by stimulation of striatum oriens in the same cell. Paired pulse ratio of the EPSC decreased during the fast superfusion and a rightward shift was observed in the peak of the amplitude distribution histogram of the evoked-EPSCs in low [Ca2+ ]o . Moreover, application of either A1 receptor antagonist DPCPX or ecto-ATPase blocker ARL67156 augmented the EPSC amplitude, and subsequent fast superfusion did not cause additional augmentation of the EPSC amplitude in the presence of DPCPX or ARL67156. These results suggest that major cause for the augmenting effect may be increase in glutamate release from the presynaptic terminal due to decrease in concentration of adenosine normally existing nearby the synapse and inhibiting the transmitter release.
To investigate the Ca2+ clearance mechanisms in presynaptic terminals, we measured Ca2+ dynamics during nerve stimulation in frog neuromuscular junctions. During 100 Hz tetanus for 4 sec. in a normal Ringer’s solution (1.8 mM Ca2+ , 0 mM Mg2+ ), [Ca2+ ]i increased in two phases, steep rising for about 0.2 s then slow increasing. When mitochondria were inhibited by rotenone and oligomycin, slow phase disappeared and [Ca2+ ]i increased monotony with rapid rate. Membrane voltage imaging of mitochondria with TMRE suggest that Ca2+ uptake to mitochondria accompanied by depolarization of membrane potential. When tetanus frequency was reduced to 20 Hz, [Ca2+ ]i dynamics did not affected by rotenone and oligomycin, however, CCCP increased [Ca2+ ]i during tetanus. The effect of rotenone and oligomycin appeared when [Ca2+ ]i increased more than 0.6 M during 40 Hz tetanus. Inhibition of Ca2+ uptake to ER did not change [Ca2+ ]i during tetanus at 40 Hz. Each inhibition of plasma membrane Ca2+ pump and Na+ /Ca2+ exchanger increased [Ca2+ ]i during tetanus at 20 Hz.
P1-a1Ø Enhancement by phospholipase A2 activation of glycin-
P1-a13 Differential distribution of parvalbumin positive bou-
ergic and GABAergic inhibitory transmission in rat substantia gelatinosa neurons
Tao Liu, Tsugumi Fujita, Hai-Yuan Yue, Kotaro Mizuta, Lian-Hua Piao, Terumasa Nakatsuka, Eiichi Kumamoto Department of Physiology, Faculty of Medicine, Saga University, Saga, Japan Phospholipase A2 (PLA2 ) activation enhances glutamatergic excitatory transmission in substantia gelatinosa (SG) neurons. The present study examined a detail of the effect of melittin on inhibitory transmission in SG neurons in adult rat spinal cord slices by using the whole-cell patch-clamp technique. Melittin enhanced the amplitude and frequency of GABAergic and glycinergic spontaneous IPSCs (sIPSCs). The GABAergic but not glycinergic transmission enhancement was suppressed by tetrodotoxin or glutamate-receptor antagonists. These actions were reduced in extent by PLA2 inhibitors. These results indicate that PLA2 activation enhances glycinergic transmission in SG neurons and that GABAergic transmission is enhanced by the facilitation of excitatory transmission by PLA2 activation and subsequent increases in glutamate-receptor activities resulting in the excitation of SG neurons. It remains to be examined what kinds of neurotransmitters are involved in the GABAergic transmission enhancement.
tons for two populations of pyramidal cells A. Matsushita, N. Ichinohe, A. Knight, K.S. Rockland RIKEN BSI, Saitama, Japan In rat perirhinal cortex, parvalbumin (PV+) boutons form a honeycomb at the border of layers (L) 1 and 2, with PV+ walls. By double labeling for PV and MAP2 (for dendrites), we found that apical dendrites of L2 pyramidal cells occurred mainly in the PV+ walls. As distal dendrites of L5 pyramidal cells occupy PV-sparse hollows, the dendrites of L2 cells are predicted to have more PV+ contacts than distal dendrites of L5 cells. Here, we used confocal analysis to quantify PV+ boutons (double labeled for VGAT) in contact with EGFP-expressing pyramidal cells in a 60 m-wide zone of L2. L2 dendrites (n = 11) had an average of 19.5 contacts (range = 7–38), but L5 dendrites (n = 11) had an average of only 5.0 (range = 0–10). This confirms a differential density of PV+ boutons for L2 vs. L5 cells at the level of the L2 honeycomb. Boutons were sometimes clustered, but typically single, with 7–30 m gaps. A next question concerns the relative perisomatic density of PV+ boutons for these two populations. We expect a population-specific proximal-distal ratio of PV+ boutons, significant for modes of synaptic integration.
Abstracts / Neuroscience Research 58S (2007) S1–S244
P1-aØ8 Characterization of a transneuronal cytokine family Cbln
Iijima 1 ,
Miura 2 ,
Matsuda 1 ,
P1-a11 Cbln1 directly induces excitatory presynaptic assembly in cerebellar Purkinje cells
Takatoshi Eriko Keiko Yuichi Kamekawa 1 , Masahiko Watanabe 2 , Michisuke Yuzaki 1 1 Department of Physiology, School of Medicine, Keio University, Tokyo, Japan; 2 Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan
Aya Ishida 1 , Keiko Matsuda 1 , Tetsuro Kondo 1,2 , Takatoshi Iijima 1 , Michisuke Yuzaki 1 1 Department of Physiology, Keio University, Tokyo, Japan; 2 National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan
Cbln1, a member of the C1q and tumor necrosis factor superfamily, plays crucial roles as a cerebellar granule cell-derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although other Cbln family members Cbln2–Cbln4 have distinct spatial and temporal patterns of expression throughout the central nervous system, their biochemical and biological properties have remained largely uncharacterized. Here, we demonstrated that Cbln2 and Cbln4 were secreted glycoproteins like Cbln1. In contrast, Cbln3 was not secreted when expressed alone, and retained in the endoplasmic reticulum or cis-Golgi. In wild-type granule cells expressing Cbln1 and Cbln3, Cbln3 proteins were secreted and reached postsynaptic sites on Purkinje cell dendrites, while Cbln3 was dramatically degraded in cbln1-null granule cells. These results indicate that Cbln2–Cbln4 may also serve as a trans-neuronal regulator of synaptic functions in various brain regions.
Cbln1 is a hexameric glycoprotein secreted from cerebellar granule cells. Although Cbln1−/− mice show impaired synapse formation and plasticity at parallel fiber-Purkinje cell synapses, its direct function has not been assessed. Here, we examined the effect of exogenous Cbln1 on Purkinje cell synapses. We found that Cbln1 in the medium increased synaptophysin-positive puncta that colocalized with dendrites of cultured Purkinje cells. Whole-cell patch-clamp recordings revealed that incubation of cerebellar slices with Cbln1 for as short as 8 h could restore the reduced frequency, but not the amplitude, of miniature excitatory postsynaptic currents in Cbln1−/− Purkinje cells. Interestingly, mutant Cbln1, in which two cysteine residues were replaced with alanine to prevent hexamerization, could not rescue the structural and functional properties of synapses in Cbln1−/− Purkinje cells. These results indicate that Cbln1 forms a hexamer and directly induces excitatory presynaptic assembly.
Research funds: KAKENHI (18700324)
P1-aØ9 Change in superfusion rate alters excitatory synaptic transmission in rat hippocampal slice Shingo Kimura, Satoshi Kawasaki, Shuji Watanabe, Reiko Fujita, Kazuhiko Sasaki Department of Physiology, School of Medicine, Iwate Medical University, Morioka, Japan
P1-a12 Ca2+ clearance mechanisms in presynaptic terminals: Ca2+ uptake to mitochondria started when [Ca2+ ]i increased to a critical level Naoya Suzuki, Yuji Motou Department of Physics, School of Science, Nagoya University, Nagoya, Japan
In CA1 pyramidal neurons of hippocampal slice, increase in rate of superfusion of the medium (fast superfusion) markedly augments the amplitude of EPSC evoked by stimulation of Schaffer collateral. However, the fast superfusion did not affect both AMPA-induced current response and IPSC evoked by stimulation of striatum oriens in the same cell. Paired pulse ratio of the EPSC decreased during the fast superfusion and a rightward shift was observed in the peak of the amplitude distribution histogram of the evoked-EPSCs in low [Ca2+ ]o . Moreover, application of either A1 receptor antagonist DPCPX or ecto-ATPase blocker ARL67156 augmented the EPSC amplitude, and subsequent fast superfusion did not cause additional augmentation of the EPSC amplitude in the presence of DPCPX or ARL67156. These results suggest that major cause for the augmenting effect may be increase in glutamate release from the presynaptic terminal due to decrease in concentration of adenosine normally existing nearby the synapse and inhibiting the transmitter release.
To investigate the Ca2+ clearance mechanisms in presynaptic terminals, we measured Ca2+ dynamics during nerve stimulation in frog neuromuscular junctions. During 100 Hz tetanus for 4 sec. in a normal Ringer’s solution (1.8 mM Ca2+ , 0 mM Mg2+ ), [Ca2+ ]i increased in two phases, steep rising for about 0.2 s then slow increasing. When mitochondria were inhibited by rotenone and oligomycin, slow phase disappeared and [Ca2+ ]i increased monotony with rapid rate. Membrane voltage imaging of mitochondria with TMRE suggest that Ca2+ uptake to mitochondria accompanied by depolarization of membrane potential. When tetanus frequency was reduced to 20 Hz, [Ca2+ ]i dynamics did not affected by rotenone and oligomycin, however, CCCP increased [Ca2+ ]i during tetanus. The effect of rotenone and oligomycin appeared when [Ca2+ ]i increased more than 0.6 M during 40 Hz tetanus. Inhibition of Ca2+ uptake to ER did not change [Ca2+ ]i during tetanus at 40 Hz. Each inhibition of plasma membrane Ca2+ pump and Na+ /Ca2+ exchanger increased [Ca2+ ]i during tetanus at 20 Hz.
P1-a1Ø Enhancement by phospholipase A2 activation of glycin-
P1-a13 Differential distribution of parvalbumin positive bou-
ergic and GABAergic inhibitory transmission in rat substantia gelatinosa neurons
Tao Liu, Tsugumi Fujita, Hai-Yuan Yue, Kotaro Mizuta, Lian-Hua Piao, Terumasa Nakatsuka, Eiichi Kumamoto Department of Physiology, Faculty of Medicine, Saga University, Saga, Japan Phospholipase A2 (PLA2 ) activation enhances glutamatergic excitatory transmission in substantia gelatinosa (SG) neurons. The present study examined a detail of the effect of melittin on inhibitory transmission in SG neurons in adult rat spinal cord slices by using the whole-cell patch-clamp technique. Melittin enhanced the amplitude and frequency of GABAergic and glycinergic spontaneous IPSCs (sIPSCs). The GABAergic but not glycinergic transmission enhancement was suppressed by tetrodotoxin or glutamate-receptor antagonists. These actions were reduced in extent by PLA2 inhibitors. These results indicate that PLA2 activation enhances glycinergic transmission in SG neurons and that GABAergic transmission is enhanced by the facilitation of excitatory transmission by PLA2 activation and subsequent increases in glutamate-receptor activities resulting in the excitation of SG neurons. It remains to be examined what kinds of neurotransmitters are involved in the GABAergic transmission enhancement.
tons for two populations of pyramidal cells A. Matsushita, N. Ichinohe, A. Knight, K.S. Rockland RIKEN BSI, Saitama, Japan In rat perirhinal cortex, parvalbumin (PV+) boutons form a honeycomb at the border of layers (L) 1 and 2, with PV+ walls. By double labeling for PV and MAP2 (for dendrites), we found that apical dendrites of L2 pyramidal cells occurred mainly in the PV+ walls. As distal dendrites of L5 pyramidal cells occupy PV-sparse hollows, the dendrites of L2 cells are predicted to have more PV+ contacts than distal dendrites of L5 cells. Here, we used confocal analysis to quantify PV+ boutons (double labeled for VGAT) in contact with EGFP-expressing pyramidal cells in a 60 m-wide zone of L2. L2 dendrites (n = 11) had an average of 19.5 contacts (range = 7–38), but L5 dendrites (n = 11) had an average of only 5.0 (range = 0–10). This confirms a differential density of PV+ boutons for L2 vs. L5 cells at the level of the L2 honeycomb. Boutons were sometimes clustered, but typically single, with 7–30 m gaps. A next question concerns the relative perisomatic density of PV+ boutons for these two populations. We expect a population-specific proximal-distal ratio of PV+ boutons, significant for modes of synaptic integration.