expression during development and plasticity (II, IV)
Previously we have shown that KCC2 expression appears as earlier as 3DIV in dissociated cultures before synapse formation, and is up-regulated following neuronal maturation. To examine whether neuronal activity and synaptic transmission affect the developmental up-regulation of KCC2 in dissociated and organotypic cultures (see Ganguly et al., 2001), we used chronic treatment on hippocampal cultures with the action-potential blocker TTX (10μM); NMDA and non-NMDA antagonist, APV (40μM) and NBQX
(10μM); as well as the GABAA receptor blocker PTX (50μM). Measurements of KCC2 immunostaining intensity did not reveal any signifi cant alteration in KCC2 developmental up-regulation in cultures treated with various combinations of the blockers (II; Fig. 5). The results were further confi rmed by western blot analysis.
These data show that neither neuronal spiking nor ionotropic glutamatergic and GABAergic transmission are required for the developmental expression of KCC2 in mouse hippocampal neurons in vitro.
The data presented do not rule out the possibility that expression of KCC2 could be modulated by trophic factors, such as brain-derived neurotrophic factor (Aguado et al., 2003) or insulin-like growth factor1 (Kelsch et al., 2001). Indeed, when studying the expression of KCC2 during plasticity, we observed down-regulation of KCC2 mRNA and protein levels by spontaneous activity. In these experiments, hippocampal slices were exposed to a solution devoid of Mg 2+ (0- Mg 2+). After 1-3 hours, RT-PCR from the slices showed that there is a progressive fall of KCC2 mRNA that starts within 30min exposure.
Hippocampal slices in Mg 2+ -free solution usually generate continuous interictal-like spontaneous activity (Anderson et al., 1986; Mody et al., 1987), which commences at ~10-15 min and shows a progressive increase in frequency during the 1-3hr recording sessions. Western blot analyses showed a pronounced decrease of KCC2 protein during 0- Mg 2+ exposure for 1-3 hr (IV; Fig. 1). The down-regulation of KCC2 expression was not caused by the withdrawal of Mg 2+ per se but by the consequent discharge activity, because pharmacologically blocking the activity with NBQX plus AP-5 retained KCC2 mRNA as well as protein at a level that was indistinguishable from parallel controls.
This activity-induced down-regulation displayed an area-specifi c pattern shown by KCC2 immunofluorescent staining in the slices. The decrease was most prominent in the CA1 region than other regions (IV; Fig. 2).
BDNF-TrkB-mediated signaling has been found to be a major molecular cascade responsible for changes in protein expression required for plasticity such as LTP and epileptogenesis. To investigate the molecular mechanism for activity-induced downregulation of KCC2, we superfused acute hippocampal slices with 0-Mg2+ solution for 3 hr in the presence or absence of the tyrosine kinase inhibitor K252a, which prevents TrkB activation by inhibiting autophosphorylation of the receptors. KCC2 mRNA and protein levels remained at control levels in the presence of K252a (IV; Fig. 6). Control experiments showed that K252a had no effect on 0-Mg2+ induced interictal-like spontaneous activity. Similar results were obtained when TrkB receptor bodies (TrkB-Fc) were applied to slices to scavenge endogenously released BDNF. These results demonstrated that TrkB activation by endogenous BDNF is required for the activity-induced down-regulation of KCC2 .
BDNF-TrkB intracellular signaling is activated through the autophosphorylation of tyrosine residues of the intracellular domain of TrkB. There are two tyrosine phosphorylation residues creating docking sites for recruiting signaling adaptor proteins linked to PLCγ (Tyr 816), and Shc/FRS-2 (Tyr 515) (Bibel and Barde, 2000; Minichiello et al., 2002). These two docking sites mediate two different signaling cascades: PLCγ activates the CaM kinase/IP3 kinase signal pathway, and Shc/FRS-2 the Ras/MAPK pathways. To further examine which signaling pathway,
downstream of TrkB, is involved in the downregulation of KCC2, we analyzed two different transgenic mice, one with a targeted mutation in the PLCγ docking site of TrkB (TrkBPLC/PLC), and the other with a targeted mutation in the Shc binding site (TrkBSHC/SHC). Interestingly, KCC2 protein levels were not significantly affected in TrkBSHC/SHC mutant slices, indicating that the activation of Shc pathway is necessary for KCC2 down-regulation.
Strikingly, KCC2 protein levels in the TrkBPLC/PLC mutant slices were elevated by the exposure to BDNF, indicating that in the absence of PLCγ cascades, the Shc pathway is able to upregulate KCC2 (IV;
Fig. 8).
Similar results were obtained from experiments in which TrkB mutant slices were exposed to the 0-Mg2+ solution for 3 hr. In addition, immunostaining for pCREB demonstrated that CREB activation (Finkbeiner et al., 1997; Bibel and Barde, 2000; Minichiello et al., 2002) may be involved in the mechanism for KCC2 downregulation in 0-Mg2+ treated slices. Scavenging endogenous BDNF also showed suppressed CREB activation in slices exposed to the 0-Mg2+ solution. The robust increase in CREB phosphorylation observed in the CA1 region after 1 hr coincides with the down-regulation of KCC2 in the same region. These data support the involvement of CREB in regulating KCC2 transcription under spontaneous activity.
These results show that the expression of KCC2 is regulated by neuronal activity.
Consequently, neuronal hyperactivity leads to compromised Cl- extrusion which is required for GABAergic postsynaptic hyperpolarization. This prediction was demonstrated experimentally by measuring the reversal potential of GABAA-mediated IPSPs with a somatic
chloride load. The driving forces of IPSPs between control slices and 0-Mg2+
treated slices were signifi cantly different.
In treated slices depolarizing reversal potentials was measured as compared to control. Our results provide evidence for the fast qualitative change in GABAergic transmission from hyperpolarizing to depolarizing under epileptic-like conditions (Kapur et al., 1995; Nabekura et al., 2002; Payne et al., 2003; see also Kaila et al., 1997). Interestingly, a recent report suggests that a Ca2+-dependent decrease in K-Cl cotransport activity is associated with coincident presynaptic and postsynaptic spiking (Woodin et al., 2003).
This effect may share the same mechanism as indicated in our studies.
Our results revealed a direct link between BDNF-TrkB signaling and downregulation of KCC2 and the underlying intracellular signaling
cascades. We found that both the PLCγ and Shc/FRS-2 activated signaling cascades are required for KCC2 down-regulation.
Experiments from transgenic TrkBPLC/PLC and TrkBSHC/SHC mice suggested that the Shc pathway is crucial for both the down-regulation and up-down-regulation of KCC2.
Down-regulation of KCC2 appears to take place if the Shc pathway is activated in conjunction with the PLCγ cascades, whereas an up-regulation is triggered by the Shc pathway acting in the absence of the PLCγ cascades. These may partly explain the different effects of BDNF on KCC2 mRNA expression in immature and mature neurons (Aguado et al., 2003; Rivera et al., 2002). We speculate that similar mechanisms for long-term plasticity are important under normal and pathophysiological conditions as well as in the maturation of functional neuronal networks.
Conclusions
1. The spatiotemporal expression patterns of distinct CCCs during embryonic development suggests that Cl- regulatory mechanisms are critically involved in the control of neuronal development.
2. Developmental expression of KCC2 in vivo and in vitro follows neuronal maturation and is strongly correlated with synaptogenesis, but neither neuronal spiking nor ionotropic glutamatergic and GABAergic transmission are required for the developmental up-regulation of KCC2 expression in mouse cultured cortical neurons.
3. KCC2-/- cortical neurons have abnormal morphology of dendritic protrusions and the number of functional excitatory synapses is reduced in KCC2-/- neurons. The interaction between KCC2 and dendritic cytoskeleton protein, 4.1N, is required for spine development.
4. The expression of KCC2 is regulated in diverse manifestations of neuronal plasticity. We show that interictal-like activity down-regulates KCC2 mRNA and protein expression in hippocampal slices, which leads to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF acting on TrkB, with down-stream cascades involving both Shc/FRS-2 and PLCγ cAMP response element-binding protein signaling.
Acknowledgments
This study was carried out at the Institute of Biotechnology and Department of Environmental and Biological Sciences under the supervision of Dr. Claudio Rivera and Professor Kai Kaila during 2000-2007.
First of all, I would like to express my deepest thanks to Claudio who took me into his group.
Claudio has endless kindness and patience in helping me deal with every detail of my experiments among these years. More importantly, he has given me the freedom and opportunities to think and test different aspects of the subject. Along my Ph.D study, his continued encouragement accelerated my enthusiasm in science. Without his help, I would have not been able to complete my Ph.D studies.
I want to thank Kai who gave me the opportunity to be his student and led me into the interesting fi eld of neuroscience. It is enjoyable to be his student because of his patience and scientifi c advices. Without his guidance and encouragement, I would have not been able to fi nish my Ph.D study.
I would also give my special thank to professor Mart Saarma, the Director of the Institute of Biotechnology, who also supervised my Ph.D study. Mart encouraged me continuously and provided valuable advices and suggestions to improve my thesis work. In addition, I am grateful to him for providing excellent facilities and resources for my research work.
I warmly thank Professor Dan Lindholm and Professor Kid Tornquist for reviewing my thesis and providing constructive criticisms to the thesis book.
Special thanks to Miika Palviainen and Marjo Heikura, for their technical supports, in particular for their pleasant help, all the fun in the lab and long-term friendship. Warmly thanks to Sergey for teaching me electrophysiology method. Thanks to Anastasia Ludwig, Anastasia Shulga, Hannele Lahtinen, Judith Thomas-Crusells, for relax work atmosphere and collaborations.
I am grateful to Docent Matti Airaksinen for collaborations and giving me valuable advices and suggestions in my thesis work. Thanks Janne Tornberg for pleasant collaboration and help.
I would like to thank Stanislav Khirug and Peter Blaesse for collaboration. All the members in the group have been a great help. Professor Juha Voipio, Eva Ruusuvuori, Junko Yamada.
Deeply thanks to Katri Wegelius for providing kind help with my study and living in Finland.
Many Thanks to Leonard Khiroug, Julia Kolikova, Ramil Afzalov, Kari Keinanen, Sarah.
Coleman, Sari Lauri for pleasant collaborations.
Many thanks to the Neurogroup members, both former and present, have been a great help, Liying Yu, Jianmin Yang, Urmas Arumae, Congyun Zheng, Maili Jakobson, Maria Lindahl, Satu Akerber, Eila Kujamaki, Pavel Uvarov, Jari Rossi, Paivi Lindfors, Yunfu Sun, Xinqun Liang, Maxim Bespalov, Mikhail Paveliev, Paivi Lindholm, Pia Runeberg-Roos, Maria Lumen, Liina Lonka.
Outside of the lab, I would like to thank Li Tian, for her friendship and help during these years.
Finally, I would like to thank my husband, Chunlin, for his love, collaboration and support;
and our son, Yanzhe, for bringing me endless happiness.
Hong Li, November 2007, Helsinki
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