Elsevier

Neuroscience

Volume 143, Issue 3, 13 December 2006, Pages 757-767
Neuroscience

Cellular neuroscience
Developmental localization of potassium chloride co-transporter 2 in granule cells of the early postnatal mouse cerebellum with special reference to the synapse formation

https://doi.org/10.1016/j.neuroscience.2006.08.044Get rights and content

Abstract

In the adult CNS, GABA is the predominant inhibitory neurotransmitter, mediating the hyperpolarization of membrane potential and regulating the glutamatergic activity. In the immature CNS, on the other hand, GABA mediates depolarization and is involved in controlling morphogenesis. This developmental shift in GABA actions from depolarization to hyperpolarization occurs as a result of decreasing the intracellular chloride ion (Cl) concentration ([Cl]i) which is regulated by the potassium (K+)-Cl co-transporter 2 (KCC2). To clarify the time-course of changes in the GABA actions during development, we examined the developmental localization of the KCC2 in the granule cells of the postnatal mouse cerebellum using specific antibodies against KCC2. The granule cell precursors and migrating granule cells were devoid of immunoreactivity against KCC2 antibodies. At postnatal day 3 (P3), the KCC2-immunolabeling was negative in the internal granular layer, although synaptophysin-positive mossy fiber terminals were detected. At P5, we first detected the KCC2-immunolabeling at the somata of granule cells and their dendrites before granule cells received inhibitory input from Golgi cells. Almost all KCC2-positive dendrites (more than 98%) attached to and formed synapses with mossy fiber terminals. As development proceeded, the number of KCC2-positive granule cells increased, and all granule cells became positive by P21. These results suggested that GABAergic transmission on granule cells might shift from excitation to inhibition after the synapse formation, and the excitatory synapse-formation and related factors might be the triggers for the expression and localization of the KCC2 in the granule cells. Furthermore, it was also suggested that formation of the GABAergic synapses and GABAergic transmission were not necessary for the KCC2-expression in the mouse cerebellar granule cells in vivo.

Section snippets

Animals

We examined mice from the C57Bl/6CrSlc mice of P0, P3, P5, 7, 10, 12, 14, 21 and 90 (as an adult). At each age, at least five mice (three for light microscopic analysis, two for electron microscopic analysis) were killed for immunohistochemistry.

Establishment of the KCC2 antibody

A cysteine residue was introduced at the amino terminal of the synthesized peptide, PVS SEG IKD FFS MKP EWE NLN, which are amino acid residues 1022–1042 of the mouse KCC2 (AF332064.1, GI:14193695). The peptide was conjugated with keyhole limpet

Immunohistochemical localization of KCC2 in the adult mouse cerebellar cortex

The specificity of the rabbit and guinea-pig KCC2 antibodies was examined by immunoblot analysis and immunohistochemistry. Each immunoblot showed a major band around the predicted molecular weight (140 kD), and the band was abolished by addition of the peptide for immunization into the primary antibody solution (Fig. 1A).

The immunohistochemical staining with rabbit KCC2-antibody showed that the immunolabeling was observed in the all three layers (Fig. 1B). In the molecular layer and Purkinje

Discussion

In the present study, we found that the KCC2 was first localized at the granule cell somata and dendrites, which formed synapses with synaptophysin-positive mossy fiber terminals at P5 as summarized in Fig. 6. The KCC2-positive granule cells increased in number during development, and all granule cells became positive by P21.

Acknowledgments

We are grateful to Hideki Nakamura at the Central Research Department of Hokkaido University School of Medicine for the technical assistance. We also thank to Dr. Hitoshi Komuro at Cleveland Clinic Foundation and Sachiko Yoshida at Toyohashi University of Technology for the valuable discussion.

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