Developmental changes in KCC1, KCC2 and NKCC1 mRNAs in the rat cerebellum

doi:10.1016/S0165-3806(02)00345-0

Developmental Brain Research

Volume 136, Issue 2, 30 June 2002, Pages 93-100

https://doi.org/10.1016/S0165-3806(02)00345-0 Get rights and content

Abstract

Cation chloride cotransporters are considered to play pivotal roles in controlling the intracellular and extracellular ionic environments of neurons, hence controlling neuronal function. To establish how these cotransporters are involved in cerebellum development, we investigated the expression of KCC1, KCC2 and NKCC1 mRNAs in the developing rat cerebellum using in situ hybridization histochemistry. In the external germinal layer, where premature cells exist, we found substantial KCC1 and NKCC1 mRNA expression on P7 and P14, while KCC2 mRNA was not detected. In contrast, KCC2 mRNA was already expressed in Purkinje cells on P1. We also observed KCC2 mRNA expression in postmigratory granule cells after P7. The expression of KCC1, KCC2, and NKCC1 mRNAs reached adult patterns by P21. In the adult cerebellum, KCC2 mRNA was expressed in most neurons, including Purkinje cells, granule cells, and stella/basket cells, while KCC1 and NKCC1 mRNAs were only detected in granule cells and glial cells. These findings suggest that in the rat cerebellum KCC2 mRNA expression is induced when neurons arrive their final destinations.

Introduction

Cation chloride cotransporters are considered to play key roles in controlling the intracellular and extracellular ionic environments of neurons, hence controlling neuronal function [8]. To date, seven members of the cation-Cl⁻ cotransporter gene family have been reported, and they are designated by their ion selectivity as KCC (KCC1–KCC4) for K⁺ dependent Cl⁻ cotransporters, NCC for Na⁺ dependent (NCC1/TSC1), or NKCC (NKCC1 and NKCC2) for cotransporters that depend on the transmembrane gradients of Na⁺ and K⁺ [5], [6], [7], [11], [12], [14], [20], [23].

In the nervous system, four members of this gene family, i.e. K-Cl cotransporter 1 (KCC1), K-Cl cotransporter 2 (KCC2), K-Cl cotransporter 3 (KCC3) and Na-K-2Cl cotransporter 1 (NKCC1) are expressed [5], [7], [12], [15], [16], [22]. Of these, KCC1, KCC2 and NKCC1 have been well investigated. Under physiological conditions, KCC1 and 2 appear to mainly extrude Cl⁻ out of the cell [15]. In contrast to the ubiquitous presence of KCC1, expression of KCC2 is detected only in the nervous system and seems to be neuronal specific [19], [22]. In neurons, KCC2-mediated Cl⁻ extrusion induced during postnatal development has been reported to be responsible for the change in Cl⁻ reversal potential that converts GABA from a depolarizing to a hyperpolarizing neurotransmitter [14], [19]. NKCC1 is a transporter that promotes accumulation of Cl⁻ into the cell and whose activity is regulated by Na⁺ gradient [20], [21]. Taken together, the balance between the above mentioned Cl⁻ extrusion system (KCC1 and KCC2) and the Cl⁻ accumulation system (NKCC1) might also play a pivotal role in regulating the intracellular Cl⁻ concentration of a given cell in the nervous system.

The rat cerebellum is anatomically immature at birth and considerable neurogenesis and synaptogenesis are known to take place postnatally [1]. Although the distributions of KCC1, 2 and NKCC1 have been reported using immunohistochemistry and in situ hybridization [4], [9], [10], [15], [18], [17], [19], [22], little information is available on their expression profiles in the developing cerebellum. We therefore investigated developmental changes in KCC1, 2 and NKCC1 mRNA expression in the developing cerebellum.

Section snippets

In situ hybridization

Male Wistar rats of various ages (at postnatal days 1, 7, 14, 21, 49; n=3 at each time point) were decapitated under diethylether anesthesia. The fresh brains were quickly removed and immediately frozen on powdered dry ice. Serial sections (20 μm thick) were cut on a cryostat, thaw-mounted onto silan-coated slides, and stored at −80 °C. After being warmed to room temperature, slide-mounted sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 15 min (all steps were

General expression patterns

To investigate KCC1, KCC2, and NKCC1 mRNA expression in the developing rat cerebellum, we used three probes already used in our previous report [9], where the specificity of the probes was confirmed. Positive cells were divided into four categories (very strong, strong, moderate, and low) by visual comparison of the extent of silver grain accumulation. Fig. 1 shows the overview of developmental changes in KCC1, KCC2, and NKCC1 mRNA expression. On the first postnatal day (P1), KCC2 mRNA was

Discussion

The rat cerebellum is anatomically immature at birth and considerable neurogenesis and synaptogenesis are known to take place postnatally [1]. Thus it is used for investigating mechanisms underlying neuronal development. In the present study, we observed that the expression of KCC1, KCC2 and NKCC1 mRNAs was very strictly regulated with respect to time and space. The main findings of this study are: (i) premature cells in the external germinal layer abundantly expressed KCC1 and NKCC1 mRNAs but

Acknowledgements

Part of this work was supported by the Ministry of Education, Science and Culture of Japan and the Ministry of Health and Welfare of Japan.

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Research reportDevelopmental changes in KCC1, KCC2 and NKCC1 mRNAs in the rat cerebellum

Abstract

Introduction

Section snippets

In situ hybridization

General expression patterns

Discussion

Acknowledgements

Trends Neurosci.

Dev. Brain Res.

J. Biol. Chem.

J. Biol. Chem.

Prog. Neurobiol.

Neuroscience

Biochem. Biophy. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Neurogenesis and neuronal migration

Research report
Developmental changes in KCC1, KCC2 and NKCC1 mRNAs in the rat cerebellum