Elsevier

Brain Research

Volume 984, Issues 1–2, 12 September 2003, Pages 149-159
Brain Research

Research report
Differential development of cation-chloride cotransporters and Cl homeostasis contributes to differential GABAergic actions between developing rat visual cortex and dorsal lateral geniculate nucleus

https://doi.org/10.1016/S0006-8993(03)03126-3Get rights and content

Abstract

A recent study suggested that γ-aminobutyric acid (GABA) plays differential roles in activity-dependent plasticity between the visual cortex (VC) and the dorsal lateral geniculate nucleus (dLGN). In the present study, to investigate differential GABAergic functions in postnatal visual system development, the development of [Cl]i, cation-Cl cotransporter expression, and the [Ca2+]i responses evoked by GABA were compared between VC and dLGN during the early stages of development. Using rat brain slices from postnatal days (P) 0–17, GABA-evoked [Ca2+]i responses and resting [Cl]i were measured by means of optical imaging of Ca2+ and Cl, respectively. Changes in the expression of cation-Cl cotransporters (viz. the outwardly-directed K+-Cl cotransporter, KCC2, and the inwardly-directed Na+,K+-2Cl cotransporter, NKCC1) were examined in VC and dLGN by in situ hybridization. At birth, the excitatory actions of GABA were powerful in VC, but missing in dLGN (as indicated by neuronal [Ca2+]i transients), and the resting [Cl]i was significantly higher in VC than in dLGN. Signals for KCC2 mRNA expression were significantly higher in dLGN than in VC at P0. This suggests that extrusion of Cl from neurons is stronger in dLGN than in VC at P0, so that a GABAergic excitatory effect was not observed in dLGN because of more negative equilibrium potential for Cl. The present study indicates clear differences in the molecular and physiological bases of Cl homeostasis and GABA actions between the developing VC and dLGN. Such differential GABAergic actions may underlie the distinct mechanisms involved in VC and dLGN development within the visual system.

Introduction

In the mammalian visual system, activity-dependent refinements of functional and anatomical connections occur in VC and dLGN during the early stages of development. In VC, these refinements lead to the development of ocular dominance columns [14], [23], [24], [25], [40], while in dLGN they lead to the segregation of the initially intermixed retinal ganglion cell axons from the two eyes [1], [29]. If the sensory input is blocked during the critical period, ocular dominance columns do not form normally [34], [38], [39]. Moreover, using GAD65-knockout mice, in which GABAergic inhibitory transmission is reduced, Hensch et al. found that they did not respond to a 4-day period of monocular vision in the critical period unlike wild-type which responded by a shift of ocular dominance [12]. In contrast, in dLGN the critical period for segregation was unaffected in GAD65-knockout mice [13]. These reports suggest that γ-aminobutyric acid (GABA) may play differential roles in activity-dependent plasticity between VC and dLGN. GABAA-receptor-mediated responses depend mainly on the cross-membrane Cl gradient, which is itself determined by the intracellular Cl concentration ([Cl]i). Differential GABAergic functions could thus be explained by differences in Cl homeostasis between brain areas [36].

Of various Cl regulators, cation-chloride cotransporters are considered to play a critical role in Cl homeostasis [18], [36]. Under physiological conditions, KCC2 (a K+-Cl cotransporter) appears to extrude Cl from the cell [16], [19], [32], [36], while NKCC1 (a Na+, K+-2Cl cotransporter) is a candidate for the promotion of Cl accumulation within the cell [5], [31], [35]. Thus, differential GABAergic roles in synaptic plasticity between the developing VC and dLGN could be attributable to differences in [Cl]i caused by differential expressions of KCC2 and NKCC1.

We therefore examined the differential development of resting [Cl]i, KCC2 and NKCC1 mRNA expressions and GABA-evoked [Ca2+]i increases which is essential for synaptogenesis [2], [3], [22], [27] between VC and dLGN during the early stages of development in the rat.

Section snippets

Materials and methods

All experiments conformed to guidelines on the ethical use of animals for research in Hamamatsu University School of Medicine, and all efforts were made to minimize both the number of animals used and any suffering caused.

GABA-induced [Ca2+]i transients in VC and dLGN at various developmental ages

Changes in [Ca2+]i induced by 100 μM of GABA were measured at the center of cell bodies in VC (of cortical-plate cells at P0 and P3, and of pyramidal neurons in layer II/III at P6, 10, 13, and 17). In dLGN, measurements were made from presumed relay neurons at the same ages.

Cortical plate neurons in VC (P0–P3) exhibited significant increases in [Ca2+]i in response to GABA application. In contrast, at P0 dLGN neurons displayed no apparent increases in [Ca2+]i (Fig. 1). These GABA-evoked [Ca2+]i

Discussion

The present study indicates GABA would increase [Ca2+]i during the early developmental period in VC, but not in dLGN. This absence of a GABAergic action in dLGN would not be due to a lack of GABAA receptors because a functional expression of GABAA receptors has been demonstrated in the developing dLGN [28]. Thus, the basis for the differential nature of the [Ca2+]i responses to GABA is likely to lie in differences in the resting [Cl]i between VC and dLGN during the early postnatal period.

Acknowledgements

We thank Dr. R. Timms for language-editing the manuscript. This work was supported by Grants-in-Aid for Scientific Research #13210065, 14017041 and 15016051 on Priority Areas—Advanced Brain Science Project from the Ministry of Education, Culture, Sports and Science and Technology, Japan, and by a grant from the Ministry of Health, Welfare and Labor, Japan to A.F.

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