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