Elsevier

Neurochemistry International

Volume 61, Issue 4, September 2012, Pages 506-516
Neurochemistry International

Ontogenetic changes in the distribution of the vesicular GABA transporter VGAT correlate with the excitation/inhibition shift of GABA action

https://doi.org/10.1016/j.neuint.2012.03.018Get rights and content

Abstract

GABA is the major inhibitory neurotransmitter in the adult CNS and is among others involved in the synchronization of large neuronal networks. During development, GABA acts as a morphogenetic factor and has transient excitatory actions in many brain regions. One distinct protein, the vesicular GABA transporter (VGAT), has been identified accumulating GABA into presynaptic vesicles prior to its exocytotic release. The function of VGAT and its distribution is well defined in the adult, but its contribution to the transient excitatory action at putative GABAergic nerve terminals in the immature brain and its potential roles in putative glutamatergic nerve terminals remain elusive. We have studied VGAT expression in the brain from late embryonic stages through several postnatal stages until adulthood. Quantitative immunoblotting and immunolabeling of tissue sections at the light microscope and the electron microscope levels show an abrupt augmentation in VGAT staining in the cerebral cortex during the first three postnatal weeks, resembling the increase in other proteins involved in GABA synthesis and recycling in the same time frame – such as GAD65, GAD67, GAT1 (Slc6a1) and SN1 (Slc38a3) – and coincides with the synaptogenetic spurt. Dynamic changes in the expression of VGAT are seen in many cellular populations and in several layers in different brain regions. However, mossy fiber terminals (MFT) elude staining for VGAT. We also demonstrate that VGAT+ nerve terminals undergo a developmental reorganization so that from targeting primarily the dendrites of the principal neurons in several brain regions in the immature brain, they target the soma of the same cells in the adult. This shift in the targeted subcellular compartment coincides with the conversion of the chloride gradient across neuronal membranes and suggests that it may be important for the shift of GABA action from excitation to inhibition and for the establishment of the potent synchronization of neuronal networks.

Highlights

VGAT levels increase abruptly after birth. ► VGAT is differentially regulated during the development. ► VGAT containing nerve terminals re-organise their targeting during development. ► Hippocampal mossy fiber terminals are not VGAT+ at any developmental stage.

Introduction

GABA is the main inhibitory neurotransmitter of the mature CNS. During development, GABA doubles as a trophic factor and has been shown to be important for normal neuronal migration, axonal sprouting and synaptogenesis (Taylor and Gordon-Weeks, 1991, Behar et al., 1994, Behar et al., 1996, LoTurco et al., 1995, Barker et al., 1998, Haydar et al., 2000, Demarque et al., 2002, Nguyen et al., 2003). Interestingly, the primary action of GABA in the immature brain is excitatory (Mueller et al., 1984, Cherubini et al., 1990, LoTurco et al., 1995, Chen et al., 1996, Wang et al., 2001). This effect is widely believed to be due to transient expression of a sodium–potassium-chloride co-transporter, NKCC1, while the chloride extruder, the potassium chloride co-transporter (KCC2), is yet not induced. As a result Cl is accumulated in immature neurons and activation of ionotropic GABAA receptors instigate efflux of Cl with subsequent membrane depolarization (Rivera et al., 1999, for reviews see Payne et al., 2003, Rivera et al., 2005). One consequence of this excitatory effect of GABA is its contribution to the generation of recurrent large and long lasting depolarizing potentials, known as giant depolarizing potentials (GDPs) (Ben-Ari et al., 1989, Ben-Ari et al., 1997, Sutor and Luhmann, 1995, Feller, 1999, Ben-Ari, 2001, Dezawa et al., 2004, Cherubini et al., 2011). GDPs are a highly conserved phenomenon observed in many brain regions and they play an important role in the activity-dependent wiring of the developing network. Thus, GABA plays multiple indispensable roles in brain ontogeny. Furthermore, GABA, GABA synthesizing enzymes (GAD65 and GAD67) (Dupuy and Houser, 1996) and GABA transporters (GAT1–3) (Yan et al., 1997, Minelli et al., 2003b, Vitellaro-Zuccarello et al., 2003) are present at early developmental stages – even before synapse formation – further bolstering morphogenetic roles of GABA during the development of the CNS.

Prior to its release, GABA is accumulated into synaptic vesicles by the vesicular GABA transporter VGAT/VIAAT (McIntire et al., 1997, Sagné et al., 1997). Accordingly, VGAT is enriched in most GABAergic and glycinergic nerve terminals (Chaudhry et al., 1998, Dumoulin et al., 1999). GABA uptake by VGAT resembles glutamate uptake by the vesicular glutamate transporters (VGLUT1–3), which utilizes the electrochemical gradient for H+ established by a vacuolar H+-ATPase across the vesicle membranes (for review see Chaudhry et al., 2008a). In both cases the uptake is also dependent on chloride (Juge et al., 2009). However, there are also important differences: VGAT and VGLUTs belong to distinct families and have different topologies – screening for VGAT homologs resulted in the molecular identification of a novel family of plasma membrane amino acid transporters (Slc38), including SN1 (Chaudhry et al., 1999, Chaudhry et al., 2002), and the C-terminal part of VGAT is localized intravesicularly (Martens et al., 2008, Antonucci et al., 2011). In contrast, the VGLUTs are homologous to bacterial toxin extruders and their C-terminus is localized extravesicularly (for review see Chaudhry et al., 2008b). Thus, these vesicular proteins undergo differential membrane trafficking (Martens et al., 2008, Hnasko and Edwards, 2011). Yet the transient excitatory contribution by VGAT from putative GABAergic nerve terminals during postnatal development is in the adult replaced by the VGLUTs (Fremeau et al., 2002, Boulland et al., 2009).

VGAT expression during brain development has partially been shown in selected brain regions (Marty et al., 2002, Minelli et al., 2003a, Takayama and Inoue, 2004, Takayama and Inoue, 2010). However, the functional roles of GABA during brain development and its mode of action require a complete understanding of ontogeny of its release machinery, ultrastructural cellular changes taking place to establish functional synapses by VGAT+ nerve terminals and identification of the targeted subcellular domains. In the current study we present a detailed and extended developmental profile of VGAT in the rat brain. Generally VGAT expression increases during development, however, in several brain regions expression levels of VGAT is already high at birth and in some cases it decreases in the course of brain development. Electron microscopy confirms that VGAT is present in morphologically inhibitory-like nerve terminals while VGAT staining is excluded from the hippocampal MFT at P4, P7 and in the adult. In specific areas of the brain, such as the hippocampal formation, we observe a shift in the localization of the VGAT+ nerve terminals: At early postnatal stages, they are present in dendritic layers while later in the development they target the perikaryon of the principal cells. This re-distribution of GABAergic nerve terminals occurs at around P7 which is also the stage at which the postsynaptic response to GABA stimulation switches from membrane depolarization to membrane hyperpolarization. Thus, our data suggest that redistribution of GABAergic nerve terminals together with changes in the expression of chloride transporters is responsible for the conversion of the GABA action from excitation towards inhibition.

Section snippets

Materials and methods

Sodium dodecyl sulfate (SDS) of high purity (>99% C12 alkyl sulfate), SuperSignal West Pico Chemiluminescent Substrate and Dimethyl pimelimidate-2HCl (DMP) were from Pierce (Rockford, IL, USA). 10% Criterion precast gels, nitrocellulose sheets (ECL), electrophoresis equipment, molecular mass markers, Precision plus all blue or Precision plus dual colors, were from Bio-Rad (CA, USA). Biotinylated anti-rabbit and streptavidin–biotinylated horseradish peroxidase complex were from Amersham

VGAT expression increases during brain development

VGAT expression at different brain developmental stages was assessed with immunoblots of brain homogenates from various developmental stages: 15 and 18 days old embryos (E15 and E18, respectively) and 0, 7, 14, 21, 36 and 56 days after birth (P0, P7, P14, P21, P36 and P56, respectively). At E18 a broad and diffuse band, typical for transporter proteins (Hazell et al., 2001, Boulland et al., 2002, Minelli et al., 2003a) is observed at around 55 kDa, the predicted molecular mass for VGAT (Chaudhry

VGAT is present from late embryonic period but shows differential developmental changes in different subregions of the brain

Developmental increase of VGAT has been shown in the mouse cerebellum (Takayama and inoue, 2004) as well as in the mouse and rat somatosensory cortex (Minelli et al., 2003a, Takayama and Inoue, 2010). Also in the rat hippocampus it has been reported that VGAT punctae increase in the pyramidal cell layer from P7 to P21 (Marty et al., 2002). We have now studied developmental expression of VGAT in detail in many regions of the rat brain and show that VGAT is already present from the late embryonic

Acknowledgements

We are grateful to Ray Miyalou-Koussou for technical help and to Prof. Jon Storm-Mathisen for comments on the manuscript. This work was supported by the Research Council of Norway.

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