Research reportGAD and GABA transporter (GAT-1) mRNA expression in the developing rat hippocampus
Introduction
GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian brain. In the hippocampus, GABA is produced in and released from inhibitory interneurons with diverse anatomical and functional characteristics [24]. In order to mark the position of GABAergic neurons, many authors have used the expression of glutamate decarboxylase, the GABA synthesizing enzyme [26], [54]. GAD occurs in two isoforms, termed GAD65 and GAD67 [18], [29]. Most hippocampal interneurons express both subtypes indicating a particularly stable production of GABA in this region [20], [26].
GABA is removed from the extracellular space by specialized membrane-bound transporter proteins. This sodium-dependent GABA uptake limits the duration of inhibitory postsynaptic potentials [11], [41] reduces background GABA levels in the extracellular environment [22], [30] and may also serve as an alternative pathway for the release of GABA, when operating in reverse direction [44]. There are four phylogenetically related GABA transporters, termed GAT-1 to GAT-4 [4], [8], [25], [33]. GAT-1 is by far the dominating isoform in the rat hippocampus where it is expressed in neurons and — at low levels — also in astrocytes [5], [36], [37], [40]. The glial GABA transporter GAT-3 can only be found at very low density in the hippocampus [35], [40].
Both GAD and GAT-1 are present before birth [13], [14], [21], [28] but undergo specific changes in spatial distribution during postnatal development. In the dentate gyrus, many neurons expressing GAD67 are present in the developing stratum moleculare around birth and appear to travel through the granule cell layer during the first two postnatal weeks before they reach their final position in the hilus [15]. In the cornu ammonis, interneurons are also developed before birth but it is unknown whether they follow a similar migration pathway as in the dentate. Less information is available about the distribution of GAT-1 mRNA during postnatal development. Quantitative studies show an up-regulation of the neuronal mRNA content during the first two postnatal weeks, followed by a down-regulation to adult levels [55]. These changes have been correlated to the altered subcellular distribution of GAT-1 protein, which is present on cell somata and dendrites at early postnatal states and subsequently becomes concentrated at presynaptic terminals of interneurons [56].
It is unclear, whether neuronal GABA transporters are present in all (and exclusively in) GABAergic interneurons. Several groups have reported mismatches between the number of cells expressing GAT-1 and GAD mRNA, respectively [17], [22], [49], [58], indicating that GAT-1 may also be present in the non-GABAergic target cells of inhibitory synapses. During development, GAT-1 may be transiently expressed in certain cell populations, as has been shown for cerebellar Purkinje cells [57].
We therefore compared the expression pattern of GAD and GAT-1 mRNA in the rat hippocampus during postnatal development. Our in situ hybridizations revealed that both transcripts largely mark the same population of putative interneurons but also display some significant differences in certain hippocampal subfields. The observed changes in the position of putative interneurons support the concept that GABAergic neurons migrate from the dentate molecular layer to the hilus during the first two postnatal weeks [15].
Section snippets
Animals and tissue preparation
Wistar rats (male and female) of different age (P1, 3, 7, 16 and adult, i.e. aged 7–9 weeks) were used. They were fed and housed under standard conditions (12-h dark/light cycle) with free access to water and rodent food pellets. All animal procedures were approved by the local government of Berlin. Rats were anaesthetized with chloralhydrate (360 mg/kg body weight, i.p.) and perfused through the aorta with ice-cold saline (ca. 250 ml). The brains of the youngest animals (P1, P3 and P7) were
Results
Hybridization with either probe (GAD or GAT-1) revealed strongly labeled putative interneurons in all hippocampal subfields and at all ages tested, beginning at P1. Besides the isolated somata, we also found a diffuse background of GAD and GAT-1 mRNA in the densely packed projection cell layers (stratum granulare and stratum pyramidale) with characteristic differences between both mRNA probes. In addition, we observed an age-dependent diffuse background staining of the principal cell layers.
Discussion
Using the expression of GAT-1 and GAD mRNA we described the maturation of the inhibitory system in the rat dentate gyrus and cornu ammonis. Our findings show a largely parallel development of neurons stained for either transcript with some significant differences in certain subregions. Cell counts revealed a characteristic shift in the position of cell somata during early ontogenesis, consistent with the proposed migration of immature interneurons from the dendritic layers of the dentate
Acknowledgements
This work was supported by a grant from the Deutsche Forschungsgemeinschaft, grant number SFB 515 B1 to AD.
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