Research ReportDevelopmental expression of GABA transporter-1 and 3 during formation of the GABAergic synapses in the mouse cerebellar cortex
Introduction
In the adult central nervous system (CNS), γ-amino butyric acid (GABA) is a predominant neurotransmitter, mediating fast inhibitory synaptic transmission and regulating the excitatory activity of neurons [22], [25]. Recent studies have revealed that GABA serves as a trophic factor during brain development, inducing morphogenesis and regulating cell proliferation, cell migration, axonal growth, synapse formation, steroid-mediated sexual differentiation and cell death [4], [23], [26], [41].
In the cerebellar cortex, four of the five types of neurons, Purkinje, stellate, basket and Golgi cells release GABA as a neurotransmitter [13], [21], [27]. The stellate cell axons make many GABAergic synaptic contacts with the dendritic shafts of Purkinje cells in the molecular layer. The basket cell axons form GABAergic synapses with Purkinje cell bodies and the initial segment of the Purkinje cell axons in the Purkinje cell layer. Golgi cell axons form inhibitory synapses with granule cell dendrites at the peripheral part of the synaptic glomeruli in the granular layer. GABAergic inputs regulate the neuronal activity of Purkinje and granule cells, which organize the major stream of neural circuitry in the cerebellar cortex. During formation of the GABAergic network in the cortex, mice open their eyes and start moving around [16], [39], [42]. Eye opening and increasing motor activity imply extensive development of motor control, coordination and learning. Furthermore, elimination of GABAergic input from the Golgi cells in the cerebellar granular layer causes overexcitation of granule cells resulting in severe ataxia during the acute phase [43]. These results suggest that GABAergic input regulates glutamatergic excitation and might play a crucial role in expression of cerebellar functions such as motor skill learning [13], [20], [21].
We are morphologically investigating the development of the GABAergic transmission system in the cerebellar cortex. We previously demonstrated the extrasynaptic localization of GABA in developing cerebellar GABAergic neurons, including Purkinje, stellate, basket and Golgi cells [33], [34], [35]. Before formation of GABAergic synapses, GABA is localized in the cytoplasm of the dendrites and cell bodies of GABAergic neurons and is concentrated in the vesicles within the axon varicosities and growth cones. This suggested that GABA could be extrasynaptically released in the developing cerebellum in two ways: (1) exocytosis of GABAergic vesicles from axon varicosities and growth cones and (2) cytosolic GABA-diacrine from dendrites and cell bodies. Physiological and biochemical studies have demonstrated that the non-vesicular form of GABA is released via the plasma membrane by the reverse transporter action of plasma membrane GABA transporters (GATs) [2], [3], [11], [37], [38], [41]. In the present study, we examined the localization of GATs in the dendrites and cell bodies of developing cerebellar GABAergic neurons, where GABA is extrasynaptically localized, to reveal the GABA-diacrine system during cerebellar development.
In previous studies, we also demonstrated the developmental change in expression and localization of the various synaptic elements, including GABA and vesicular GABA transporter (VGAT), in the presynaptic axons and GABAA receptors in the postsynaptic membrane during formation of GABAergic synapses [32], [33], [34], [35], [36]. In the present study, we examined the developmental change in expression and localization of GATs to clarify the development of the GABA-uptake system in the mouse cerebellar GABAergic synapse.
GATs mediate the GABA-uptake from the synaptic cleft by exchanging Na+ and Cl− ions in the mature brain [5], [10], [17]. Molecular cloning has isolated four GATs: GAT-1, GAT-2, GAT-3 and BGT-1. Mouse GAT2, GAT3 and GAT4 (no hyphen) are species homologs of rat BGT-1, GAT-2 and GAT-3, respectively. Among them, we focused on GAT-1 and GAT-3 (mouse homologues of GAT1 and GAT4, respectively), which are localized widely in neural and glial cells [7], [8], [14], [24], [28], [29]. Furthermore, we used not the rat [44] but the mouse cerebellum as a comparison with the previous studies demonstrating the extrasynaptic localization of GABA in cerebellar GABAergic neurons [33], [35].
We found that GABA might be extrasynaptically released not by diacrine, but by only exocytosis from GABAergic axons in the developing mouse cerebellar cortex. During formation of GABAergic synapses, GABA might be reuptaken firstly into the presynaptic terminals via GAT-1 and subsequently into both presynaptic terminals via GAT-1 and astrocyte processes via GAT-3.
Section snippets
Animals
We examined mice from the C57Bl/6CrSlc strain at postnatal days 5 (P5), 7, 10, 14, 21 and 90 (as adults). At each postnatal stage, at least five mice were sacrificed for immunohistochemistry.
Establishment of a guinea pig VGAT antibody
Guinea pigs were immunized by subcutaneous injection of the VGAT peptide (CGDEGAEAPVEGDIHYQRGGA) [30] conjugates with keyhole limpet hemocyanin (KLH). The specific IgG fraction of the synthetic peptide for VGAT was affinity-purified, and the specificity of the antibody was checked as previously described
Immunohistochemical localization of GAT-1 and GAT-3 in the adult mouse cerebellar cortex
GAT-1-immunolabeling was observed throughout the cerebellar cortex (Fig. 1A). GAT-1-positive dots and short lines were scattered in the molecular layer and surrounded Purkinje cell bodies in the Purkinje cell layer. In the granular layer, the GAT-1-immunolabeling often exhibited ring-shaped profiles. Next, we performed double staining by using a guinea pig antibody against VGAT, which we newly raised, or a mouse calbindin antibody. The GAT-1-immunolabeling was localized at VGAT-positive dots in
Discussion
In the present study, we investigated the developmental changes in expression and localization of GAT-1 and GAT-3 in the mouse cerebellar cortex. GAT-1 was detected within the presynaptic axons of Golgi cells in the granular layer after P5 and those of stellate and basket cells in the molecular and Purkinje cell layers after P7. GAT-3 was observed within the astrocyte processes in the Purkinje and granular layers after P10. Neither transporter was detected within the dendrites and cell bodies
Acknowledgments
We are grateful to Yoshihiko Ogawa and Hideki Nakamura at the Central Research Department of Hokkaido University School of Medicine for technical assistance and to Dr. Takayuki Yoshida at the Department of Molecular Neuroanatomy, Hokkaido University School of Medicine for valuable discussion.
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