The roles of vesicular GABA transporter during embryonic development 胎生期発達における小胞型GABAトランスポーターの役割




    • 斉藤, 憲史 サイトウ, ケンジ



The roles of vesicular GABA transporter during embryonic development




斉藤, 憲史


サイトウ, ケンジ




博士 (学術)







&nbsp; &nbsp;This thesis describes the generation and analysis of vesicular GABA transporter (VGAT) KO mice to elucidate the functional role of VGAT during embryonic development. I have four main points of discussion: (1) generation of VGAT KO mice, (2) morphological defects in the VGAT KO mice, (3) VGAT-independent GABA release, and (4) spinal circuit formation in the absence of VGAT.<br/>&nbsp; &nbsp;In the mammalian central nervous system, inhibitory neurons release GABA and glycine as neurotransmitters. In GABAergic neurons, GABA is synthesized from glutamate by two glutamic acid decarboxylases (GADs), GAD65 and GAD67. GABA is transported into synaptic vesicles (SVs) by a VGAT and is released from axon terminals by Ca2+-dependent exocytosis. GABA activates either ionotropic GABAA or metabotropic GABAB receptors, which localize to either pre- or post-synaptic membranes. The activation of the receptors is terminated by the reuptake of GABA into axon terminals and/or glial cells by plasma membrane GABA transporters (GATs). As GABA and glycine share the same vesicular transporter and VGAT is thought to be the only vesicular transporter for inhibitory amino acids, VGAT is essential for inhibitory neurotransmission via SVs. <br/>&nbsp; &nbsp;Recent gene KO studies on inhibitory neurotransmission have elucidated not only essential roles in neural functions but also an unexpected contribution to development of non-neural tissue. For example, deletion of the GAD67 gene leads to a non-neural developmental defect, cleft palate, and loss of VGAT results in cleft palate as well as omphalocele. Omphalocele is a herniation of the gut and liver through the umbilical ring. These studies have offered a conditional gene KO technique for further understanding the role of inhibitory neurotransmission. <br/>&nbsp; &nbsp;(1) Conditional VGAT mice based on the Cre/loxP system were generated. The conditional VGAT mice were crossed with mice expressing Cre recombinase in germ cells to obtain VGAT KO mice. Western blotting revealed the VGAT protein to be successfully eliminated from the VGAT KO brain. In addition, VGAT KO mice exhibited substantial increases in overall GABA and glycine, but not glutamate, levels in the forebrain, while the expression of GABA-synthesizing enzymes did not differ between controls and KOs. <br/>&nbsp; &nbsp;(2) Although previous studies elucidated that the deletion of genes related to inhibitory neurotransmission leads to unexpected developmental defects such as cleft palate and omphalocele, rather less attention has been paid to other developmental abnormalities. To further explore the role of inhibitory neurotransmission in proper embryonic development, a comprehensive histological analysis was performed in controls and KOs. <br/>&nbsp; &nbsp;VGAT KO mice were dead at birth, and had a cleft palate and omphalocele, confirming previously reported phenotypes. Their body weight at embryonic day (E) 18.5 was significantly lower than that of wild-type littermates. Histological examination revealed a decrease in trapezius muscle mass, hepatic congestion, and little alveolar space in the VGAT KO mice.&nbsp; &nbsp;(3) In the last two decades, there is increasing evidence of neurotransmission outside synapses. Non-vesicular release is thought to be involved in this process. However, since it remains unclear to what extent the vesicular release contributes to the amount of GABA released, it is important to determine whether GABA is present in the extracellular space in the VGAT KO brain. To this end, whether or not GABA release could be confirmed in VGAT KO brain was investigated. <br/>&nbsp; &nbsp;At first, GABAAR-mediated synaptic currents were recorded using a whole-cell patch-clamp method. Electrophysiological recordings from E17.5 striatal neurons showed that the VGAT KO mice exhibited no spontaneous miniature inhibitory postsynaptic currents (IPSCs), suggesting the absence of vesicular release in the striatum. To investigate the presence of non-vesicular GABA release and the reversal of GAT-1, the amounts of GABA released from the forebrain slices were quantified. The slices were incubated in a small chamber containing artificial cerebrospinal fluid (ACSF) with or without a GAT-1 inhibitor, nipecotic acid, and the amount of GABA released into the ACSF was measured by HPLC. Without nipecotic acid, the amount of GABA in ACSF did not differ between controls and KOs. Blocking GAT-1 by nipecotic acid increased the amount of GABA in the ACSF. These results indicate that GABA can be released by VGAT-independent non-vesicular mechanisms in the embryonic mouse forebrain and that the plasma membrane GAT does not release, but rather than recovers the extracellular GABA. <br/>&nbsp; &nbsp;(4) There is increasing evidence that GABA and glycine have neurotrophic effects in the developing nervous system, in which synapse formation are still immature. In such a situation, inhibitory neurotransmission must act non-synaptically. However, it is debatable whether or not vesicular GABA release is required for the neural circuit formation. To explore this issue, the responses to dorsal-root stimulation were examined in the control and VGAT KO mice. <br/>&nbsp; &nbsp;Spontaneous IPSCs were absent in spinal cord motoneurons of VGAT KO mice. However, electrical stimulation of the dorsal root evoked excitatory, but not inhibitory, responses in the motoneurons. The latency of this excitatory response was similar to that of the control preparations. These results indicate that the sensory pathway to motoneurons is formed in the absence of GABA- and glycine-mediated synaptic responses. VGAT KO mice at E17.5-18.5 were completely immobile and stiff, and none of them responded to mechanical stimuli by pinching of the tail. Therefore, the lack of inhibitory transmission to motoneurons in VGAT KO mice likely resulted in defects in the spontaneous and stimulus-induced movements in vivo. <br/>&nbsp; &nbsp;This study provides evidence that VGAT has an essential role not only in GABA- and/or glycine-mediated neurotransmission but also in embryonic development. Another significant achievement of this study is the generation of conditional VGAT mice. This provides an opportunity to further understand roles of GABAergic neurotransmission. For example, distinct groups of inhibitory neurons contain different peptides and different calcium-binding proteins. Different classes of inhibitory neurons have precise patterns of axon targeting. These distinct subtypes of inhibitory neurons appear to contribute to specific functions in the brain. The conditional VGAT mouse provides a new tool to study the subtype-specific and circuit-specific role of inhibitory neurons in brain functions. <br/>



    • 8000000549503
  • 本文言語コード
    • eng
  • NDL書誌ID
    • 023254389
  • データ提供元
    • 機関リポジトリ