Reduced Gene Expression of KCC2 Accelerates Axonal Regeneration and Reduces Motor Dysfunctions after Tibial Nerve Severance and Suturing2024 · Gamma-aminobutyric acid and glycine (GABA/Gly) are predominantly inhibitory neurotransmitters in the mature central nervous system; however, they mediate membrane potential depolarization during development. These differences in actions depend on intracellular Cl- concentrations ([Cl-]i), which are primarily regulated by potassium chloride cotransporter 2 (KCC2). After nerve injury, KCC2 expression markedly decreases and GABA/Gly mediate depolarization. Following nerve regeneration, KCC2 expression recovers and GABA/Gly become inhibitory, suggesting that KCC2 reduction and GABA/Gly excitation may be crucial for axonal regeneration. To directly clarify their involvement in regeneration, we analyzed recovery processes after tibial nerve severance and suturing between heterozygous KCC2 knockout mice (HT), whose KCC2 levels are halved, and their wild-type littermates (WT). Compared with WT mice, the sciatic functional index-indicating lower limb motor function-was significantly higher until 28 days after operation (D28) in HT mice. Furthermore, at D7, many neurofilament-positive fibers were elongated into the distal part of the sutured nerve in HT mice only, and myelinated axonal density was significantly higher at D21 and D28 in HT animals. Electron microscopy and galanin immunohistochemistry indicated a shorter nerve degeneration period in HT mice. Moreover, a less severe decrease in choline acetyltransferase was observed in HT mice. These results suggest that nerve degeneration and regeneration proceed more rapidly in HT mice, resulting in milder motor dysfunction. Via similar microglial activation, nerve surgery may reduce KCC2 levels more rapidly in HT mice, followed by earlier increased [Cl-]i and longer-lasting GABA/Gly excitation. Taken together, reduced KCC2 may accelerate nerve regeneration via GABA/Gly excitation.
Action Sequence Learning Is Impaired in Genetically Modified Mice with the Suppressed GABAergic Transmission from the Thalamic Reticular Nucleus to the Thalamus2023 · The thalamic reticular nucleus (TRN) is a thin sheet of GABAergic neurons surrounding the thalamus, and it regulates the activity of thalamic relay neurons. The TRN has been reported to be involved in sensory gating, attentional regulation, and some other functions. However, little is known about the contribution of the TRN to sequence learning. In the present study, we examined whether the TRN is involved in reward-based learning of action sequence with no eliciting stimuli (operant conditioning), by analyzing the performance of male and female Avp-Vgat-/- mice (Vgatflox/flox mice crossed to an Avp-Cre driver line) on tasks conducted in an operant box having three levers. Our histological and electrophysiological data demonstrated that in adult Avp-Vgat-/- mice, vesicular GABA transporter (VGAT) was absent in most TRN neurons and the GABAergic transmission from the TRN to the thalamus was largely suppressed. The performance on a task in which mice needed to press an active lever for food reward showed that simple operant learning of lever pressing and learning of win-stay and lose-shift strategies are not affected in Avp-Vgat-/- mice. In contrast, the performance on a task in which mice needed to press three levers in a correct order for food reward showed that learning of the order of lever pressing (action sequence learning) was impaired in Avp-Vgat-/- mice. These results suggest that the TRN plays an important role in action sequence learning.
C
科研费 / 研究项目
GABA/グリシンの興奮性応答を介した神経再生カスケードの解析と治療法への応用2021 · GABAとグリシンは、成熟動物の主要な抑制性神経伝達物質である。両者は幼若期に興奮性応答を示す事から、形態形成に関与すると考えられてきた（Kobayashi et al. 2021, Shimizu et al. 2022）が、ノックアウトマウスの解析から、現在は否定的である。我々は、神経損傷後の運動神経軸索の再生期にもGABA/グリシンが興奮性応答を示すことを見出し（Tatetsu et al. 2012, Kim et al. 2018）、応答性の変化が損傷軸索の再生を加速するという仮説をたてて研究を行っている。 ①新しいタイプの神経損傷モデルとして、脛骨神経結紮動物を用い、損傷後の変化を解析した。その結果、（１）ミクログリアの活性化⇒KCC2発現の減少⇒軸索再生のシグナル経路が認められ、神経損傷後に共通する現象であることが示された。（２）ガラニン、コリンアセチルトランスフェラーゼ（ChAT）2つの分子の動きが神経軸索の変性と再生を示す客観的指標となることが明らかになった。この研究は、Neuroscience Research誌に掲載された（Yafuso et al. 2022）。 ②GABA/グリシンを抑制性に導く輸送体(KCC2)を半減させてたKCC2ノックアウトマウスのヘテロ接合体と野生型を比較して、運動神経切断・縫合後の運動機能の変化などを解析した。その結果、KCC2が半減し、GABA/グリシンの応答性が興奮性にシフトしたマウスにおいて、（１）運動機能の障害程度が低く回復が早い。（２）神経軸索の再生が早く進行する。２つの傾向が認められた。現在、現在動物数を増やし検討を重ねている。 ③野生型マウスとKCC2ノックアウトヘテロ接合体について神経損傷後脊髄におけるmRNA解析を行って、大きく変化する分子について解析を進めている。
