Investigating the Exocytosis and Mobility of Single GABAergic Synaptic Vesicles Undergoing Spontaneous Release in Living Neurons Using a Real-time Three-dimensional Nanometer-accuracy Tracking Microscope
Description: Neurons communicate by releasing neurotransmitters from synaptic vesicles in presynaptic terminals. Recent studies have shown that the spontaneous (i.e., activity-independent) release of neurotransmitters plays an important role in neuronal function and in synaptic development. Moreover, impaired spontaneous release of synaptic vesicles has been implicated in several neurodevelopmental and psychiatric disorders. However, our understanding of spontaneous synaptic vesicle release is quite limited. Specifically, the exocytosis and mobility of single inhibitory synaptic vesicles undergoing spontaneous release has not been investigated, even though spontaneous release occurs frequently in mature inhibitory synapses and given that inhibitory synaptic transmission plays a critical role in maintaining the delicate balance between neuronal excitation and inhibition. Importantly, changes in this balance have been shown to underlie several neurological disorders, including autism spectrum disorder, schizophrenia, and epilepsy.
Recently, we built an advanced real-time three-dimensional nanometer-accuracy tracking microscope, which allows us to track single synaptic vesicles loaded with a quantum dot (QD) with an accuracy of ~20-30 nm in three dimensions. Using this microscope, we have studied the exocytosis and mobility of single synaptic vesicles in living neurons and found that synaptic vesicles from the readily releasable pool (RRP) in excitatory synapses lie close to their fusion sites. Furthermore, we specifically labeled GABA-containing vesicles in inhibitory mature neurons and found that their evoked release is highly dynamic, providing rapid inhibitory synaptic transmission without synaptic fatigue in the neuronal network. However, the exocytosis and mobility of single inhibitory vesicles undergoing spontaneous release has not been investigated, limiting our knowledge regarding inhibitory neurotransmission and underlying neurological disorders.
Therefore, we will test our working hypothesis that the exocytosis and/or mobility of GABAergic synaptic vesicles undergoing spontaneous release may differ from GABAergic vesicles undergoing evoked release. In this proposal, we will measure the exocytosis and mobility of single GABAergic vesicles undergoing spontaneous release in rat mature hippocampal and striatal neurons using real-time three-dimensional nanometer-accuracy tracking microscope, electron microscopy, and electrophysiology. We will also examine the release sites of spontaneously releasing GABAergic synaptic vesicles. Finally, we will examine whether and how neuronal activity affects the exocytosis and/or mobility of GABAergic synaptic vesicles labeled via spontaneous release.
These results will provide new important insights into differences between spontaneously releasing GABAergic vesicles and vesicles undergoing evoked release. Thus, this project will advance our understanding of inhibitory neurotransmission and facilitate the development of new therapeutic approaches for treating a number of neurological disorders associated with altered inhibitory neurotransmission.
神經元通過從突觸前末端的突觸小泡釋放神經遞質進行交流。最近的研究表明，神經遞質的自發（即與活動無關）釋放在神經元功能和突觸發育中起著重要作用。此外，突觸小泡的自發釋放受損與幾種神經發育和精神疾病有關。然而，我們對自發突觸小泡釋放的理解非常有限。具體而言，尚未研究在抑制性神經元中自發釋放的單個囊泡的胞吐作用和流動性，儘管自發釋放經常發生在成熟的抑制性突觸中，並且鑑於抑制性突觸傳遞在維持神經元興奮和抑制之間的微妙平衡中起著關鍵作用.重要的是，這種平衡的變化已被證明是幾種神經系統疾病的基礎，包括自閉症譜系障礙、精神分裂症和癲癇症。
最近，我們構建了一種先進的實時三維納米精度跟踪顯微鏡，它使我們能夠在三個維度上以約 20-30 納米的精度跟踪裝載有量子點 (QD) 的單個突觸小泡。使用這台顯微鏡，我們研究了活神經元中單個突觸小泡的胞吐作用和移動性，發現興奮性突觸中易釋放池 (RRP) 中的突觸小泡靠近它們的融合位點。此外，我們特別標記了抑制性成熟神經元中含有 GABA 的囊泡，發現它們的誘發釋放是高度動態的，在神經元網絡中提供快速抑制性突觸傳遞而沒有突觸疲勞。然而，尚未研究自發釋放的單個抑制性囊泡的胞吐作用和流動性，這限制了我們對抑制性神經傳遞和潛在神經系統疾病的了解。
因此，我們將檢驗我們的工作假設，即自發釋放的 GABA 能突觸小泡的胞吐作用和/或流動性可能與誘發釋放的 GABA 能小泡不同。在本提案中，我們將使用實時三維納米精度跟踪顯微鏡、電子顯微鏡和電生理學測量大鼠成熟海馬和紋狀體神經元中單個 GABA 能囊泡自發釋放的胞吐作用和流動性。我們還將檢查自發釋放 GABA 能突觸小泡的釋放部位。最後，我們將檢查神經元活動是否以及如何影響自發釋放標記的 GABA 能突觸小泡的胞吐作用和/或移動性。
這些結果將為自發釋放 GABA 能囊泡和經歷誘發釋放的囊泡之間的差異提供新的重要見解。因此，該項目將促進我們對抑制性神經傳遞的理解，並促進開發新的治療方法來治療與抑制性神經傳遞改變相關的許多神經系統疾病。
Amount: HKD 1,180,000
Funding type: research
Funder scheme: General Research Fund
End date: 2026-02-28
Investigators: Hyokeun Park, Byung-Ho Kang, Wing Ho Yung
Institution: The Hong Kong University of Science and Technology
Funded outputs count: 6
Funder award ID: 16102322
OpenAlex ID: G2703381663