Dendritic spinules are thin protrusions, formed by neuronal spines, not adequately resolved by diffraction-limited light microscopy, which has limited our understanding of their behavior. Here we performed rapid structured illumination microscopy and enhanced resolution confocal microscopy to study spatiotemporal spinule dynamics in cortical pyramidal neurons. Spinules recurred at the same locations on mushroom spine heads. Most were short-lived, dynamic, exploratory, and originated near simple PSDs, whereas a subset was long-lived, elongated, and associated with complex PSDs. These subtypes were differentially regulated by Ca²⁺ transients. Furthermore, the postsynaptic Rac1-GEF kalirin-7 regulated spinule formation, elongation, and recurrence. Long-lived spinules often contained PSD fragments, contacted distal presynaptic terminals, and formed secondary synapses. NMDAR activation increased spinule number, length, and contact with distal presynaptic elements. Spinule subsets, dynamics, and recurrence were validated in cortical neurons of acute brain slices. Thus, we identified unique properties, regulatory mechanisms, and functions of spinule subtypes, supporting roles in neuronal connectivity.

树突棘是由神经元棘形成的细突起，衍射极限光学显微镜无法充分分辨，这限制了我们对其行为的理解。在这里，我们进行了快速结构照明显微镜和增强分辨率共聚焦显微镜来研究皮质锥体神经元的时空小棘动力学。尖刺在蘑菇脊柱头部的相同位置再次出现。大多数是短暂的、动态的、探索性的，并且起源于简单的 PSDs，而一个子集是长期的、拉长的，并与复杂的 PSDs 相关联。这些亚型受 Ca ^{2+ 的}差异调节瞬变。此外，突触后 Rac1-GEF kalirin-7 调节小刺的形成、伸长和复发。长寿命的小刺通常包含 PSD 片段，与远端突触前末端接触，并形成次级突触。NMDAR 激活增加了小刺的数量、长度和与远端突触前元件的接触。在急性脑切片的皮层神经元中验证了小刺亚群、动力学和复发。因此，我们确定了小刺亚型的独特特性、调节机制和功能，在神经元连接中起支持作用。