Ultrastructural Clarification Of The Peripherally Located Actin Network In The Myelinated Axons

Objective: Actin(monomeric G-actin and multimeric F-actin)is the most abundant proteins in eukaryotic cells and participates in numerous biological processes,such as cell motility,intracellular trafficking,and signaling.There is also a large amount of actin in axons.With the advancement of imaging technology,people's cognition of the cytoskeleton structure in the nervous system has gradually deepened,but so far,the ultrastructural distribution of F-actin in myelinated axons is still unclear.Because F-actin is apt to depolymerize following fixation and dehydration,the ultrastructure detection of F-actin is not easily achieved.In addition,both G-actin and F-actin are present in the nerve cells,which cannot be distinguished only by using anti-actin immunochemistry.In the present study,an ultrastructural detection method of F-actin by use of a FITC-specific antibody and a highly probe specific for F-actin,phalloidin were used to explore the subcellular distribution of F-actin in the periphery of myelinated axons,dorsal root ganglia,and spinal cord.Methods: In this experiment,10 SD rats(female)were used.The spinal cords,dorsal root ganglia,and spinal nerves were dissected out at the level of L4-L5 after cardiac perfusion.The sections were stained with FITC-conjugated phalloidin,which were further converted to peroxidase/DAB products by a FITC-anti-FITC system.The phalloidin stained sections were examined with a laser scanning confocal microscope.The DAB stained sections were photographed using an Olympus BX53 F microscope equipped with a digital imaging system.Ultrathin sections were examined and photographed with a transmission electron microscope.Results:Under the fluorescence microscope: 1.In the spinal cord,FITC-Phalloidin staining was found mainly in the peripheries of nerve cells.The neuronal somata remained unstained,except for weak fluorescence scattered in the perikaryal cytoplasm.The periphery of neurons displayed strong fluorescence signals,and blood vessels were intensely stained,which could be identified by their hollow circular appearance.Axon hillock and initial segment could not be distinguished under the fluorescence microscope.2.In the DRG,the cytoplasm of neuronal somata and the axoplasm were devoid of fluorescence,but the peripheries surrounding the neuronal somata and axons were brightly labeled.Analysis of the fluorescence microscopic images could not determine the precise localization of F-actin in the DRG axons.3.In spinal nerves,the outer and inner perimeters of myelinated fibers were moderately stained,but the intermodal axoplasm was virtually devoid of fluorescence.In the longitudinal sections,the Schmidt-Lanterman incisures were intensely labeled.The paranodal and nodal regions were prominently stained.A strongly labeled central band could be discerned in the nodal region.Under the electron microscope: 1.In the anterior horn of the spinal cord,almost all the positive sites were found as terminal boutons.The cytoplasm of neuronal cell bodies and their processes remained unstained,but diffuse staining was often seen surrounding membranous organelles.2.The cell bodies and the axon initial segments of DRG neurons were surrounded by a unique glial envelope formed by satellite cells,which were intensely labeled.3.In the DRG,less intensely stained materials are seen sparsely beneath the plasma membrane of the axon or the DRG neuron,the positive products for F-actin are located in the adaxonal region of the satellite cell envelope around the axon initial segment.,but the bulk cytoplasm remains unlabeled.4.In the myelinated nerves,the outer margin of compact myelin and the adaxonal cytoplasm of the myelin-forming cells were intensely labeled,appearing as a double train track pattern around the intermodal axon.At higher magnification,it was clear that the positive products for Factin were concentrated in the outer and inner collar cytoplasm of Schwann cells or oligodendrocytes.The subjacent axons were very faintly stained,and only diffuse staining was found in the axoplasm.5.In the Schmidt-Lanterman incisures,the positive products for F-actin were localized in the cytoplasmic channels of Schwann cells,and the surrounding membrane was also densely deposited with the positive products.In the nodal region,the Schwann cell microvilli and paranodal terminal loops are intensely labeled.6.In the vicinity of nodes of Ranvier,the Schwann cell microvilli were strongly labeled.respectively,where an F-actinpositive layer is observed beneath the axolemma.Less intense staining is also observed in the paranodal terminal loops.Conclusions: 1.In the gray matter of the spinal cord,F-actin were localized predominately in the presynaptic element around the neuronal cell bodies and dendrites,and the axon terminal around the initial segment of the axon(in the presynaptic element);2.In the dorsal root ganglion,F-actin is mainly located in the glial envelope formed by satellite cells layer around the axon initial segments of the dorsal root ganglion neurons;3.In the central and peripheral myelinated nerve fibers,F-actin is mainly located in the outer and inner collars of cytoplasm surrounding the intermodal axon,Schmidt-Lanterman incisures,the paranodal terminal loops,and the nodal microvilli.