Shuichiro Hayashi

Nodal protrusions, increased Schmidt-Lanterman incisures, and paranodal disorganization are characteristic features of sulfatide-deficient peripheral nerves.

Galactocerebroside and sulfatide are two major glycolipids in myelin; however, their independent functions are not fully understood. The absence of these glycolipids causes disruption of paranodal junctions, which separate voltage‐gated Na+ and Shaker‐type K+ channels in the node and juxtaparanode, respectively. In contrast to glial cells in the central nervous system (CNS), myelinating Schwann cells in the peripheral nervous system (PNS) possess characteristic structures, including microvilli and Schmidt‐Lanterman incisures, in addition to paranodal loops. All of these regions are involved in axo–glial interactions. In the present study, we examined cerebroside sulfotransferase‐deficient mice to determine whether sulfatide is essential for axo–glial interactions in these PNS regions. Interestingly, marked axonal protrusions were observed in some of the nodal segments, which often contained abnormally enlarged vesicles, like degenerated mitochondria. Moreover, many transversely cut ends of microvilli surrounded the mutant nodes, suggesting that alignments of the microvilli were disordered. The mutant PNS showed mild elongation of nodal Na+ channel clusters. Even though Caspr and NF155 were completely absent in half of the paranodes, short clusters of these molecules remained in the rest of the paranodal regions. Ultrastructural analysis indicated the presence of transverse bands in some paranodal regions and detachment of the outermost several loops. Furthermore, the numbers of incisures were remarkably increased in the mutant internode. Therefore, these results indicate that sulfatide may play an important role in the PNS, especially in the regions where myelin–axon interactions occur.

Specializations of plasma membranes in Pacinian corpuscles: Implications for mechano-electric transduction

SummaryPacinian corpuscles of cat mesentery were studied with freeze-fracture and thin sectioning methods after chemical fixation.Intramembranous particles (IMPs) exhibit differences in both density and pattern of distribution between the axolemma of the smooth short axis (x-axis) region and that of the axonal spine region of the long axis (y-axis) of the axon terminal. The axolemma of thex-axis has IMPs at a density of 2687±581 per μm2 (mean±S.E.M.), and these particles are 9.0±1.7 nm (mean ±S.D.) in diameter. In contrast, the axolemma of they-axis has a higher density of IMPs (3607±612 per μm2) which are larger (diameter, 10.0±1.7 nm). The particle distribution is not homogeneous inx-axis membranes as there are small patchy areas devoid of particles scattered throughout the entire surface. The E-face of the axolemma has a low density of IMPs (∼ 200 perμm2 in bothx- andy-axes). However, IMPs in the E-face are smaller (∼9nm) in thex-axis than in they-axis (∼10nm).The inner core lamellar cells have IMPs at a density of 3276±739 per μm2 and 553±169 per μm2 in the P- and E-faces, respectively. The particles are about 10 nm in diameter in both faces. Many gap junctions occur between lamellar cells especially near the clefts, suggesting that hemilamellae of each inner core half are kept at the same electrotonic potential.The outer core lamellar cells have IMPs at a density of 2239±403 per μm2 and 536±123 per μm2 in their P- and E-faces, respectively. The particles are approximately 10 nm in diameter in both faces. A noteworthy finding is that tight junctions are prominent at cell-to-cell appositions within individual lamellae, especially in the first and second (or sometimes third) innermost lamellae of the outer core. These tight junctions are considered to be a barrier to the leakage of fluid and/or ions between interlamellar spaces as well as between inner and outer cores.An intermediate cell layer is identified between the inner and outer cores. The connective tissue space of this cell layer corresponds to the endoneurium, indicating that intermediate layer cells are comparable to endoneurial fibroblasts. These cells exhibit a low density of IMPs (658±119 per μm2 in the P-face) and particles are about 9 nm in diameter.The above findings indicate that the plasmalemmata of the axon terminal of the inner and outer core cells are specialized in terms of content and distribution of IMPs. The difference in axolemmal IMPs between thex-axis and they-axis suggests that there is some separation of function in components of mechano-electric transduction between these two regions. In addition it can be assumed that the hemilamellae of each inner core half constitute an electrotonically coupled environment on each side of the axon terminal abutting thex-axis. Furthermore, the fluid of the inner core is completely segregated and probably different in composition from that of the outer core.

Improved methods for ultracryotomy of CNS tissue for ultrastructural and immunogold analyses.

We examined each step of the protocol for ultracryotomy for central nervous system tissue in order to define and overcome some of the methodological difficulties. The following three steps emerged as critical for the methods success: (1) pretreatment of grids to render them hydrophilic immediately before use; (2) careful collection of ultrathin cryosections during ultracryotomy; (3) removal of the appropriate amount of excess poly(vinyl alcohol)-uranyl acetate (PVA-UA) prior to drying after staining with PVA-UA. By taking account of the three critical steps described above, we succeeded in obtaining ultrathin cryosections, including serial sections, with excellent preservation of ultrastructure, as well as semithin cryosections which are useful for evaluating the quality of the samples and for selecting areas of interest for ultrastructural analysis. Cytoplasmic organelles in neurons and glial cells, and the fine structure of synapses and myelinated fibers were well preserved. The localization of gold particles after immunostaining for astrocytic glutamate transporter (GLAST), metabotropic glutamate receptor 1 (mGluR1) and neurofilament protein was consistent with previous reports and ultrastructure was well-preserved in all cases. These findings should be helpful to researchers wishing to carry out ultrastructural and immunogold analyses of cryosections of nervous tissue.

A re-evaluation of the cytology of cat Pacinian corpuscles

SummaryThe ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristic concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.