Ulf Karlsson

Fixation of the central nervous system for electron microscopy by aldehyde perfusion

Glutaraldehyde, formaldehyde, and osmium tetroxide perfusion fixation of the central nervous system of rats were compared and evaluated. Aldehyde fixation was considered superior to the direct osmium tetroxide fixation with respect to methodological considerations and at least equal with respect to fixation criteria. Use of gas mixture, prewash, vasodilator drugs, and low temperature perfusate were not found necessary with aldehyde perfusion. A very close cell-to-cell membrane relationship is universally found, restricting the extracellular space to considerably less than that previously reported. The possible significance of the general absence of extracellular space is discussed. Certain ultrastructural differences of osmium tetroxide and aldehyde perfusion fixation are described. An attempt also has been made to visualize the first signs of perfusion fixation through a hole in the skull during perfusion.

FIXATION OF THE CENTRAL NERVOUS SYSTEM FOR ELECTRON MICROSCOPY BY ALDEHYDE PERFUSION. II. EFFECT OF OSMOLARITY, PH OF PERFUSATE, AND FIXATIVE CONCENTRATION.

Hypertonic fixatives perfused well, but gross brain shrinkage was excessive with extreme hypertonicity. Increasing the amount of buffer in a perfusate to 1200 mOsM resulted in shrinkage of cells and large amounts of extracellular space. Increasing the fixative concentration did not cause extensive extracellular space formation but had only relatively subtle effects on fine structure. Moderate hypertonicity resulted in little gross brain volume change. Isotonic (300–320 mOsM) perfusates acted similarly to hypotonic fixatives, which caused gross swelling of the brain and poor preservation on the ultrastructural scale and would not flow well at perfusion. While extreme pHs (2 and 11) were detrimental to preservation of structure, pHs of 6 and 8 caused little change. A brief “wash” of pH 2.0 buffer immediately preceding pH 7.4 perfusion caused the appearance of large scale extracellular space, but also fair preservation of ultrastructure.

Three-dimensional studies of neurons in the lateral geniculate nucleus of the rat: I. Organelle organization in the perikaryon and its proximal branches

The results of a two- and three-dimensional study of the content of perikarya and proximal branches from neurons in the rat lateral geniculate body are reported. Techniques for the serial sectioning procedure and the serial analysis are outlined. The two neurons analysed three-dimensionally were elliptoid, multipolar with modified bipolarity, uninucleated, uninucleolated and uniciliated with a variety of organelles dispersed in the cytoplasm. Most organellesvariety of organelles dispersed in the cytoplasm. Most organelles were quantitatively analysed with respect to morphological dimensions. The cellular membranes were of particularly impressive dimensions. An almost complete, quantitatively integrated, structural view at the organelle level is given for the analysed parts of the neurons. Mitochondria, Golgi complexes and cytoplasmic dense bodies were abundant in the middle of the perikarya, while α-cytomembranes and free ribosomes had their highest concentrations at the poles of the perikarya. Also, mitochondria and cytoplasmic dense bodies were observed in relatively high concentrations in the proximal branches. The results are evaluated and discussed in relation to technique, conventional two-dimensional analysis and some biochemical data available for similar parts of neurons.

Cellular organization of the frog muscle spindle as revealed by serial sections for electron microscopy

By means of extensive group serial sectioning it was possible to obtain a detailed and topographical picture of the frog muscle spindle through motor and sensory regions. The distribution of different cell types was analyzed. On the basis of the topographical morphology of the intrafusal muscle fiber and its innervation, a descriptive nomenclature of parts of the spindle was adopted. The spindle envelope is divided into outer and inner capsules. The outer capsule encapsulated groups of intrafusal muscle fibers at their sensory regions. Inner capsule cells were found in the sensory zone, dominating and surrounding each intrafusal muscle fiber in the reticular zone. Extracellular filamentous material was found to follow the distribution of inner capsule cells. The intrafusals were found to alter their overall diameter within a wide range outside of the reticular zone, but only slightly within that zone. In this zone the diameter of all muscle fibers in a specific bundle was about equal. In the sensory zone nucleated satellite cells, e.g., intrafusal satellite cells, were found in intimate contact with the muscle fibers. They commonly occurred in the sensory compact zones but were sparse in reticular or motor compact zones. Each intrafusal muscle fiber was surrounded by sensory chains of nerve bulbs that were joined together with thin links. A general bimodal distribution of these sensory structures was found with maxima at the borders between the reticular and the sensory compact zones. Motor regions were observed situated inside the capsular envelope of the muscle spindle. The results with respect to the cellular topography of the frog muscle spindle are discussed.

Observations on the postnatal development of neuronal structures in the lateral geniculate nucleus of the rat by electron microscopy

Postnatal stages of development of the lateral geniculate nucleus from the rat were investigated by electron microscopy. As compared with the adult, a gradient of increased number of vesicle-filled processes per unit area developed in the second week after birth and before the animal had opened its eyelids. Myelinization started in this material at about the same time as the animal opened its eyelids. Extracellular space was more abundant compared to that in the adult stage. Other ultrastructural features are described and related to the adult structure, such as membrane differences and abundance of α -cytomembranes.

Comparison of the myelin period of peripheral and central origin by electron microscopy

The repeating radial periods of some peripheral and central nervous system myelin sheaths in albino rats were compared in an electron microscopic study. Essentially the peripheral myelin period appeared to be larger than the central by about 10%. This result confirmed earlier wide-angle X-ray diffraction data. Direction of sectioning was observed to affect the myelin period, probably proportionally to the hardness of the embedded tissue blocks. Microscopical procedures also tended to increase the spreading of the measurements provided the myelin period was assumed to be similar within the same part of the nervous system in the same animal species. This was elucidated by comparing differences obtained in the same and separate microscopy sessions. Possible causes of error were discussed.

Fixation of the central nervous system for electron microscopy by aldehyde perfusion: III. Structural changes after exsanguination and delayed perfusion1

Brain tissue from aldehyde-perfused rats was analyzed with the electron microscope with respect to qualitative structural changes due to exsanguination and delayed perfusion from 5 to 60 minutes. More severe alterations were generally found after 15, 30, and 60 minutes as compared with those after 5 minutes of perfusion delay. The extracellular space was found increased after 15, 30, and 60 minutes of perfusion delay. Triple-layered membranes and myelin sheath structures were generally found resistant to the experimental procedure. Most intracellular organelles changed due to the adverse fixation situation, but changes were not at any stage ubiquitous. Nuclear material generally displayed aggregation. Nuclear membrane and α-cytomembrane cisternae widened. This was especially prominent in glial cells. Sometimes attached ribosomal particles appeared to become detached. Ribosomal aggregates showed tendency for separation into single ribosome-like particles. Golgi membrane complexes formed rounded structures, occasionally with circular continuity. Mitochondria swelled with or without retention of internal material. Some mitochondria exhibited evidence of internal autolysis, while others appeared in ring forms or partially constricted. Particularly in cerebellar Purkinje cells, stacks of small membrane-bound cisternae were associated with α-cytomembrane-bound cisternae. Both types of cisternae were also often associated with constricted mitochondria. The observed structural changes warrant the conclusion that each cell type and also individual cells differ significantly in response to disturbances enforced by the perfusion delay. Fixation criteria can be constructed for each organelle system.

Three-dimensional studies of neurons in the lateral geniculate nucleus of the rat: II. Environment of perikarya and proximal parts of their branches

Lateral geniculate neurons were investigated with respect to the environment of perikarya and proximal branches using two- and three-dimensional analysis by electron microscopy. Two cells were completely analysed and reconstructed with regard to specialized neuronal contacts. No branches without α-cytomembranes could be found projecting from the perikaryon. A descriptive nomenclature was adopted for the different types of processes and contacts observed. Perikarya appeared to contact far less neuronal knobs than proximal branches. The neuronal knobs were preferentially grouped with particularly high concentrations on spines. Quantitative observations established that no clear ultrastructural differentiation of neuronal knobs into special groups could be made on this material. Specialized neuronal contacts of type V (vesicle-related) all appeared similar in ultrastructure. On the average, 30 % of the contact surface of the neuronal knobs was specialized, often in several patches. Specialized neuronal contacts V were observed between V processes and perikarya or other V processes, as were spines on perikarya and proximal neuron branches. No spine apparatuses could be found. Specialized neuronal contacts of type S (with symmetrical membrane specialization) were observed between no-V processes and perikarya, V processes or other no-V processes. The perikarya were otherwise invested by extensive cellular sheets, that were interpreted to be glial. This investment covered at least 80 % of the surface of the perikaryon, the rest mainly involved specialized neuronal contacts. A minute extracellular space was estimated for the perikaryon, but relatively more for the branches. It was primarily occuping “seams” between meeting cellular processes at the surface.

Small leptomeric organelles in intrafusal muscle fibers of the frog as revealed by electron microscopy.

By means of serial sectioning for electron microscopy, the general shape and dimensions were described for two types of small cross-striated organelles (leptomeric organelles of types I and II) from frog intrafusal muscle fibers. They consisted of regularly spaced, interconnected disks, which showed periodicities of about 1870 A and 300 A, respectively. A structural relationship was proved to exist between the end of the filaments of the type I organelle and the Z-disk of the sarcomere, thus coupling it in series with the sarcomeres. In stretch experiments it was possible to indicate that the type I periodicity changed in the same direction as the periodicity of surrounding sarcomeres. The possible role of the type I organelle is discussed. Another cross-striated organelle (leptomeric organelle of type II) is described for the first time. Structurally it resembled the type I organelle except for the shorter spacing of its granulated disks. The occurrence of the type II organelles was less frequent and less defined with respect to localization than that of type I. Its function remains obscure.

Three-dimensional studies of neurons in the lateral geniculate nucleus of the rat: III. Specialized neuronal contacts in the neuropil1

Interneuronal contact relationships were investigated by electron microscopy in the neuropil of the rat lateral geniculate nucleus, partly utilizing three-dimensional analysis technique. Specialized contacts composed of (a) V and no-V processes, (b) spines, V, no-V processes and glia and (c) spines, two V processes, one no-V process and glia were described and discussed in relation to morphological features. Morphological characteristics were found distinct for each of the two V process components participating in the most complex type of interneuronal contact. A symmetrical membrane specialization and intracellular content was found to differ for all three components. Contents of V and no-V processes were described and related to previously published data. Also, the ultrastructure of nodes of Ranvier was established using this technique of preparation. Extracellular space at the nodal region was consistently observed. Glia investment was found to be sparser on thinner no-V processes as compared with that on perikarya.