A freeze-etch study of the central myelin.

The intramembranous organization of the interparanodal myelin lamellae in CNS was investigated by means of the freeze-etching technique. The density of the IMPs is about equal on both P- and E- fracture faces of every myelin sheath, but varies between the myelin sheaths. The morphology of the myelin tight junctions is also described. The nature of the IMPs and the functional significance of their mode of distribution are discussed.

Use of lead aspartate block staining in quantitative EM autoradiography of phospholipids: application to myelinating peripheral nerve.

For quantitation of electron microscope (EM) autoradiographs, micrographs must contain clear images which are relatively free of heavy metal precipitates. Satisfactory contrast is usually obtained by staining individual ultra-thin sections with lead citrate. It was recently reported that sequential block staining of tissue with ferrocyanide-reduced osmium tetroxide and lead aspartate produced excellent contrast for EM autoradiography, with sections relatively free of lead precipitate. This protocol avoids the manipulation involved in staining individual ultra-thin sections. We have adapted this method to quantitative EM autoradiographic studies, primarily of phospholipid metabolism in peripheral nerve. We show that block staining with lead aspartate provides: (a) ultrastructural contrast of routinely high quality for myelinated peripheral nerve; (b) high (greater than 98%) retention of glycero-labeled lipid during dehydration and embedment; and (c) a distribution of de novo tritiated glycerol-labeled lipid in ultra-thin sections that is quantitatively identical to the distribution recorded for samples stained by the more laborious post-embedment method. During a 2-hr labeling period in vivo, tritiated glycerol is incorporated into phosphatidylcholine (44%), phosphatidylethanolamine (22%), other phospholipids (16%), and neutral lipids (15%). The analysis of grain distribution in developing sciatic nerve labeled for 2 hr with tritiated glycerol demonstrates that myelinating Schwann cells play the major role in synthesis of endoneurial lipids. Lipid synthesis in myelinated fibers is localized in perinuclear regions of Schwann cell cytoplasm. These regions lie external to compact myelin. Unmyelinated fibers and other endoneurial cells independently incorporate glycerol into lipids.

Co-localization of the myelin-associated glycoprotein and the microfilament components, F-actin and spectrin, in Schwann cells of myelinated nerve fibres.

The myelin-associated glycoprotein (MAG) is an intrinsic membrane protein that is specific for myelinating cells. MAG has been proposed to function in the PNS as an adhesion molecule involved in Schwann cell-axon contact and maintenance of cytoplasmic channels within the myelin sheath. In this report we show that the microfilament components, F-actin and spectrin, co-localize with MAG in periaxonal membranes, Schmidt-Lanterman incisures, paranodal myelin loops, and inner and outer mesaxons of myelinating Schwann cells. F-actin was localized light microscopically by rhodamine-labelled phallicidin binding. Spectrin and MAG were localized by light microscopic and ultrastructural immunocytochemistry. The findings indicate that plasma membrane linkage of F-actin in Schwann cells is likely to occur via spectrin, and raise the possibility that microfilaments interact with the cytoplasmic domain of MAG. An interaction between MAG and microfilaments would be consistent with the proposed function of MAG as an adhesion molecule.

Neural organization of the masseter muscle in the pig.

The neural organization of the pig masseter, an architecturally and functionally compartmentalized muscle, was investigated by using dissection, glycogen depletion, evoked electromyography, and counts of axon numbers at various levels along the masseteric nerve. The masseteric nerve enters the muscle as two rostral branches, which also supply the zygomatico-mandibularis, and a more caudal main branch, which soon divides into four terminal nerves with variable distributions. Stimulation of filaments containing roughly 50 extrafusal motor axons resulted in glycogen depletion of 5-20% of the muscle fibers in very small subvolumes of the masseter; the affected subvolumes were delimited by perimysium. Electromyography after stimulation of various branches of the nerve confirmed the distributions deduced from anatomy and further indicated that axons do not branch between the rostral and main nerve branches but may occasionally do so among the more distal terminal branches of the main branch. The proximal trunk of the masseteric nerve contains about 3,500 myelinated fibers with a bimodal size distribution. Approximately 1,000 of the larger fibers were estimated to be extrafusal motor axons. Along the proximal trunk of the nerve, fibers were constantly rearranged; coupled with the observation that the locations of motor unit territories were usually not related to the position of the stimulated axons within the nerve, this suggests that the nerve trunk is not strictly ordered somatotopically.

Demonstration of GABA-like immunoreactivity in myenteric plexus of frog stomach.

The GABAergic innervation of the frog stomach was studied by means of an indirect immunohistochemical method. Whole mount preparations were obtained from frog stomachs after the animals had been perfused with a mixture of picric acid, glutaraldehyde and glacial acetic acid. Samples were incubated with an antiserum specific for GABA coupled to BSA with glutaraldehyde. Anti-rabbit IgG-HRP was processed by the two step method (Eckert and Ude 1983). GABA-positive varicose fibers and also nerve cell bodies were revealed within the myenteric plexus. The density of GABA-immunoreactive neurons was not higher than 4-8 cell/cm2, which is approximately 1% of the total nerve cell number in the myenteric plexus.

Membrane heterogeneity in sensory receptors of rat vibrissae: Merkel receptors differ from other nerve endings.

The cupric/ferrocyanide reaction was used to analyse cation-binding sites in rat vibrissae. The heminode of the last myelinated segment had moderate staining. No staining of any complex receptor was found except for Merkel receptors. Both the Merkel cell and associated nerve ending were stained except for their apposed cell membranes, suggesting either reciprocal membrane specialization at that site, or prevention of cupric/ferrocyanide penetration into the space between the two cells.

The effects of dorsal rhizotomy and spinal cord isolation on calcitonin gene-related peptide-labeled terminals in the rat lumbar dorsal horn.

In the present study, the origin of calcitonin gene-related peptide (CGRP) to the dorsal horn in the rat lumbar spinal cord is investigated. CGRP immunoreactivity is examined following multiple unilateral and bilateral dorsal rhizotomies and isolated cord preparations (spinal cords are isolated by transecting the cord in two places and cutting all dorsal roots between the transections). Seven to 11 days after surgery, unilateral multiple dorsal rhizotomies result in a drastic decrease in CGRP-stained terminals on the operated side; following bilateral dorsal rhizotomies and isolated cord preparations, one or two CGRP varicosities remain in the dorsal horn in each section. The numbers of CGRP-immunostained varicosities observed in the latter two preparations are not significantly different, suggesting that few if any axons descending from the brain contribute to the CGRP terminal population in the spinal cord dorsal horn. Based on these data, we hypothesize that dorsal root ganglion cells are the only source of CGRP to the rat lumbar dorsal horn.

Propriospinal fibers in the white matter of the cat sacral spinal cord.

The propriospinal system, which consists of those neurons completely contained within the spinal cord, is important because it underlies much spinal behavior. To provide quantitative data on this system, the present study determines numbers of axons in the isolated S2 cat spinal cord and compares these figures with the normal. The conclusion is that 60% of the fibers in the spinal cord at this location are propriospinal. Findings of particular interest are that the great majority of unmyelinated propriospinal axons are found in the dorsal part of the lateral funiculus, and that there are large numbers of descending myelinated fibers in the dorsal funiculi. These data will serve as a basis for evaluating axon numbers that follow various experimental regimens purporting to result in neural sprouting.

The relationship between structural features of peripheral nerves and suture methods for nerve repair.

Based on techniques for identifying and distinguishing motor, sensory, and mixed fasciculi in peripheral nerves, the authors propose guidelines for selecting suture methods for nerve repair. When many mixed fasciculi are known to exist at the nerve lesion, epineurial repair is preferable; fascicular (perineurial) repair is more suitable when pure motor and sensory fasciculi are clearly recognized. Generally, epineurial repair is indicated for more proximal injuries, with fascicular repair most appropriate for more distal sites. A greater ratio of epineurial connective tissue to intrafascicular nervous tissue implies an inclination toward fascicular repair.

Immunohistochemical demonstration of nociceptors in the capsule and synovial folds of human zygapophyseal joints.

Substance P, a physiologically potent neuropeptide, is considered to participate in nociceptive transmission of nerve impulses. Substance P immunofluorescent nerves are demonstrated in the inferior joint recess capsule and its synovial folds in human lumbosacral zygapophyseal joints. This may have clinical significance in spinal pain.

Immunocytochemical localization of 5'-nucleotidase in myelinated peripheral nerves from the rat.

In sciatic and trigeminal nerves from the rat, 5'-nucleotidase immunostaining was observed on the surfaces of the myelinated fibers and in the membranes encircling the outermost loops of the myelin sheaths, the paranodal loops, and perhaps the inner loops, but neither in the compact myelin nor in the axoplasm. These results, which were consistent with previous biochemical data regarding sciatic nerve, suggest that the function of 5'-nucleotidase in myelinated fibers in the peripheral nervous system may be to promote diffusion of adenosine between the glial and neuronal compartments.

Brain-specific hyaluronate-binding protein. A product of white matter astrocytes?

The distribution of glial fibrillary acidic (GFA) protein and hyaluronectin, a hyaluronate-binding protein isolated from human brain, was compared in brain, spinal cord and optic nerves of pigs and dogs by indirect immunofluorescence with monoclonal antibodies. In spinal cord white matter the localization of the two proteins was similar, both antigens forming a mesh surrounding myelinated axons. A similar distribution of the two proteins was also observed in the periventricular glia as well as in the glia limitans of spinal cord and optic nerves. Cerebral white matter was hyaluronectin-positive, but the GFA-positive stellate astrocytes did not stain with hyaluronectin antibodies in this location. Hyaluronectin antibodies did not stain grey matter, the granular layer of the cerebellum excepted. The astrocytes identified with GFA antibodies in hyaluronectin-negative grey matter were: the fibrous astrocytes forming the glia limitans on the surface of the cerebral hemispheres; the protoplasmic astrocytes of cerebral isocortex and basal ganglia; the fibrous astrocytes of cerebral allocortex (hippocampus); Bergmann radial glia in the molecular layer of the cerebellar cortex; and fibrous astrocytes of spinal cord anterior and posterior horns. It is concluded that the hyaluronectin fraction reacting with the monoclonal antibodies is a brain-specific protein probably produced by white matter astrocytes. We propose to call this fraction brain-specific hyaluronectin, to be distinguished from other fractions reacting with polyclonal antibodies and with different localizations.

Morphometric analysis of experimental spinal cord injury in the cat: the relation of injury intensity to survival of myelinated axons.

The pattern of axonal destruction and demyelination that occurs in experimental contusion injury of cat thoracic spinal cord was studied by line sampling of axons in 1 micron thick plastic sections with the light microscope. Injuries were produced by a weight-drop apparatus, with the vertebral body (T9) below the impact stabilized by supports under the transverse processes. The effects of two combinations of weight and height were examined: 10 or 13 g dropped 20 cm onto an impact area of 5 mm diameter. Animals were kept for 3-5 months after injury, then fixed by perfusion for histological analysis. The number of surviving myelinated axons was found to vary both with the weight used and with the size of the spinal cord. A measure of impact intensity was derived from the calculated momentum of the weight at impact divided by the cross sectional area of the cord (interpolated from dimensions measured rostral and caudal of the lesion following fixation). At impact intensities greater than 0.02 kg-m/s/cm2 there was practically no survival of axons at the center of the injury site, combined with almost complete breakdown of the pial margin. Between 0.08 and 0.2 kg-m/s/cm2 the number of surviving axons varied between 100,000 and 2,000, approximating a negative exponential function (r = -0.88). The number of axons surviving in the outer 100 microns of the cord varied practically linearly (r = -0.82) between near normal and less than 1% of normal over the same range of injury intensity. The number of surviving axons decreased with depth from the pia, also approximating a negative exponential function, with a 10-fold decrease in density over approximately 500 microns. The average slope of this relation with depth remained similar over the range of injury intensity examined, though the slope appeared inversely related to variation in axonal survival for different individuals at a given intensity. It is argued that the loss of axons is probably determined primarily by mechanical stretch at the time of impact. Its centrifugal pattern may be explained by longitudinal displacement of the central contents of the cord, reflecting the viscoelastic "boundary layer" properties of parenchymal flow within the meningeal tube. This is illustrated with reference to the behavior of a gelatin model under compression. The preferential loss of large caliber axons and the characteristic shift to abnormally thin myelin sheaths (resulting from post-traumatic demyelination) both varied in extent independently of injury intensity and overall axonal survival.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple visual areas in the caudal superior temporal sulcus of the macaque.

Anatomical and physiological evidence indicates that, in addition to area MT, much of the cortex in the caudal superior temporal sulcus (STS) of the macaque has visual functions. Yet the organization of areas outside of MT remains unclear, and there are even conflicting data on the boundaries of MT itself. To examine these issues, we recorded form neurons throughout this region in three monkeys. Anterograde or retrograde tracers were injected into MT at the conclusion of recording to identify its projection fields. Based on differences in their visuotopic organization, neuronal properties, receptive field size, myeloarchitecture, and pattern of connections with MT, several visual areas were distinguished within the caudal STS. Area MT, defined as the heavily myelinated portion of the striate (VI) projection zone in STS, contained a systematic representation of only about the central 30 degrees--40 degrees of the contralateral field. The far peripheral field was represented medial to MT in MTp, which we had previously found receives projections from far peripheral V1 and V2 (Ungerleider and Desimone: J. Comp. Neurol. 248:147-163, 1986). Like MT, MTp contained a high proportion of directionally selective cells, and receptive field size in MTp was the size expected of MT fields if the latter were to extend into the periphery. Areas MST (medial superior temporal) and PP (posterior parietal) were found medial to MT and MTp. Both MST and PP had a high proportion of directionally selective cells, but only MST received a direct projection from MT. Cells in MST had larger receptive fields than those in either MT or MTp but nonetheless displayed a crude visuotopic organization. Receptive fields of cells in PP were even larger, some including the entire contralateral visual field. Furthermore, unlike cells in MST, some in PP responded to auditory or somesthetic stimuli in addition to visual stimuli. Area FST, which has a distinctive myeloarchitecture, was found anterior to MT in the fundus of the STS, for which it is named. FST received a direct projection from MT, but only about a third of its cells were directionally selective. Receptive fields of cells in FST were large, often included the center of gaze, and often crossed into the ipsilateral visual field. Area V4t (transitional V4) and a portion of V4 were found lateral to MT within the STS, and both received direct projections from MT. V4t has a distinctive, light myelination. Both areas had a low incidence of directionally selective cells, and both contained coarse representations of the lower visual field.(ABSTRACT TRUNCATED AT 400 WORDS)

Projections to the superior temporal sulcus from the central and peripheral field representations of V1 and V2.

In a series of three studies, we have begun to explore the sequence of visual information processing along the pathway from striate cortex (V1), through MT, into the parietal lobe. In this first study, we sought to establish the relationships among MT, the heavily myelinated zone of the superior temporal sulcus (STS), and the V1 and V2 projection fields in the STS. Autoradiographic material from seven hemispheres of six macaques injected with tritiated amino acids into either V1 or V2 was analyzed in detail, and the results were plotted onto two-dimensional reconstructions of the STS. Autoradiographic material from eight additional macaques with V2 injections was also examined. The results indicate that the central visual field representations of both V1 and V2 project into the heavily myelinated zone in the lower bank and floor of the STS, confirming prior studies, whereas the far peripheral representations of both V1 and V2 project into the cortex medial to this zone on the upper bank of the sulcus. There is no evidence that this medial cortex is a separate area that receives projections from V1 and V2 in parallel with the projections these areas send to the heavily myelinated zone. Rather, there seems to be a single projection field of V1 and V2 whose central representation lies within the heavily myelinated zone and whose most peripheral representation lies medial to it. Because of the difference in myelination between the central and peripheral field representations as well as visuotopic anomalies between them, we retain the term "MT" for the heavily myelinated zone and apply the term "MTp" to the far peripheral projection zone. Both MT and MTp are required to process the complete outputs of V1 and V2 within the STS and thus should probably be regarded as two distinctive parts of a single visual area. The difference in myelination between MT and MTp suggests that there is a difference in visual processing between the central and peripheral visual fields. The average size of MT is estimated to be 62 mm2, and the average size of MT and MTp combined to be 76 mm2, which is consistent with estimates derived from several other studies.

Cortical connections of visual area MT in the macaque.

We have identified the cortical connections of area MT and determined their topographic organization and relationship to myeloarchitectural fields. Efferents of MT were examined in seven macaques that had received injections of tritiated amino acids, and afferents were examined in one macaque that had received injections of two fluorescent dyes. The injection sites formed an orderly sequence from the representation of central to that of peripheral vision in the upper and lower visual fields. In addition to connections with the striate cortex (V1), connections were found between MT and a variety of extrastriate areas, including V2, V3, V3A, V4, V4t, VIP, MST, FST, possibly PO, and, finally, the frontal eye field. The connections of MT with V1, V2, and the dorsal and ventral portions of V3 were topographically organized and consistent with the visuotopic arrangement reported previously in these areas. V2 could be distinguished from V3 by the distinctive myeloarchitectural appearance of the former. Connections with areas V4 and V4t also displayed at least a coarse visuotopic organization, in that the central representation of MT projected laterally in these areas and the peripheral representation projected medially. The lower visual field representation of V4 was located dorsally, on the prelunate convexity, while the upper field representation was located primarily on the ventral aspect of the hemisphere. V4t had a distinctively light myeloarchitecture and received projections from only the lower field representation of MT. The remaining connections of MT were with areas located entirely in the dorsal half of the hemisphere. There were widespread connections with areas MST and FST in the superior temporal sulcus, with some evidence for a crude visuotopic organization in MST. Connections were also found with area VIP in the intraparietal sulcus, with area V3A on the annectent gyrus, possibly with area PO in the dorsomedial prestriate cortex, and, finally, with the frontal eye field on the anterior bank of the lower limb of the arcuate sulcus. Area FST and parts of both MST and VIP had a distinctive myeloarchitecture. The pattern of laminar connections with V1, V2, and V3 indicated that MT projects "back" to these areas and they project "forward" to MT. That is, the projections to these areas from MT terminated in both the supragranular and infragranular layers and the projections to MT from these areas originated predominantly from cells located above granular layer IV (above layer IVC in V1).(ABSTRACT TRUNCATED AT 400 WORDS)

Correlation between the morphology and the lipid and protein compositions in the peripheral nervous system of individual 8-day-old normal and trembler mice.

The hereditary, hypertrophic interstitial neuropathy which afflicts the trembler mouse manifests itself about two weeks after birth. Consequently, the identification of these mutant mice was not possible before this age, except when double mutants were available. We show that the trembler mice can be easily distinguished from their normal littermates before the clinical symptoms appear by using an HPTLC/densitometry technique that allows the simple and rapid analysis of the polar lipids extracted from one sciatic nerve. The results presented in this paper demonstrate important differences between the polar lipid compositions of sciatic nerves from 8-day-old normal and trembler littermates, whose phenotypes were confirmed by the morphological analysis of the contralateral sciatic nerves. The small amount of material that is needed for this identification makes it possible to use the remaining nerve material for other studies. Furthermore, important differences between the sciatic nerve protein compositions of normal and trembler mice, identified according to their polar lipid composition, were also observed and these differences can, therefore, also be employed for the identification of the mutants before the manifestation of the clinical symptoms of the trembler neuropathy.

A quantitative developmental study of the peripheral nerve lipid composition during myelinogenesis in normal and trembler mice.

The quantitative evolution of 10 polar lipids was examined in the sciatic nerves of normal and trembler mice between the ages of 3 days and 60 days. In normal nerves, the polar lipids accumulated slowly until the age of 9 days. A period of rapid accumulation then took place until 18 days of age, after which the phospholipids plateaued, while the glycolipid content continued to increase at a slower rate. The results obtained for the sciatic nerves of trembler mice show that the accumulation of all the polar lipids studied, except phosphatidylcholine and hydroxysulfatides, is abnormal from the earliest stages of postnatal development, and strongly support the view that the primary disorder in the trembler peripheral nervous system is one of dysmyelination. With the exception of cardiolipin, all the lipids in the trembler nerves stopped accumulating at the age of 18 days. The cerebrosides were the lipids the most affected severely at all ages.

Localizations of ruthenium red positive material in rabbit peripheral nerves.

The penetration and distribution of ruthenium red in the axon-myelin-Schwann cell complex of developing rabbit peripheral nerve fibers are investigated. Ruthenium red positive material is established in the axoplasm, axolemma, periaxonal space, major dense lines and intraperiod lines of the compact myelin, mesaxons, split peripheral myelin lamellae, Schmidt-Lanterman and longitudinal incisures, paranodal loops and axo-glial contacts, Schwann cell cytoplasm and basal lamina, nodal extracellular matrix, desmosome-like structures, endoneural collagen. Some features of the distribution of the contrast material in the developing myelin sheath are described. Regional differences of the axolemma and of the Schwann cell cytoplasm and plasmalemma are established. The prevalence of glycoproteins or glycolipids in the ruthenium red stained material in its different localizations is discussed on the basis of trypsin and hyaluronidase digestion performed.

Structural organization of the optic tectum of Barbus meridionalis Risso. II. Outer strata (SFGS, SO and SM).

The present work deals with the histological organization of the three outer strata of the optic tectum of Barbus meridionalis. As far as general features are concerned, of noteworthy interest are the absence of myelin in the SM and the ordered pattern of myelinic distribution of the SO. The SFGS shows a more irregular appearance than the SM and SO. It was only possible to observe with clarity several neuronal types in the SO and SFGS, whereas in the SM no clear neuronal types could be seen. The SFGS was seen to contain pyramidal neurons with spiny dendrites in the SM, together with fusiform, multipolar and horizontal neurons. In this latter type we have described neurons with ascending axons emerging from the soma or from the base of a principal dendrite. The SO contains neurons whose dendrites are distributed mainly on the horizontal plane, in each case showing certain particular features.