Observations on a transient phase of focal swelling in degenerating unmyelinated nerve fibers.

This study describes the nature and time-course of a swelling phase during the degeneration of unmyelinated nerve fibers, as observed in highly organized cultures of rodent sensory ganglia. Observations were made on nerve fascicles after they were cut and during nutritional deprivation. About 12 hr after nerve transection, large, clear vacuoles appear throughout fascicles distal to the cut. These vacuoles are most numerous at 24 hr and then gradually subside; after 48 hr, only small granules mark the severed fascicles. Electron microscopy shows that the vacuoles are, in fact, massive focal dilations of unmyelinated axons. Similar focal dilations in unmyelinated axons are observed if cultures are not refed for 5-7 days; under these conditions glucose concentrations fall below 20 mg/100 ml and degenerative changes begin to appear in neuronal somas. If the gas-tight assembly is opened and the culture refed, there is rapid disappearance of axonal dilations (usually within 1 hr) and recovery of many of the damaged neurons. Cooling (4 degrees C) prevents this reversal, suggesting that an active process is involved. It is postulated that the swellings result from the failure of active axolemmal ion-pumping mechanisms prior to loss of selective permeability in the axon membrane. The reasons for the focal nature of the swellings is unknown. A literature review indicates that a phase of focal swelling has frequently been observed during the degeneration of unmyelinated nerve fibers in vivo.

Spatial patterns of threadlike elements in the axoplasm of the giant nerve fiber of the squid (Loligo pealii L.) as disclosed by differential interference microscopy and by electron microscopy.

The giant nerve fiber of the squid (Loligo pealii L.) has been investigated in situ, and in fresh and fixed preparations, by differential interference microscopy and electron microscopy. A continuous, three-dimensional network, composed of threadlike elements, was disclosed in the axoplasm. The threadlike elements in the axoplasm are twisted as a whole into a steep, right-handed helix. In a peripheral ectoplasmic region, the elements are more parallel to one another and more densely packed than in a central endoplasmic core. The threadlike elements can be resolved into a hierarchy of decreasing order of size. Successive levels of the hierarchy are formed by the association of smaller elements into larger ones. The following levels in the hierarchy of network elements have been distinguished: 1-3-micro-wide threads, 0.1-0.35-micro-wide strands, and 70-250-A-wide unit-filament strands. The differential interference microscope selects, from the network, threads oriented at a specific angle to the long axis of the axon. The specific angle depends upon the orientation of the long axis of the axon relative to the direction of shear. It is postulated that the network configuration is expressed in the solid-state properties of the axoplasm essential for the normal functioning of the nerve fiber.

Configuration of a filamentous network in the axoplasm of the squid (Loligo pealii L.) giant nerve fiber.

High-resolution electron microscopy is integrated with physicochemical methods in order to investigate the following preparations of the giant nerve fibers of the squid (Loligo pealii L.): (1) Thin sections of fibers fixed in four different fixatives; (2) fresh axoplasm stained negatively in solutions of different pH and composition; (3) chemically isolated threadlike elements of the axoplasm. A continuous, three-dimensional network can be identified in all these preparations of the axoplasm. The network is composed of coiled or looped unit-filaments approximately 30 A wide. The unit-filaments are intercoiled in strands approximately 70-250 A wide. The strands are oriented longitudinally in the axoplasm, often having a sinuous course and cross-associations. Microtubules are surrounded by intercoiled unit-filaments and filamentous strands. Calcium ions cause loosening and disintegration of the network configuration. UO(2) (++) ions of a 1% uranyl acetate solution at pH 4.4 display a specific affinity for filamentous protein structures of the squid giant nerve fiber axoplasm, segregating the filamentous elements of the axoplasm in a coiled, threadlike preparation. The uranyl ions combine probably with the carboxyl groups of the main amino acids of the protein-glutamic and aspartic acids. It is proposed that by coiling/decoiling and folding/unfolding of the unit-filaments, shifts in physicochemical properties of the axoplasm are maintained.

The organization of synaptic axcplasm in the lamprey (petromyzon marinus) central nervous system.

The fine structure of synapses in the central nervous system of lamprey (Petromyzon marinus) ammocoetes has been investigated. Both synapses within the neuropil and synaptic links between giant fibers (including Müller cells) and small postsynaptic units are described. The distribution of neurofilaments and microtubules in nerve profiles over a wide diameter range is described, and the possible role of these structures in intracellular transport is discussed. Electron micrographs indicate that small lucent "synaptic vesicles" occur sparsely throughout the axoplasm and in regular arrays in association with microtubules in the vicinity of synapses. Within a synaptic focus, immediately adjoining the presynaptic membrane, vesicles are randomly arranged and are not associated with microtubules. Neurofilaments are present, generally in large numbers, but these are not associated with vesicles or other particulates. The structural findings are considered in terms of current concepts of fast and slow transport in neurons and the mechanochemical control of intracellular movement of materials.

Rapid degeneration of ampullary electroreceptor organs after denervation.

Electroreceptors (ampullary organs) of the transparent catfish (Kryptopterus bicirrhus) lie in the epidermis, and contain spherical receptor cells that receive purely afferent innervation from the lateral line nerve. Section of this nerve causes rapid degenerative changes to occur in the receptors. Fine structural alterations occur in the receptor cell synapses and nerve fiber 6-12 h postoperatively. Disruption of the receptor cells begins by 18 h and most are lost by 48 h. By 72 h supporting cells and secretory cells also show marked degeneration, and by 96 h they may be totally lost. The rapid degeneration of the electroreceptor organs of Kryptopterus should make them a useful preparation for analysis of neurotrophic functions.

Unmyelinated axons in the posterior funiculi.

Electron microscopy of the dorsal funiculus in the rat reveals that most axons in this pathway are unmyelinated. These axons have not previously been counted, nor are they considered in modern studies on the organization of the dorsal funiculus. Because of the importance of this pathway in somatic sensation, it is important to understand that these fibers exist and that they are present in greater numbers than the well-studied myelinated axons.

The morphometric myelinated fiber composition of D11 as compared to L3, L4, and L5 ventral spinal roots of man.

As compared to the diameter frequency distribution of myelinated fibers of L3, L4 and L5 ventral spinal roots (VSRs) which have two well developed peaks [large-diameter axons (AL) and intermediate-diameter axons (AI)] and a poorly developed or non-existent peak of small-diameter axons (AS), D11 VSRs contain small AL and AI peaks and a prominent AS peak. Because the ratio of AL to AI myelinated fibers of D11 VSRs is similar to that in L3, L4 and L5 VSRs and it is known that D11 VSRs contain many pre-ganglionic autonomic fibers it seems likely that AS is composed predominantly of preganglionic autonomic fibers. The mean diameter of the AS peak in D11 VSRs is approximately 2.4 micrometer. Average numbers of AS, AI and AL myelinated fibers for D11 VSRs are 3835, 738, and 1545 per root. These values should be useful as controls for morphometric studies of various neuropathies. The average diameter positions of the peak of AL, AI and AS of D11 VSRs as compared to those of L3, L4 and L5 are displaced to smaller diameter categoried which may reflect the smaller axon diameters of neurons which are shorter.

Lumbar motoneurons of man: I) number and diameter histogram of alpha and gamma axons of ventral root.

The numbers of large and intermediate diameter myelinated fibers of 17 ventral lumbar (L3, L4 and L5) spinal roots (vsr) of man (ages 17-81 years) have been evaluated using the improved methods of histologic processing and morphometry now available, semi-automatic methods of measurement and programmed calculation and plotting. A population of small diameter myelinated fibers as found in thoracic (T11) vsr, which are presumed to be preganglionic sympathetic fibers, were essentially absent from L3, L4 and L5 roots. On the average, there were 5716 (70%) large diameter fibers and 2625 (30%) intermediate diameter fibers in the L3 ventral spinal root. Comparable figures for L4 vsr were 4900 (70%) and 2152 30%) and for L5 vsr were 5043 (75%) and 1559 (25%). On the assumption that these lumbar roots contain few if any small diameter myelinated pre-ganglionic sympathetic axons, the large diameter fibers correspond to alpha motoneuron axons and the small diameter fibers to gamma motoneuron axons. With age, a decrease in the number of myelinated fibers was estimated to be 350 fibers per decade (approximately 5% per decade).

Propriospinal fibers in the rat.

Segments of rat spinal cord were isolated by transecting in two places and sectioning all dorsal roots between the transections. Following this procedure, the areas of the gray and white matter are decreased by approximately 50% compared to normal. We feel, for reasons elaborated in the discussion, that the white matter of the isolated segments contains almost exclusively propriospinal axons. If this is accepted, then the axonal counts in this paper provide estimates of the numbers of propriospinal axons in rat spinal cord. In the isolated segments, the lateral funiculi contain 21,000 myelinated and 31,000 unmyelinated axons and the ventral funiculi 10,500 myelinated and 1,500 unmyelinated axons. The number of these fibers is approximately 33% of the number in unoperated spinal cords. Thus approximately one-third of the axons in rat sacral lateral and ventral funiculi are propriospinal, a lower figure than would have been predicted from classical work. The ratio of myelinated to unmyelinated fibers is higher for propriospinal fibers than for the other axons in these funiculi. Thus the propriospinal axons, as a group, are slightly larger than the other axons in these funiculi. This is against currently accepted thinking which generally regards the propriospinal fibers as the finest in the white matter of the cord. Finally, the quantification of propriospinal systems in these funiculi allows more precision in our thinking about the organization of the spinal cord.

The cell bodies of origin of sympathetic and sensory axons in some skin and muscle nerves of the cat hindlimb.

Cell bodies of sensory and sympathetic axons projecting to skin and skeletal muscle of the cat hindlimb have been labeled retrogradely with horseradish peroxidase (HRP) in order to study location, size, and numbers of the somata of these neurons. HRP was applied to the freshly transected axons of nerves supplying hairy skin (superficial peroneal, SP; sural, Su), hairy and hairless skin of the paw (medial plantar, MP), or skeletal muscle (gastrocnemius-soleus, GS). Serial sections of lumbosacral dorsal root and sympathetic ganglia were studied after standard histochemical processing. Additionally, the numbers of myelinated fibers in the same nerves were determined. All sensory somata and 99.4% of sympathetic cell bodies were located ipsilaterally. Sensory somata were commonly restricted to two adjacent dorsal root ganglia (usually L6-7 for SP, MP; L7-S1 for Su, GS). Although sympathetic somata were more widely distributed rostrocaudally, their maximum frequency always occurred in the segmental ganglia immediately rostral to the sensory outflows, i.e., corresponding to rami communicantes grisei. Dimensions of sympathetic somata varied little between populations projecting to different tissues and were unimodally distributed. The size distributions of sensory somata were characterized by a peak between 10 and 20 microns radius, similar to sympathetic somata, and a varying smaller number of cells ranging up to 60 microns radius. Each nerve had a characteristic distribution profile of afferent somata. A population of very small cells was only present in GS, while the largest sensory somata in GS and MP were bigger than those in SP and Su. Numerical analysis of the data disclosed the characteristic composition of both myelinated and unmyelinated fibers in each nerve studied.

Postnatal loss of axons in normal rat sciatic nerve.

Myelinated and unmyelinated axons were counted in sciatic nerves of newborn, 5-day-old, 14-day-old, and adult rats. Myelinated axons increase from essentially none at birth to approximately 8,000 in adulthood, but total axon numbers decrease steadily from 33,954 at birth to 22,872 in adulthood. Thus there is a significant postnatal loss of axons from rat sciatic nerve. This loss is, in our opinion, not associated with the death of the cells that give rise to these axons. This is thus an example of a regressive event that probably is of importance in normal neural development, namely the postnatal elimination of axons unaccompanied by death of the neurons that give rise to axons. These findings presumably imply a considerable amount of proximal peripheral axon branching, and the postnatal elimination of axons in the sciatic nerve presumably results from a reduction of this branching. Thus postnatal elimination of processes on, for example, somatic muscle cells may be at least partially the result of long axon elimination rather than local withdrawal of presynaptic processes, as is usually thought to be the case. In addition, an increased number of axons resulting from early postnatal manipulations may indicate cessation of axon loss rather than formation of new axons.

Connections of the visual cortex in the hedgehog (Paraechinus hypomelas). II. Corticocortical projections.

Cortical subdivisions based on cytoarchitectural and myeloarchitectural observations of normal tissue were correlated with the topography of corticocortical connections in the visual system of the Pakistani hedgehog. Large subpial aspiration lesions were made in both visual and non-visual cortical regions to determine the areal limits of the corticocortical connections of the visual cortex. Subsequently, discrete electrolytic lesions were placed within the visual cortex. After appropriate survival periods, the brains were processed and stained with the Fink-Heimer technique. The results of these studies show that the visual cortex may be subdivided into four distinct regions from lateral to medial; the lateral parastriate cortex, the lateral and medial part of striate cortex, and the medial parastriate cortex. Within these regions, interhemispheric connections between visual cortices arise mainly in the lateral striate and lateral parastriate regions and terminate in a single band within the lateral portion of the cytoarchitecturally defined striate cortex. These corticocortical projections, therefore, substantially overlap with the geniculostriate projections. Lateral striate cortex and lateral parastriate cortex project in a reciprocal fashion that correlates well with the physiologically defined mirror image representation of two retinotopic maps of the binocular visual field on cortex. These connections are reflected about a line that is closely correlated with the medial edge of the band of commissural axon terminals that is located within the lateral striate cortex, instead of corresponding exactly with the striate-parastriate border as they do in other mammals. Medial striate cortex projects to medial parastriate cortex, indicating that the monocular portion of V I is related to a separate secondary area of cortex on the medial wall of the hemisphere.

Human brain in tissue culture. I. Acquisition, initial processing, and establishment of brain cell cultures.

This paper details the in vitro techniques used to establish cells in culture from the brains of 40 patients, most of whom had chronic neurologic disease. The clinical and pathologic features of these patients are given. The success in establihsing cell lines was dependent upon the origin of tissue (biopsy vs. autopsy), the site of removal from the brain, and various environmental and technical manipulations in vitro.

A method for automatic classification of large and small myelinated fibre populations in peripheral nerves.

The statistical analysis of morphometric data collected from biopsies of human superficial peroneal nerve is complicated by the heterogeneity of the population of myelinated fibres. In order to make separate statistical analyses of the subpopulations of large and small fibres we have developed a computer program (written in PASCAL) for their automatic separation. The method is based on a dynamic centres clustering algorithm and was applied to the multifactorial space defined by the principal component analysis of the morphometric variables: axonal diameter, myelin sheath thickness, circularity index and g-ratio. The classification technique was applied to measurements obtained from 5 control nerves, and to simulated data, and in each case it gave consistent Gaussian subpopulations with no need for the introduction of supplementary variables.

Immuno-electron microscopic study of calcitonin gene-related peptide (CGRP) in axis cylinders of the vagus nerve. CGRP is present in both myelinated and unmyelinated fibers.

The localization of immunoreactive calcitonin gene-related peptide (IR-CGRP) in vagus nerve fibers of the rat was studied with pre-embedding electron immunocytochemistry. IR-CGRP was localized in a small number (approximately 5%) of myelinated fibers and approximately 50% of unmyelinated fibers of the vagus. The immumoreactivity was observed in the dense cored vesicles (75-150 nm) and in the axoplasm, but absent in the clear vesicles.

Tissue culture analysis of neurogenesis: myelination and synapse formation are retarded by serum deprivation.

Explants from newborn mouse cerebellum were cultured in nutrient media containing either adequate (30%) or low (15% or 7.5%) serum content. By light microscopic observation, delayed and inhibited myelination was detected in cultures fed with low serum media (experimental cultures). Specific activities of two enzymes related to myelin synthesis, 2',3'-cyclic nucleotide-3'-phosphohydrolase and 3-hydroxy-3-methylglutaryl coenzyme A reductase, were also reduced in experimental cultures. Morphometric analysis of electron micrographs showed that the size of presynaptic endings and total area occupied by synapses in experimental cultures were substantially reduced, while synaptic density per unit area increased. Reflecting the results of synaptic underdevelopment, the levels of two neurotransmitter enzymes, choline acetyltransferase and glutamic acid decarboxylase, were also decreased in experimental cultures.

Selective ovarian sympathectomy in the rabbit.

Evidence implicates the ovarian nerves in the regulation of hormonal and ovulatory functions. A technique for selectively denervating the in situ rabbit ovary is therefore of considerable investigative value. The ovarian artery was stripped of its adventitia and nerve bundles. Three weeks later, fluorescent histochemistry confirmed the complete absence of adrenergic nerves in the vessel walls and ovarian stroma of the experimental ovary, as compared to the abundant fluorescent structures present in the contralateral control ovary. The surgically treated ovaries demonstrated no significant ischemic or trophic change on gross and routine histologic examination.

Studies on the function of the denervated rabbit ovary: human chorionic gonadotropin-induced ovulation.

The described method for selective sympathetic denervation of the in vivo rabbit ovary involved stripping the ovarian artery of its nerve bundles and adventitial tissue. The ovary was then entirely free of fluorescent-staining adrenergic nerves. This technique was used to study the effects of ovarian denervation on HCG-induced ovulation. After HCG was administered to 22 rabbits which had previously undergone unilateral ovarian denervation, the ovaries were observed for follicular maturation and rupture. Control ovaries demonstrated a mean of 5.6 stimulated follicles/ovary; denervated ovaries had a mean of 5.4. An average of 3.5 follicles/control ovary ruptured; an average of 3.1 follicles/denervated ovary ruptured. Furthermore, the time course of ovulation after HCG did not differ between denervated and intact ovaries. These results indicate that HCG-induced ovulation in the rabbit is not interrupted by ovarian denervation.

Myelinated retinal nerve fibers.

In a series of 3,968 consecutive autopsies, myelinated nerve fibers of the retina were presented in 39 (0.98%) cases and bilateral in three (7.7%) affected cases; thus, 42 (0.54%) of the 7,936 eyes examined were affected. Myelinated nerve fiber lesions appeared as white or gray-white striated patches corresponding in shape to the distribution of retinal nerve fibers and demonstrated frayed borders. Myelinated retinal nerve fibers were continuous with the optic nerve head in 14 (33%) and discontinuous with the optic nerve head in 28 (66%) affected eyes. By light microscopy and electron microscopy, myelinated retinal fibers were marked by a ganglion cell axon surrounded by concentric lipoprotein lamellae that formed the myelin sheath. Glial cells were often prominent near the myelin sheaths, but other components of the sensory retina were morphologically normal. Clinically, 32 patients with myelinated retinal nerve fibers had comparable overall features, visual field defects less extensive than expected on the basis of ophthalmoscopic appearance, and normal findings on fluorescein angiography. Four patients had a syndrome characterized by ipsilateral extensive myelinated retinal nerve fibers, anisometropic myopia, amblyopia, and strabismus.

Computerized image recognition for morphometry of nerve attribute of shape of sampled transverse sections of myelinated fibers which best estimates their average diameter.

A computerized image recognition method was used to measure various attributes of shape of cross-sections of myelinated nerve fibers. Measurements were made at intervals over 1/2 internode of each fiber on 20 fibers from each of 4 sural nerves from rats. Diameters were computed in 6 different ways from the computer measurements and compared for bias, precision, and accuracy between sections and to the diameter of an idealized cylinder reconstructed for each fiber from multiple actual cross sections. The diameter computed from cross-sectional areas of transversely sectioned myelinated fibers, converted into a circle, showed the highest precision, greatest accuracy and least bias. Fibers were classified by shape and the frequency was determined in defined regions (I1 = paranodal, I3= nuclear and I2=region between I1 and I3) of the 1/2 internode. A crenated shape is highly characteristic of the I1 region. The boomerang shape was found most frequently in I3 whereas the circular shape was found most frequently in I2. Epileptical and boomerang shapes of myelinated fibers within fascicles which have been orientated carefully to obtain transverse sections, are not due to obliquity of section. Therefore, using the minor axis to determine the diameter of such profiles, as we had done previously in our laboratory, is in error. We conclude from these studies, that in carefully orientated transverse sections of nerve trucks, the diameter calculated from measurement of area converted to a circular shape is the best among the various estimates of myelinated fiber diameter and is the most suitable one for use in computerized image recognition systems for nerve morphometry. It seems reasonable to extrapolate this general conclusion to myelinated fibers of man.