Structure of the giant fibers of earthworms.

The median giant fiber and the pair of lateral giant fibers that run the length of the ventral nerve cord in earthworms were thought to arise by fusion of the axons of several nerve cells in each segment. The structure of these giant fibers has now been examined with a fluorescent dye injected into single fibers. Each giant axon connects to one cell body in each segment; the giant fibers are not fused axons. In each segment, the median giant fiber has three branches and each lateral giant fiber has five branches. These branches are presumably dendritic. No structural differences between the giant fibers in anterior and posterior regions of the worm seem to account for the functional polarity of the giant fiber system observed in behavioral studies.

Motoneuron morphology and synaptic contacts: determination by intracellular dye injection.

The structure and dendritic connections of an identified crustacean motoneuron were analyzed by intracellular injection of dye. Some processes of the neuron end in the ganglionic neuropil, but most terminate on axons which pass through the ganglion in specific, identifiable tracts. The former processes are ipsilateral to the soma, while the latter, as well as their connections, display bilateral symmetry. Structural and functional evidence suggests that the demonstrated contacts are synaptic junctions, and that the approach can therefore be used to study patterns of synaptic organization in complex neural networks.

The changing view of neural specificity.

The generation of specific patterns of neuronal connections has usually been regarded as a central problem in neurobiology. The prevailing view for many years has been that these connections are established by complementary recognition molecules on the pre- and postsynaptic cells (the chemoaffinity theory). Experimental results obtained in the past decade, however, indicate that the view that axon guidance and synaptogenesis proceed according to restrictive chemical markers is too narrow. Although a more rigid plan may prevail in some invertebrates, the formation of specific connections in vertebrates also involves competition between axon terminals, trophic feedback between pre- and postsynaptic cells, and modification of connections by functional activity.

Pars intermedia: unitary electrical activity regulated by light.

In the pars intermedia of frogs in the dark two types of spontaneously firing neuronal units have been found; one can be inhibited by and the other is indifferent to increases in illumination. The receptor for the light-inhibited units appears to be the pineal organ. Transection experiments indicate that the axons to the two kinds of units in the pars intermedia are separately grouped in the floor.

Magnitude and location of surface charges on Myxicola giant axons.

The effects of changes in the concentration of calcium in solutions bathing Myxicola giant axons on the voltage dependence of sodium and potassium conductance and on the instantaneous sodium and potassium current-voltage relations have been measured. The sodium conductance-voltage relation is shifted along the voltage axis by 13 mV in the hyperpolarizing direction for a fourfold decrease in calcium concentration. The potassium conductance-voltage relation is shifted only half as much as that for sodium. There is no effect on the shape of the sodium and potassium instantaneous current-voltage curves: the normal constant-field rectification of potassium currents is maintained and the normal linear relationship of sodium currents is maintained. Considering that shifts in conductances would reflect the presence of surface charges near the gating machinery and that shape changes of instantaneous current-voltage curves would reflect the presence of surface charges near the ionic pores, these results indicate a negative surface charge density of about 1 electronic charge per 120 A2 near the sodium gating machinery, about 1 e/300 A2 for the potassium gating machinery, and much less surface charge near the sodium or potassium pores. There may be some specific binding of calcium to these surface charges with an upper limit on the binding constant of about 0.2 M-1. The differences in surface charge density suggest a spatial separation for these four membrane components.

Morphology and electrophysiological properties of squid giant axons perfused intracellularly with protease solution.

Squid giant axons were perfused intracellularly with solutions containing various kinds of proteases (1 mg/ml). Except for a 10 micro layer inside the axolemma the axoplasm was removed by a 5 min perfusion with Bacillus protease, strain N' (BPN'). The resting and action potentials were unchanged and the axon maintained its excitability for more than 4 hr on subsequent enzyme-free perfusion. After perfusion with protease solution for 30 min the axoplasm was almost completely removed. The excitability was maintained, but the action potential became prolonged and rapidly developed a plateau of several hundred milliseconds. The change was not reversible even when the enzyme was removed from the perfusing fluid. Two other enzymes, prozyme and bromelin, also removed the protoplasm without blocking conduction. Trypsin suppressed within 3 min the excitability of the axon. It is suggested that the proteases alter macromolecules in the excitable membrane and thus affect the shape of the action potential.

A new approach toward analyzing peripheral nerve fiber populations. I. Variance in sheath thickness corresponds to different geometric proportions of the internodes.

The thickness of the myelin sheath is known to increase with axon caliber, but there is also a superimposed, slight variation in sheath thickness depending on whether a fiber of a given caliber has very long or very short internodes. This relationship between myelin sheath thickness and the geometric proportion of the internode has been shown in subserial sections of isolated nerve fibers. It allows a prediction of sheath thickness from the quotient internode length/axon caliber, or conversely, a prediction of internode foreshortening from sheath thickness. We applied this new approach to the analysis of sciatic fiber populations of frogs, mice, rats and cats. The geometric proportions of these fibers were defined by the quotient internode length/fiber caliber. This quotient was compared with minor variation in sheath thickness as determined with a computer-assisted technique measuring large numbers of fibers in low-power electron micrographs. The method also calculated fiber shrinkage and recalculated all data for circular fiber profiles. The data obtained confirmed previous electron microscopic measurements showing that there is a slight reduction in sheath thickness when a fiber of a given caliber has relatively short internodes, and vice versa. A population of very thin, thinly myelinated fibers was also revealed. Sheath thickness and the geometric proportions of internodes in frogs differed markedly from those in mammals.

A new approach toward analyzing peripheral nerve fiber populations. II. Foreshortening of regenerated internodes corresponds to reduced sheath thickness.

The new approach used in this study is based on the concept that axon caliber is not the only factor affecting the thickness of the myelin sheath. It is necessary to consider the entire geometric proportions of the internode, since sheath thickness corresponds to the relationship between axon caliber and the length of the internode. This type of analysis was applied to the regenerated internodes in rat sciatic nerves. Survival periods of 4, 9, 18 and 36 weeks were studied after lesions had been placed in young adult rats. The data show significantly thinner sheaths for regenerated fibers as compared with normal nerves, consistent with previous observations. This reduction in sheath thickness, however, corresponded quantitatively to the degree of foreshortening of internodes in the regenerated nerves. An average reduction of 10 in the quotient internode length/fiber caliber corresponded to a reduction of about 0.015 in the relative thickness of the sheath (quotient axon diameter/fiber diameter). This means that regenerated myelin sheaths are not truly hypoplastic; rather, they are adapted to the reduced internode length, and have the same relationship found for normal fibers. In partially damaged nerves there was a clear distinction in terms of sheath thickness between regenerated fibers and undamaged fibers. Demonstration of this phenomenon by scatter diagrams opens new possibilities for the quantitative assessment of neuropathies.

Projections from the cochlear nucleus to the inferior colliculus in normal and neonatally cochlea-ablated gerbils.

The distribution of the projection from one cochlear nucleus (CN) within each inferior colliculus (IC) was studied in adult, normal gerbils and adult gerbils subjected to unilateral ablation of the contralateral cochlea at 2 days of age. The projection was studied by using the Fink-Heimer technique for impregnating degenerating axons and their terminal processes with silver. Following an extensive, unilateral lesion of the CN, degeneration was seen in both ICs of all animals. In normal animals, degeneration was both more widespread and heavier in the contralateral than in the ipsilateral central nucleus of IC (ICC). Degeneration was most widespread in the rostral and lateral parts of both ICCs and in the ventral part of the contralateral ICC. Degeneration was observed in 26% of the area examined in ipsilateral ICC and in 73% of the area examined in contralateral ICC. In cochlea-ablated animals there was a much greater similarity in the area of degeneration in the ICC ipsilateral (57%) and contralateral (67%) to the CN lesion. The same regional distributions of degeneration were observed as in the normal animals except that the distribution of degeneration in the ipsilateral ICC more closely resembled the normal contralateral than the normal ipsilateral profile. We conclude that the normal distribution of projections from the CN within the ipsilateral ICC is substantially modified by neonatal ablation of the contralateral cochlea.

Primary sensory ganglion cells projecting to the principal trigeminal nucleus in the mallard, Anas platyrhynchos.

The trigeminal and glossopharyngeal ganglia of the adult mallard were studied following HRP injections into the principal trigeminal nucleus (PrV). The PrV consists of the principal trigeminal nucleus proper (prV) and the principal glossopharyngeal nucleus (prIX). After an injection into the prV, the labeled cells were found in the ipsilateral trigeminal ganglion. After an injection into the prIX, labeled cells were found in the ipsilateral distal glossopharyngeal ganglion, but not in the proximal ganglion of the IX and X cranial nerve (pGIX + X). In Nissl preparations, two types of ganglion cells in the trigeminal ganglion, pGIX + X, and distal ganglion of N IX could be distinguished: larger light cells and smaller dark cells. We could not determine whether the HRP-labeled cells belonged to both types or to one of them; but because all the labeled cells were over 20 microns, we concluded that the smallest cells (10-19 microns) in the trigeminal ganglion and distal ganglion of N IX did not project to the PrV. The labeling of the cells in the distal ganglion of N IX (average 34.5 microns) was uniformly moderate. In the trigeminal ganglion there were two types of labeled cells: heavily labeled cells (average 29.1 microns) and moderately labeled cells (average 35.1 l microns). These two types of labeling (moderate and heavy) may reflect two types of primary sensory neurons: cells with ascending, nonbifurcating axons, and cells with bifurcating axons. We speculate that the former are proprioceptive neurons and the latter tactile neurons. Labeled bifurcating axons in the sensory trigeminal complex gave off collaterals to all parts of the descending trigeminal nucleus except to the caudalmost laminated spinal part.

Distribution of phenylethanolamine N-methyltransferase cell bodies, axons, and terminals in monkey brainstem: an immunohistochemical mapping study.

Adrenaline (epinephrine) is an important candidate transmitter in descending spinal control systems. To date intrinsic spinal adrenergic neurons have not been reported; thus adrenergic input is presumably derived from brainstem sites. In this regard, the localization of adrenergic neurons in the brainstem is an important consideration. Maps of adrenergic cell bodies and to a lesser extent axons and terminal fields have been made in various species, but not in monkeys. Thus, the present study concerns the organization of adrenergic systems in the brainstem of a monkey (Macaca fascicularis) immunohistochemically mapped by means of an antibody to the enzyme phenylethanolamine N-methyltransferase (PNMT). PNMT-immunostained cell bodies are distributed throughout the medulla in two principal locations. One concentration of labeled cells is in the dorsomedial medulla and includes the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (X), and an area ventral to X in a region of the reticular formation (RF) known as the central nucleus dorsalis (CnD) of the medulla. A few scattered cells are observed in the periventricular gray just ventral to the IVth ventricle and on midline in the raphe. The second major concentration of PNMT-immunostained cells is located in the ventrolateral RF, lateral and dorsolateral to the inferior olive (IO), including some cells in the rostral part of the lateral reticular nucleus (LRN). Terminal fields are located in the NTS, X, area postrema (AP), and the floor of the IVth ventricle in the medulla and pons. A light terminal field is also observed in the raphe, particularly raphe pallidus (RP). A heavy terminal field is present in locus coeruleus (LC). Fibers labeled for PNMT form two major fiber tracts. One is in the dorsomedial RF extending as a well-organized bundle through the medulla, pons, and midbrain. A second tract is located on the ventrolateral edge of the medulla and caudal pons. Fibers in this tract appear to descend to the spinal cord. A comparison with maps of other catecholamine neurons in primates is discussed, confirming that the distribution of the adrenergic system in monkeys is similar to that described in the human.

Sympathetic postganglionic unmyelinated axons in the rat peripheral nervous system.

We determined the contribution made to the unmyelinated axon population of the rat peripheral nervous system by sympathetic paravertebral ganglion cells. Sympathectomy, achieved by administration of guanethidine to neonatal rats, led to atrophy of the sympathetic paravertebral ganglion chain, a 95% decrease in peripheral nerve norepinephrine, and loss of 20 to 26% of the unmyelinated axons in a cutaneous nerve (sural), a muscular nerve (nerve to soleus), and a mixed nerve (sciatic). These data indicate that up to a quarter of the total population of peripheral nerve unmyelinated axons are sympathetic ganglia-derived.

Unmyelinated axon subpopulations in the rat peripheral nervous system.

Using the neurotoxin capsaicin, we examined subpopulations of unmyelinated axons in mixed (sciatic), cutaneous (sural), and muscular (nerve to soleus) nerves. Administration of capsaicin to neonatal rats caused reduction of the sciatic nerve immunoreactive (IR)-substance P (by 45%) and IR-somatostatin (by 84%) contents. This correlated with a substantial reduction in unmyelinated axons in the sciatic and sural nerves (45% and 65%, respectively), although there was no significant decrease in unmyelinated axons in the nerve to soleus. In a parallel study, we have shown that sympathetic ganglia-derived unmyelinated axons account for about 20 to 25% of the total unmyelinated axon population in both the sural nerve and the nerve to soleus. Thus, in the sural nerve, the majority of unmyelinated axons are dorsal root ganglia-derived, contain either substance P or somatostatin, and are capsaicin-sensitive; whereas in the nerve to soleus, the majority of unmyelinated axons are dorsal root ganglia-derived but are insensitive to capsaicin and do not contain substance P or somatostatin. These latter unmyelinated axons presumably contain a yet to be defined neurotransmitter and may be the axons connecting with muscular ergoreceptors, a subpopulation of unmyelinated axons that are biochemically and functionally distinct from the unmyelinated axons of cutaneous nerves.

Distal axonopathy: one common type of neurotoxic lesion.

Neurotoxic chemicals commonly produce retrograde degeneration of the axons of long and large nerve fibers in the central and peripheral nervous system. This produces a clinical picture of polyneuropathy in man and animals in which sensory and motor disturbances develop in the feet and hands then progress with time to the legs and arms. Distal axonopathy, as the underlying pathologic process is termed, is one of four principal types of neurotoxic diseases, the others including degeneration of neurons (neuronopathy), myelin sheaths (myelinopathy) and damage to the neurovasculature (neurovasculopathy). In the experimental animal, these four types of neurotoxic diseases can be distinguished by examining selected areas of brain and nerve tissues prepared by contemporary methods of tissue fixation. These procedures may form the basis of a new and sensitive assay for neurotoxicity.

The course of axons through the retina and optic nerve head.

By identifying degenerating axons in tissue specimens from 22 primate eyes, it was possible to demonstrate the normal course of axon fibers. Nerve fiber bundles from a group of retinal ganglion cells travel together with little tendency to disperse laterally. In addition, axons are stratified such that processes from more central ganglion cells are successfully added to the inner strata of the retinal nerve fiber layer. Within and behind the lamina cribrosa, areas of degeneration following retinal photocoagulation were well circumscribed and confined to a group of adjacent axon bundles. This degree of retinotopic organization of axons within the nerve head and retinal fiber layer is believed to be consistent with the premise that isolated lesions within the lamina cribrosa could cause well-organized paracentral scotomas such as those characteristic of early glaucoma.

The histology of retinal nerve fiber layer bundles and bundle defects.

The fiber bundle striations recognized clinically in normal monkey eyes appear to be bundles of axons compartmentalized within glial tunnels formed by Müller's-cell processes, when viewed histologically. The dark boundaries that separate individual bundles are the broadened foot endings of these cells near the inner surface of the retina. Within one week after focal retinal photocoagulation, characteristic fundus changes could be seen in experimental eyes. In histologic sections of the involved retina, there was marked cystic degeneration of the retinal nerve fiber layer. Within one month, atrophy of distal axon segments was complete. With the drop-out of damaged axons and thinning of individual fiber bundles, retinal striations became less prominent. The resulting fundus picture in these experimental eyes is similar to fiber bundle defects that can be seen clinically in various neuro-ophthalmic disorders.

The organization of nerve fiber bundles in the primate optic nerve head.

Horseradish peroxidase was injected into various areas of the optic disc of monkeys to label retrograde axoplasmic transport and permit identification and mapping of ganglion cells projecting through the areas of injection. Peripheral ganglion cell axons project through the deeper layers of the peripapillary nerve fiber layer (NFL) and enter the peripheral optic nerve. Peripapillary ganglion cell axons project through those coming from peripheral locations to superficial positions in the NFL, then to the central optic nerve. The Bjerrum portion of the optic nerve head includes approximately the central 30 degrees of the superior and inferior temporal quadrants of the disc. The course of nerve fiber bundles corresponding to those most susceptible to glaucoma (Bjerrum area) is regularly and predictably organized horizontally and vertically in the nerve head.

Nerve fibre morphometry: a comparison of techniques.

This study compared different approaches to measuring nerve axon and fibre diameters and areas from transverse sections. A mock photomicrograph and mock tissue section, each with 100 identical, circular 'fibres' was constructed. Three measurement protocols were investigated: (A) circular approximation from minimum diameter; (B) circular approximation from the mean of orthogonal diameters; and (C) calculation of diameter and area from a digitized circumference. For each protocol, all 100 fibres on the photomicrograph were repeatedly measured using a digitizing tablet. Similarly, the fibres on the mock tissue section were measured using a digitizing tablet and microscope with camera lucida. The variance for these data was calculated. Protocols were compared on the basis of variability and the amount of digitizing time required. For diameter measurements, protocol B showed significantly lower variability than A or C (P less than 0.05), with only a modest increase in digitizing time over A. For area measurements, protocols B and C showed significantly lower variability than A (P less than 0.05), again with a modest increase in digitizing time. Measurements made using the microscope and camera lucida showed significantly lower variability than those made from the photomicrograph, but took more time. These data suggest that for diameter measurements, a mean of orthogonal diameters approach is best, and that for area measurements, a traced circumference approach is best as it is more flexible than the orthogonal diameter approach. While the microscope and camera lucida setup is more time-consuming to use, it eliminates the need for photomicrograph production.

An economic anterograde axonal tracing method using Phaseolus vulgaris agglutinin (PHA) P-form.

Phaseolus vulgaris leucoagglutinin (PHA-L) has been demonstrated to be an excellent neuroanatomical anterograde tracer. So far there are relatively few applications of this technique, mainly due to the cost of the lectin. Instead of PHA-L, we have successfully used PHA-P, which is a crude and inexpensive form of PHA-L. The sensitivity of the present method, examined in the rat striatonigral pathway, is as high as that of the conventional method. Furthermore, a large amount of PHA-P, injected into the cat eye, demonstrated the retinofugal projection in detail.

Neural anatomy of the human anterior cruciate ligament.

The histology of the anterior cruciate ligament was studied by a modified technique of the Gairns gold chloride stain for neural elements. Three morphological types of mechanoreceptors and free nerve-endings were identified: two of the slow-adapting Ruffini type and the third, a rapidly adapting Pacinian corpuscle. Rapidly adapting receptors signal motion and slow-adapting receptors subserve speed and acceleration. Free nerve-endings, which are responsible for pain, were also identified within the ligament. These neural elements comprise 1 per cent of the area of the anterior cruciate ligament.