Acetylcholinesterase activity of motor and sensory nerve fibers in the spinal nerve roots of the rat

Summary The histochemical and cytochemical distribution of acetylcholinesterase activity in the anterior and posterior spinal nerve roots and ganglia of the rat was demonstrated by the Karnovsky method using acetyl and butyrylthiocholine as substrates and eserine and DFP as inhibitors. Light and electron microscopic examination of transverse frozen sections enabled the simultaneous visualization of end product in relationship to the various fiber components of each nerve root. While the enzymatic activity of the anterior roots was consistantly observed in the large extrafusal and small intrafusal motor fibers a relatively greater amount of precipitate occurred in aggregates of myelinated and unmyelinated fibers believed to represent preganglionic sympathetic nerves. In contrast, no significant enzymatic activity could be demonstrated in the myelinated nerve fibers of the posterior root. In the sensory sytem, the limited enzymatic precipitate was largely restricted to the unmyelinated afferent fibers and to their small cell bodies in the dorsal root ganglia. The ultrastructural distribution of enzymatic activity was located in the granular endoplasmic reticulum and perinuclear spaces of the ganglion cells. Within peripheral nerves this end product occurred between the apposing axonal and Schwann cell membranes and along the membranous aspect of occasional axoplasmic vesicles of both myelinated and unmyelinated nerve fibers.This study was supported by grants NB 04161-04 and NB 04161-05 of the National Institute of Neurological Diseases and Blindness. — The author would like to thank MissMaria C. la Valle for her skillful technical assistance.     

The chorda tympani and glossopharyngeal nerves in the adult hamster

The numbers and diameters of axons in the intact chorda tympani(CT) and lingual branch of the glossopharyngeal nerve (GN) arequantified with the use of electron microscopic photomontages.The cross-sectional diameters of the CT and GN average 68 and86 microns, respectively. The intact CT contains {small tilde}1050 fibers, 63% are unmyelinated and 37% are myelinated. TheGN contains {small tilde} 1600 fibers, 79% are unmyelinatedand 21% are myelinated. Both nerves are made up of relativelysmall unmyelinated and myelinated fibers, although the GN showsa broader distribution of diameters for its myelinated fibersdue to the presence of general somatosensory fibers. Followingde-efferentation, there is a 48% reduction in the number ofunmyelinated fibers in the CT. Fifty-two per cent of the unmyelinatedfibers are sensory. The number of myelinated fibers is not significantlyreduced and nearly all of the myelinated fibers are sensory.Sixty-seven per cent of the fibers within the CT are sensory.The de-efferented CT contains an equal number of unmyelinatedand myelinated axons and a total of {small tilde} 700 fibers.Comparable data in the rat indicate that its intact and de-efferentedCT are organized differently in regards to the numbers of sensoryand motor, and myelinated versus unmyelinated fibers. The findingsof the present study, together with the available data fromother species, suggest that anatomical differences in the make-upof the major gustatory nerves do not contribute in any obviousway to the known differences in the response properties betweenthe rat and hamster CT, and that the number of myelinated fibersin the visceral motor component of the CT varies considerablyacross species.     

Electron-microscopic analysis of the mouse facial nerve near the geniculate ganglion

An electron-microscopic analysis of the mouse facial nerve near the geniculate ganglion shows that there are, on the everage, 603 more nerve fibers in the portion of the nerve distal to the geniculate ganglion than there are in the part proximal to the ganglion. The average distal increase in the number of unmyelinated fibers is 444 and that in the myelinated fibers is 165. The somatic motor nerve fibers and the parasympathetic fibers in the mouse facial nerve may not contribute to the distal excess. It is possible that the increase in the number of unmyelinated fibers distal to the geniculate ganglion is mainly due to the presence of postganglionic sympathetic fibers in the facial trunk distal to the geniculate ganglion and the greater petrosal nerve. The distal increase in the number of myelinated fibers may be mainly contributed by the sensory fibers.     

Adenosine triphosphatase activity of cutaneous nerve fibers

Summary The histochemical study of Mg⁺⁺-activated adenosine triphosphatase (Mg⁺⁺-ATPase) activity was carried out on the peripheral nerves of mouse digital skin by light and electron microscopy. Under the light microscope, the ATPase activity was clearly demonstrated on the nerve fibers as a fine network in the subepidermal regions. Under the electron microscope, the reaction product of enzyme activity was located in the interspace between axolemma and the surrounding Schwann cells of the unmyelinated nerve fibers. No reaction product was observed in the space between the axolemma and the Schwann cells associated with myelinated nerve fibers. Demonstrable activity was absent at the nodes of Ranvier as well as on the para- and internodal regions of these myelinated axons. The part of the axolemma lacking a Schwann cell sheath failed to show a reaction product. The perineural epithelial cells surrounding the nerve fibers displayed reaction product in the caveolae. These results suggest a functional difference in the axon-Schwann interface of myelinated as compared to unmyelinated nerve fibers. The function of the perineural epithelial cell would be expected to be a regulatory one in transferring materials across the epithelium to keep the proper humoral environment around nerve fibers.     

Evidence that contact with connective tissue matrix is required for normal interaction between Schwann cells and nerve fibers

Explants of fetal rat sensory ganglia, cultured under conditions allowing axon and Schwann cell outgrowth in the absence of fibroblasts, occasionally develop nerve fascicles that are partially suspended in culture medium above the collagen substrate. In these suspended regions, fascicles are abnormal in that Schwann cells are decreased in number, are confined to occasional clusters along the fascicle, provide ensheathment for only a few axons at the fascicle periphery, and do not form myelin. When these fascicles are presented with a substrate of reconstituted rat-tail collagen, Schwann cell numbers increase, ensheathment of small nerve fibers occurs normally, and larger axons are myelinated. We conclude that, for normal development, Schwann cells require contact with extracellular matrix as well as axons. The Schwann cell abnormalities in suspended fascicles are similar to those observed in nerve roots of dystrophic mice.     

Heterophilic binding of L1 on unmyelinated sensory axons mediates Schwann cell adhesion and is required for axonal survival.

This study investigated the function of the adhesion molecule L1 in unmyelinated fibers of the peripheral nervous system (PNS) by analysis of L1- deficient mice. We demonstrate that L1 is present on axons and Schwann cells of sensory unmyelinated fibers, but only on Schwann cells of sympathetic unmyelinated fibers. In L1-deficient sensory nerves, Schwann cells formed but failed to retain normal axonal ensheathment. L1-deficient mice had reduced sensory function and loss of unmyelinated axons, while sympathetic unmyelinated axons appeared normal. In nerve transplant studies, loss of axonal-L1, but not Schwann cell-L1, reproduced the L1-deficient phenotype. These data establish that heterophilic axonal-L1 interactions mediate adhesion between unmyelinated sensory axons and Schwann cells, stabilize the polarization of Schwann cell surface membranes, and mediate a trophic effect that assures axonal survival.     

Quantitative studies on the motor root of the mouse facial nerve. An electron-microscopic study.

Fiber count analysis was performed with the electron microscope on the motor root of the facial nerve in six mice. On an average, 3,433 (84.9%) of the total nerve fibers (4,046) were myelinated and 592 (14.6%) unmyelinated. The motor root consisted mostly of large myelinated fibers (large fiber group), but a part of the root consisted of small myelinated and unmyelinated fibers (small fiber group). The nerve fibers of the small fiber group appear to correspond with those of the intermediate nerve, and to pass through the motor root and enter the intermediate nerve near the geniculate ganglion.     

Axon typing of rat muscle nerves using a double staining procedure for cholinesterase and carbonic anhydrase.

We developed a method for detecting activity of axonal cholinesterase (CE) and carbonic anhydrase (CA)--markers for motor and sensory nerve fibers (NFs)--in the same histological section. To reach this goal, cross-sections of muscle nerves were sequentially incubated with the standard protocols for CE and CA histochemistry. A modified incubation medium was used for CA in which Co++ is replaced by Ni++. This avoids interference of the two histochemical reactions because Co++ binds unspecifically to the brown copper-ferroferricyanide complex representing CE activity, whereas Ni++ does not. Cross-sections of the trapezius muscle nerve containing efferent and afferent NFs in segregated fascicles showed that CE activity was confined to motor NFs. Axonal CA was detected solely in sensory NFs. The number of labeled motor and sensory NFs determined in serial cross-sections stained with either the new or the conventional technique was not significantly different. Morphometric analysis revealed that small unreactive NFs (diameter less than 5 microns) are afferent, medium-sized ones (5 microns less than d less than 7 microns) are unclassifiable, and large ones (d greater than 7 microns) are efferent. The heterogenous CE activity of thick (alpha) motor NFs is linked to the type of their motor units. "Fast" motor units contain CE reactive NFs; "slow" ones have CE negative neurites.     

A quantitative electron microscope analysis of peripheral nerve in the urodele amphibian in relation to limb regenerative capacity

An approximate 1:1 ratio of myelinated to unmyelinated fibers was established in counts from electron micrograph montages in nerves of the newt, Triturus (Notophthalmus) viridescens. The number of myelinated fibers correspond to the number counted with the light microscope after osmium fixation. Light microscope counts of silver impregnated sections yielded a value slightly higher suggesting that, except for bundles of unmyelinated fibers, the silver technique revealed mainly myelinated fibers. The results were used to reassess previous quantitative studies on the relation between number of nerve fibers and the control which nerves exert on regeneration. For a truer estimate of the number of axons affecting regeneration, fiber values previously reported should now be doubled to include the large number of unmyelinated fibers. However, calculations show that the unmyelinated fibers contribute less than 3% of the total neuroplasm in the peripheral nerve. Finally, counts made of Schwann cells and fibroblasts show that the latter are few in number.     

Distribution of the 75-kD Low-Affinity Nerve Growth Factor Receptor in the Primate Peripheral Nervous System

Disruption of the 75-kD low-affinity nerve growth factor (NGF) receptor (p75) has been shown to result in sensory and sympathetic nervous system deficits (Lee et al., 1992a,b). In order to establish precisely which subsets of neurons are capable of responding to neurotrophins (NTs) through the low-affinity NGF receptor, p75 was localized in the primate autonomic and somatic sensory nervous systems. In the autonomic system, cell bodies of some parasympathetic and enteric neurons expressed detectable levels of p75, whereas all sympathetic neurons expressed the protein. In the sensory system, some, but not all, cell bodies were labeled in cranial and spinal sensory ganglia and in the mesencephalic nucleus. Some peripheral and central projections of the sensory neurons were also labeled. Centrally, most of the labeled processes were found in regions containing primarily small unmyelinated fibers, including lamina II of Rexed and areas of the solitary tract and nucleus. Peripherally, labeled processes were associated with unmyelinated nerves and specialized structures such as taste buds and Meissner corpuscles, but not with myelinated processes. This study indicates that the subset of neurons in the autonomic nervous system likely to be capable of responding to neurotrophins is broader than generally thought, and that p75-ex-pressing neurons tend to be clustered. Moreover, in the sensory nervous system p75 is expressed by most cell bodies, but expression in their projections is restricted both peripherally and centrally to unmyelinated processes and nerve terminals.     

Composition of the medial and posterior articular nerves of the mouse knee joint

The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 +/- 28 nerve fibers consisting of 63 +/- 24 unmyelinated and 12 +/- 6 myelinated fibers. The PAN was composed of 195 +/- 50 nerve fibers, namely 129 +/- 28 unmyelinated and 66 +/- 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 microm with a maximum between 0.3 and 0.6 microm. The myelinated nerve fibers in the MAN ranged from 1 to 8 microm with a peak between 2 and 5 microm. In the PAN, their diameters ranged from 1 to 12 microm with a clearly visible peak at 4-5 microm and a plateau at 8-9 microm that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.     

Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman microspectroscopy

Detection of peripheral nerve tissues during surgery is required to avoid neural disturbance following surgery as an aspect of realizing better functional outcome. We provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues, including myelinated and unmyelinated nerves, against adjacent tissues that employ spontaneous Raman microspectroscopy. To investigate the Raman spectral features of peripheral nerves in detail, we used unfixed sectioned samples. Raman spectra of myelinated nerve, unmyelinated nerve, fibrous connective tissue, skeletal muscle, tunica media of blood vessel, and adipose tissue of Wistar rats were analyzed, and Raman images of the tissue distribution were constructed using the map of the ordinary least squares regression (OLSR) estimates. We found that nerve tissues exhibited a specific Raman spectrum arising from axon or myelin sheath, and that the nerve tissues can be selectively detected against the other tissues. Moreover, myelinated and unmyelinated nerves can be distinguished by the intensity differences of 2,855 cm^?1, and 2,945 cm^?1, which are mainly derived from lipid and protein contents of nerve fibers. We applied this method to unfixed section samples of human periprostatic tissues excised from prostatic cancer patients. Myelinated nerves, unmyelinated nerves, fibrous connective tissues, and adipose tissues of the periprostatic tissues were separately detected by OLSR analysis. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.     

Sex Differences in Sensory and Motor Branches of the Pudendal Nerve of the Rat

The morphology of the pudendal nerve was quantified in adult male and female rats. The sensory branch of the pudendal nerve was about three times as large in cross section in males as in females, and the motor branch was about five times as large. Electron microscopy was used to determine the ultrastructural bases of these gross size differences. Differences that were found included greater packing density of both myelinated and unmyelinated axons in females, larger myelinated and unmyelinated axons in males, larger myelin sheaths of sensory axons in males, more numerous myelinated axons in both branches of males, and more numerous unmyelinated axons in the sensory branch of males. There was also some indication that myelinated sensory axons were more likely to branch in the dorsal clitoral nerve of females than in the homologous nerve of males. Morphological differences in the structure of pudendal axons, their associated Schwann cells, and the extracellular matrix as well as differences in sensory and motor axonal number all have potential implications for the sexual differentiation of the central nervous system and behavior.     

Morphometric comparison between human and rat abducens and oculomotor nerves

A morphologic and morphometric comparison between normal human and rat extraocular muscle nerves was performed using a computer-assisted method to obtain scatter diagrams of relative sheath thickness (g ratio = quotient axon diameter/fiber diameter). Human and rat extraocular muscle nerves (nervus abducens and ramus medialis n. oculomotorii) were excised immediately before the nerve branching at the entering point into the muscle. There was no difference in the absolute number of myelinated fibers between the oculomotor and abducens nerves in both species. The distribution of myelinated fibers was classified according to their g ratios into a two-stage density cluster analysis. Two main populations of nerve fibers for human oculomotor and rat oculomotor and abducens nerves and three main populations for human abducens nerve were differentiated morphometrically and mathematically, differing in their relative sheath thicknesses. There are distinct differences between scatter diagrams of human and rat extraocular muscle nerves, in correlation with the basically different oculomotor functions of these two species. The morphometric differences between human and rat extraocular muscle nerves suggest a difference in the myelination process and the presence of functionally different nerve fibers, strongly indicated by the populations and subpopulations of myelinating nerve fibers peculiar to extraocular muscle. The existence of more than two different types of myelinated fibers in the human nerves implies that the traditional classification based on fiber caliber must be reviewed and a comparison of different classes of nerve and muscle fibers should be performed.     

Fiber size and sensory endings of the middle cardiac nerve of the domestic fowl (Gallus domesticus)

The middle cardiac nerve, a branch of the vagus, innervates the ventricles of the avian heart. Of 533 myelinated sensory fibers, the size range was 2 micron. The ratio of myelinated to unmyelinated sensory fibers ranged from 2.17 to 3.48. Sensory endings resembled a network pattern with no distinct receptor-like endings. Frequency of nerve population increased from apex to base of the heart.     

Nerve growth factor receptor (NGFR) immunoreactivity in skeletal muscles of the rat.

The peroxidase-antiperoxidase (PAP) method, and a specific monoclonal antibody (192-IgG) were used to determine the localization of nerve growth factor receptor (NGFr) in the skeletal muscles of the adult rats. The rectus femoris and the gastrocnemius (medialis and lateralis) muscles were analyzed. Occurrence of NGFr immunoreactivity was observed in: 1) a subpopulation of myelinated nerve fibers within muscle nerve trunks; 2) the vascular adventitia and nerve-like profiles around the blood vessels; 3) the outer capsule and the surface of the intrafusal muscle fibers of muscle spindles. Conversely, images, suggesting the presence of NGFr on muscle fibers or in motor end-plates, were not found. Our results suggest the presence of NGF-binding sites in sensory and sympathetic nerve fibers, and/or their target tissues localized on the skeletal muscles of the rat, whereas the motor nerve fibers lack of NGFr. The dependence of sympathetic neurons, proprioceptive primary sensory neurons, and motoneurons innervating the mammalian muscles upon NGF or other neurotrophic factors is discussed.     

Composition of the medial and posterior articular nerves of the mouse knee joint

The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 &#45 28 nerve fibers consisting of 63 &#45 24 unmyelinated and 12 &#45 6 myelinated fibers. The PAN was composed of 195 &#45 50 nerve fibers, namely 129 &#45 28 unmyelinated and 66 &#45 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 &#119 m with a maximum between 0.3 and 0.6 &#119 m. The myelinated nerve fibers in the MAN ranged from 1 to 8 &#119 m with a peak between 2 and 5 &#119 m. In the PAN, their diameters ranged from 1 to 12 &#119 m with a clearly visible peak at 4-5 &#119 m and a plateau at 8-9 &#119 m that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.     

Pathological changes of human unmyelinated nerve fibers: a review

In the cutaneous nerves, unmyelinated nerve fibers outnumber the myelinated ones but are scarcely analyzed, especially at autopsy. This indifference toward the pathology of unmyelinated nerve fibers may be due to the necessity of electron microscopic analyses and, more importantly, the obscurity of pathological alteration of unmyelinated nerve fibers in aging as well as in peripheral nerve disorders. The aim of this article is to review (1) the normal appearance including postmortem changes, (2) the age-related changes, and (3) the pathological alteration in various neuropathic and non-neuropathic conditions, of unmyelinated nerve fibers in the sural nerve. For the complete analyses of sural nerve, qualitative and quantitative estimation of unmyelinated nerve fibers in all specimens should be recommended and it sometimes has an important diagnostic value.     

Limb muscle regeneration in peripheral nerve autografts

In a previous study we demonstrated regenerative growth of extraocular muscle within transplanted peripheral nerve autografts. The present study addresses the feasibility of inducing regeneration of limb muscle within autologous peripheral nerve implants in the gluteus medius of beagles. In six anesthetized animals, a 2-cm segment of the left infraorbital sensory nerve was removed from the nose and implanted between the cut ends of several muscle fascicles in the left gluteus medius. After 4 weeks, the nerve grafts were removed and examined by light and electron microscopy. Muscle fibers were seen surrounded by the epineurium of the implanted nerve along its entire length, growing in parallel with the long axis of the nerve. The regenerating fibers were closely associated with the basal lamina of degenerating myelinated and unmyelinated axons. This study suggests that limb muscle, like extraocular muscle, is capable of organized regenerative growth within peripheral nerve autografts.     

扬子鳄视神经的研究

本文研究了扬子鳄的视神经。结果明明,视神经中可见有髓纤维和无髓纤维。有髓纤维分布均匀,无髓纤维常聚集成团;胶质细胞核,在视神经中可看到两种类型,有髓纤维总数为２００,０００－３００,０００根,纤维直径范围为０．４１－６．６６μｍ,只有一个峰值,峰直径为１．３１μｍ;纤维轴突径与纤维直径之比（ｄ／Ｄ）约为０．７３－０．７５。经统计分析,同个体左右侧神经纤维数目有差异,同一神经中周围区与中央区数目分布