Unmyelinated Axon Profiles Research Articles

Characterization of the optic nerve and retinal ganglion cell layer in the dysmyelinated adult Long Evans Shaker rat: evidence for axonal sprouting.

Myelin in the central nervous system (CNS) is hypothesized to help guide the growth of developing axons by inhibiting sprouting of aberrant neurites. Previous studies using animal models lacking CNS myelin have reported that increasing capacity for sprouting axons is negatively correlated with the degree of myelination. In the present study, we investigated the optic nerves of the recently identified Long Evans Shaker (LES) rat with prolonged dysmyelination of adult axons to determine whether the lack of myelin basic protein (MBP) in adult LES rats could manifest as increases in the population of CNS axons. We observed numerous small, unmyelinated axon profiles (<0.3 microm in diameter) clustered in bundles alongside normal caliber axons in dysmyelinated LES rats but not in normal myelinated Long Evans (LE) rats. These putative axon profiles resembled sprouting axons previously described in the CNS. Moreover, the high number of small putative axon profiles could not be accounted for by any significant increases in the number of ganglion cells and displaced amacrine cells in the ganglion cell layer when compared with normal rats as evaluated by using a variety of techniques. This finding suggests that the observed clusters of putative axon profiles were not due to developmental abnormalities in the retina but to the lack of myelin in the optic nerves of LES rats. The adult LES rat, therefore, may serve as a useful model to study the role of myelin in regulating axon development or axon regeneration after CNS injury in the adult mammalian system.

Septal cholinergic deafferentation of the dentate gyrus results in a loss of a subset of neuropeptide Y somata and an increase in synaptic area on remaining neuropeptide Y dendrites

Removal of cholinergic septal inputs using the immunotoxin 192 IgG-saporin reduces the number of interneurons containing neuropeptide Y (NPY)-immunoreactivity in the rat dentate gyrus by approximately 30% [Milner et al., J. Comp. Neurol. 386 (1997) 48–59]. The goal of the present study was to determine if NPY-containing neurons that survive deafferentation have any distinguishing morphological and/or microenvironmental features. For this, 2 or 24 weeks after intracerebroventricular injections of 192 IgG-saporin, NPY-immunolabeled neurons in the hilus of the dentate gyrus were examined by electron microscopy. Neither the size nor morphological traits of NPY-labeled perikaryal or dendritic profiles from lesioned compared to control rats at either time-point differed significantly. However, at both time-points, NPY-containing somatal profiles from immunolesioned rats compared to controls had a reduced percentage of their plasmalemmal surface apposed to unmyelinated axon profiles and an increased percentage of their surface occupied by astrocytic profiles. At the 24 week time-point, these differences were statistically significant. The primary contributing factor for these changes was the absence of a subgroup of NPY-labeled somatal profiles in lesioned rats compared to controls which was: (a) distinguished by frequent appositions of unmyelinated axons (from 15 to 35%) to the plasmalemmal surface; and (b) located primarily in the central hilar region. Unlike NPY-containing somata, changes associated with NPY-labeled dendritic profiles were exclusively related to associated presynaptic profiles at the 24 week time-point. In lesioned rats compared to controls at this time-point, NPY-containing dendritic profiles had a concurrent increase in the percentage of the plasmalemmal surface occupied by active zones and the size of terminals contacting them. The present results combined with those of our earlier study suggest that septal cholinergic deafferentation results in: (a) the loss of a distinct subpopulation of hippocampal NPY-containing neurons; and (b) an increase in total active zone area suggesting a strengthening of synaptic connections to the surviving population of NPY-containing neurons in the long term.

Ultrastructural analysis of NMDA, AMPA, and kainate receptors on unmyelinated and myelinated axons in the periphery

The present study determines the proportions of unmyelinated cutaneous axons at the dermal–epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal–epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Aδ and Aβ, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive. J. Comp. Neurol. 391: 78–86, 1998. © 1998 Wiley-Liss, Inc.

Ultrastructural analysis of NMDA, AMPA, and kainate receptors on unmyelinated and myelinated axons in the periphery.

The present study determines the proportions of unmyelinated cutaneous axons at the dermal-epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal-epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Adelta and Abeta, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive.

Post-irradiation bladder dysfunction: development of a rat model.

The aim of this research was to establish a small animal model for the functional and morphological study of post-irradiation bladder dysfunction. Young adult female Wistar rats were X-irradiated with single doses of 10, 15, 20 or 25 Gy. Filling cystometry was performed to assess changes in reservoir function: the volume infused to produce a rise in intravesical pressure of 5 cmH2O was calculated as an index of compliance. A biphasic reduction in this index was noted in animals receiving 15-25 Gy; the first reduction developed at about 4 weeks, and the second started at 3-4 months and persisted at 6 months. Bladder tissue was taken at this time (6 months post-irradiation) for morphological study. Histological examination demonstrated an increased mast cell density in the irradiated bladders, but was otherwise non-specific; fibrosis was discernible in only half of the 18 animals studied. Electron microscopy showed focal degeneration of smooth muscle cells, and in some areas there was selective degeneration of unmyelinated axon profiles. The biphasic reduction in the compliance index is consistent with the timing of the symptoms of the acute and late irradiation reactions reported by radiotherapy patients. Changes in axon profiles and mast cell density may be of functional significance.

Postlarval growth of the peripheral gustatory system in the channel catfish, Ictalurus punctatus

The phenomenon of postlarval cell addition to the peripheral nervous system of fish has been reported for some sensory systems, but has yet to be characterized for the gustatory system. Many fishes, such as catfish, possess taste buds scattered across their body surface, and presumably, the number of taste buds increases during growth of the animal. The present study was undertaken in order to examine the process of growth in the peripheral gustatory system and to determine whether the degree of convergence of receptors onto primary sensory afferents changes during growth. The recurrent facial nerve of channel catfish was used for these studies since this nerve contains no general cutaneous components and innervates taste buds along the fish's body surface. Electron micrographs were made of cross sections of this nerve taken from individuals ranging in size from 5.1 to 39.5 cm standard length. In addition, estimates were made of the number of taste buds innervated by this nerve by determining taste bud density along selected regions of the flank and fins in large and small fish. As catfish get larger, the number of both myelinated and unmyelinated axon profiles in the recurrent facial nerve increases, but at a slower rate than the number of taste buds innervated by this nerve. Thus, on average, the number of taste buds innervated by each fiber increases as the fish enlarges; on average there are 2 taste buds per axons profile in small fish and nearly 14 taste buds per axon profile in large fish. The rate of addition of new axon profiles to the nerve is estimated at roughly 70 per day over the range of sizes studied. Although generation of new ganglion cells and axons may contribute to this increase, several lines of evidence indicate that axonal branching occurs. In addition, the mean axon diameter for both myelinated and unmyelinated axons increases during postlarval growth. The finest myelinated fibers (0.2 micron) in small animals were significantly smaller than the finest myelinated fibers (0.7 micron) in larger animals.

Fibre composition of the ventral roots L7 and S1 in the owl monkey (Aotus trivirgatus)

The ventral roots L7 and S1 of the owl monkey Aotus trivirgatus, were examined by electron microscopy. On average, these roots contain 2950 and 1837 myelinated axons respectively. In both roots the myelinated axons have bimodal size distributions, but the S1 root contains more small myelinated axons. Both roots contain a substantial proportion of unmyelinated axon profiles (UAP). In the L7 root the proportion of UAP decreases as the spinal cord is approached, from 19% distally to 5% in the juxtamedullary rootlets. Unmyelinated and very small myelinated CNS-type axons have not been observed in the L7 transitional region. The average S1 root contains some 40% unmyelinated axons at all examined proximo-distal levels. Unmyelinated/very small myelinated axons are easily found on the CNS side of the S1 transitional region, in direct relation to motoraxon bundles. Bundles of unmyelinated and small myelinated axons occur in the ventral pia mater of both segments. The unmyelinated axons in the L7 root of the owl monkey appear to be arranged like those in the feline L7 ventral root, possibly representing afferents. It is likely that most unmyelinated and small myelinated axons in the ventral root S1 are autonomic efferents.

Changes in myelinated and unmyelinated axon numbers in the proximal parts of rat sural nerves after two types of injury

Counts of myelinated and unmyelinated axon profiles have been made from normal, uninjured rat sural nerves and from nerves injured 6 months earlier in one of two ways. In one group of rats the nerve was simply cut and left to regenerate, leading to the development of a neuroma in continuity, while in the second group the nerve was cut but then ligated as well to prevent regeneration; this led to stump neuroma formation. After nerve transection and regeneration, with subsequent formation of a neuroma in continuity, there was no change in the number of myelinated axon profiles found 25 mm proximal to the old injury site when compared with control, but there was an 18% reduction (P < 0.05) in the number of unmyelinated axon profiles. Immediately proximal to the injury site the picture was similar, with there still being the same number of myelinated axon profiles as in control material but here the reduction in unmyelinated axon numbers was slightly greater at 24% (P < 0.05). In the proximal part of nerves that had been cut and stump neuroma formation induced there was a large increase (33%) in myelinated axon profiles over and above control values (P < 0.001) but the number of unmyelinated profiles was the same as in controls. Closer to the stump neuroma the number of myelinated axon profiles had increased yet further to be 88% (P < 0.001) above control while the number of unmyelinated ones remained no different from control. Our interpretation of these results is that after nerve transection and regeneration there is no loss of peripheral neurons supporting myelinated axons but some loss of those supporting unmyelinated ones. If a cut nerve is prevented from regenerating and a stump neuroma forms, however, a vigorous sprouting response is triggered in neurons with myelinated axons while those supporting unmyelinated axons are possibly prevented from dying. The reaction of peripheral neurons to injury is such that the number of axons they support varies along the nerve as one goes disto-proximally away from the injury site. Thus discrepancies in results from different laboratories have come about because material for axon counting has been taken from different points along the nerve relative to the injury site and also because the material has been taken from nerves injured in different ways.

Unmyelinated axons in the feline trigeminal motor root

The trigeminal motor root was studied in the electron microscope at different proximodistal levels in eight adult cats. Counts at a level halfway between the trigeminal ganglion and the pontine junction showed that the root contains about 9% (n = approximately 300) unmyelinated axon profiles at this level. Small groups of unmyelinated axons occur on both sides of the PNS-CNS border, in the surrounding pia mater, and in perivascular spaces of the CNS compartment. Examination of serial sections from the PNS-CNS transitional region showed that some unmyelinated axons actually cross the PNS-CNS border. The functional significance of these fibres remains unknown.

Presence of unmyelinated axons in the spinal root of the feline accessory nerve

The spinal root of the accessory nerve was studied electron microscopically at different levels in 3 adult cats. It was found that this nerve contains several unmyelinated axons. In the main nerve trunk the proportion of unmyelinated axon profiles was 27% at the level of the foramen magnum. In juxtamedullary root fascicles near the PNS/CNS transition the proportion of unmyelinated axons was lower and these axons tended to occupy superficial positions in the fascicles. No intrafascicular unmyelinated axons were found in the immediate vicinity of the PNS/CNS transition but bundles of unmyelinated axons occurred in the surrounding pia mater. The findings suggest that unmyelinated axons in the spinal accessory nerve contribute to the pial vasomotor and/or sensory innervation.

Electron microscopic and immunohistochemical evidence that unmyelinated ventral root axons make u-turns or enter the spinal pia mater.

The occurrence of unmyelinated and small myelinated axons and of nerve fibres with a substance P-like immunoreactivity was studied in juxtamedullary L7 ventral root fascicles and surrounding pia mater of kittens and cats. Electron microscopic analysis of thin transverse serial sections from this region in adult cats showed that the number of unmyelinated axon profiles decreases rapidly as the CNS is approached, reaching zero near or at the CNS/PNS border. No unmyelinated axons were found to enter the CNS through this root. At least to some extent, the disappearance of unmyelinated ventral root axons from the juxtamedullary root fascicles was due to presence of intrafascicular axonal hairpin loops and to a shift of axons from ventral root fascicles to the pia mater. It was also found that, in the L7 ventral root, the content of unmyelinated axons in the pia mater is lower in kittens than in cats. Fluorescence microscopic examination of specimens incubated with substance P antiserum showed that some looping axons and ventral root-pia mater axons were substance P immunoreactive. These observations suggest the hypothesis that sensory unmyelinated axons might grow through the L7 ventral root and enter the pia mater during postnatal development. Moreover they show that the occurrence of sensory unmyelinated axons in ventral roots does not necessarily contradict the law of Magendie .

Invasion of the L7 ventral root and spinal pia mater by new axons after sciatic nerve division in kittens

In young kittens the left sciatic nerve was divided followed by nerve suture, distal stump resection, or resection combined with proximal stump capping. The kittens were killed 2 to 3 months postoperatively. In other kittens bilateral L7 ventral rhizotomy or neuroma excision was carried out 80 to 90 days after nerve resection, followed by killing a few days later. Thin sections from the ventral root L7 were examined in the electron microscope. In all animals the average proportion of ventral root unmyelinated axon profiles (UAP) was above normal on the operated side, being highest in capped and resected animals and lower in sutured cases. The proportion of UAP was inversely related to the extent of functional calf muscle reinnervation. In cases with high proportions of UAP, numerous bundles of unmyelinated and small myelinated axons were present in juxtamedullary root fascicles and surrounding pia mater. After L7 ventral rhizotomy in resected animals few intact UAP occurred together with numerous signs of axon degeneration in the proximal root stump on the lesion side. After neuroma excision the proportion of ventral root UAP decreased. Therefore some proportion of the new axons should represent recurrent sprouts from peripheral sources.

Preferential loss of unmyelinated L7 dorsal root axons following sciatic nerve resection in kittens

Some time following sciatic nerve resection in kittens the number of L7 spinal ganglion neurons appears to have decreased more than the number of L7 dorsal root axons, and the proportion of unmyelinated axons is unchanged. The present results show that removal of the sciatic neuroma results in a clearly subnormal proportion of unmyelinated axon profiles. This indicates that recurrent sprouts from the neuroma had entered the dorsal root and that spinal ganglion neurons with unmyelinated axons are preferentially lost after axotomy in kittens.

Occurrence of unmyelinated axon profiles at distal, middle and proximal levels in the ventral root L7 of cats and kittens

The proportion of unmyelinated axon profiles (UAP) was estimated electron-microscopically at different proximo-distal levels in the ventral root L7 of 7 normal adult cats and 2 kittens. In 3 of the adult cats the occurrence of UAP was also determined in the juxtamedullary root fascicles, including the CNS/PNS transitional region. Three additional adult (male) cats, which had been subjected to prepubertal castration, were examined at mid-root level. Prepubertal castration was found not to interfere with the increase in L7 ventral root UAP that takes place during puberty. In the normal animals the proportion of UAP was highest distally in the ventral root and decreased as the spinal cord was approached. The same trend was seen in cats and kittens, but the values were obviously higher in adults. The content of UAP decreased markedly in the immediate vicinity of the CNS/PNS border. Very few unmyelinated axons and no myelinated CNS-type axons with sizes below the gamma range were found on the CNS side of this border. In addition groups of PNS-type unmyelinated and small myelinated axons occurred in the pia mater outside some juxtamedullary root fascicles.

Abnormally high content of unmyelinated axon profiles in the ventral root L7 of a cat with an abdominal neoplasm.

The ventral roots of L7 of a cat with an intestinal neoplasm were examined electron-microscopically at different proximo-distal levels. The proportion of unmyelinated axon profiles was 50-55% at mid-root and distal levels as compared to 30% in normal adult cats. In the juxtamedullary root fascicles the proportion was normal (average 20%), and few unmyelinated axons seemed to penetrate into the CNS through the ventral root. The number of myelinated axons was within the normal range.

SYMPATHETIC INNERVATION OF THE SWEAT GLANDS AND PILORRECTOR MUSCLES OF MACAQUES AND HUMAN BEINGS

A loose network of catecholamine-containing nerve terminals (adrenergic nerves) has been demonstrated around some of the coils of human and macaque ecerine sweat glands with the histofluorescence method [1–4]. After administration of the monamine oxidase inhibitor nialamide plus norepinephrine, fluorescent fibers appeared in nearly all the coils of the macaque glands. Systemic injection of reserpine or local injection of either 6-hydroxydopamine (6-OHDA) or 5-hydroxydopamine (5-OHDA) caused complete disappearance of the fluorescent fibers around all glandular coils. Under varying physiologic conditions, there is considerable fluctuation in the concentration of norepinephrine in the adrenergic terminals of sweat glands. Fluorescent fibers appeared sporadically around the apocrine glands of human axillae. With the aid of the electron microscope many unmyelinated varicose axon profiles were seen in the vicinity of both ecerine and apocrine glands of human beings and macaques. Most of these profiles contained many small agranular and a few large dense-cored vesicles (cholinergic terminals); other profiles had small and large dense-cored vesicles (adrenergic terminals). The sweat glands of human beings and macaques, then, are innervated with large numbers of cholinergic terminals and a few adrenergic terminals. After macaque ulnar nerves were constricted, fluorescent catecholamines were demonstrated in the proximal sides of the axons. Subsequent denervation of the ulnar nerves induced degeneration of the periglandular terminals and disappearance of the fluorescent fibers in the fifth digits. Dense plexuses of fluorescent fibers were found in the piloarrector muscles of macaques. When viewed with an electron microscope, a majority of the unmyelinated terminal axons appeared to be adrenergic: a few appeared to be cholinergic.