Selective decrease of small sensory neurons in lumbar dorsal root ganglia labeled with horseradish peroxidase after ND:YAG laser irradiation of the tibial nerve in the rat

Recent electrophysiological evidence indicates that Q-switched Nd:YAG laser irradiation might have selective effects on neural impulse transmission in small slow conducting sensory nerve fibers as compared to large diameter afferents. In an attempt to clarify the ultimate fate of sensory neurons after laser application to their peripheral axons, we have used horseradish peroxidase (HRP) as a cell marker to retrogradely label sensory neurons innervating the distal hindlimb in the rat. Pulsed Nd:YAG laser light was applied to the tibial nerve at pulse energies of 70 or 80 mJ/pulse for 5 min in experimental rats. Seven days later HRP was applied to the left (laser-treated) and to the contralateral (untreated) tibial nerve proximal to the site of laser irradiation. In control animals the numbers of HRP-labeled dorsal root ganglion cells were not significantly different between the right and the left side. In contrast, after previous laser irradiation labeling was always less on the laser-treated side (2183 +/- 513 cells, mean +/- SEM) as compared to the untreated side (3937 +/- 225). Analysis of the dimensions of labeled cells suggested that the reduction of labeled cells on the laser-treated side was mainly due to a deficit in small sensory neurons. Since the conduction velocity of nerve fibers is related to the size of their somata, our histological data imply that laser light selectively affects retrograde transport mechanisms for HRP in slow conducting sensory nerve fibers.     

Laser Effects on Myelinated and Nonmyelinated Fibers in the Rat Peroneal Nerve: A Quantitative Ultrastructural Analysis

We have recently shown that Nd:YAG laser irradiation of rat peripheral nerve differentially impairs action potential transmission in small, slowly conducting sensory fibers compared to fast conducting afferents. In addition, the number of small sensory neurons of the A-δ- and C-fiber group labeled with HRP is significantly reduced after laser irradiation, while the number of labeled large sensory neurons and motoneurons was not affected. To further evaluate this laser-induced injury, we examined three distinct regions of the laser-irradiated rat peroneal nerve using ultrastructural morphometric methods. These regions were the site of laser irradiation and zones 10 mm proximal and 5 mm distal to the injury. The contralateral nerve was sham treated. Our results indicate that for the small nonmyelinated fibers, there was a significant increase in both mean fiber size and the number of microtubules per fiber, but a decrease in the number of neurofilaments. In contrast, the number of myelinated and nonmyelinated fibers is not significantly altered at 7 days following laser irradiation, and the mean diameter and frequency distribution of myelinated nerve fibers was unchanged. This study demonstrates that selective functional alterations in laser-irradiated nerves (nerve conduction velocity, HRP transport properties) are accompanied by ultrastructural changes of axonal organelles in nonmyelinated fibers. Nd:YAG laser light might ultimately prove to be a powerful tool to selectively alter functional properties in small, slowly conducting afferent fibers, without causing degeneration at the ultrastructural level at the site of irradiation. We hypothesize further that the laser-induced functional alterations might be related to differential thermally mediated changes.     

Brain stem projections of sensory and motor components of the vagus complex in the cat: I. The cervical vagus and nodose ganglion

The motor and sensory connections of the cervical vagus nerve and of its inferior ganglion (nodose ganglion) have been traced in the medulla oblongata of 32 adult cats with the neuroanatomical methods of horseradish peroxidase (HRP) histochemistry and amino acid autoradiography (ARG). In 14 of these subjects, an aqueous solution of HRP was applied unilaterally to the central end of the severed cervical vagus nerve. In 13 other cases, HRP was injected directly into the nodose ganglion. Three of these 13 subjects had undergone infranodose vagotomy 6 weeks prior to the HRP injection. A mixture of tritiated amino acid was injected into the nodose ganglion in five additional cats. The retrograde transport of HRP yielded reaction product in nerve fibers and perikarya of parasympathetic and somatic motoneurons in the medulla oblongata. Furthermore, a tetramethyl benzidine (TMB) method for visualizing HRP enabled the demonstration of anterograde and transganglionic transport, so that central sensory connections of the nodose ganglion and of the vagus nerve could also be traced. The central distribution of silver grain following injections of tritiated amino acids in the nodose ganglion corresponded closely with the distribution of sensory projections demonstrated with HRP, thus confirming the validity of HRP histochemistry as a method for tracing these projections. The histochemical and autoradiographic experiments showed that the vagus nerve enters the medulla from its lateral aspect in multiple fascicles and that it contains three major components—axons of preganglionic parasympathetic neurones, axons of skeletal motoneurons, and central processes of the sensory neurons in the nodose ganglion. Retrogradely labeled neurons were seen in the dorsal motor nucleus of X(dmnX), the nucleus ambiguus (nA), the nucleus retroambigualis (nRA), the nucleus dorsomedialis (ndm) and the spinal nucleus of the accessory nerve (nspA). The axons arising from motoneurons in the nA did not traverse the medulla directly laterally; rather, all of these axons were initially directed dorsomedially toward the dmnX, where they formed a hairpin loop and then accompanied the axons of dmnX neurons to their points of exit. Afferent fibers in the vagus nerve reached most of the subnuclei of the nTS bilaterally, with the more intense labeling being found on the ipsilateral side. Labeling of sensory vagal projections was also found in the area postrema of both sides and around neurons of the dmnX. These direct sensory projections terminating within the dmnX may provide an anatomical substrate for vagally mediated monosynpatic reflexes. Following deefferentiation by infranodose vagotomy 6 weeks prior to HRP injections into the nodose ganglion, a number of neurons in the dmnX were still intensely labeled with the HRP reaction product. The axons of these HRP-labeled perikarya may constitute the bulbar component of the accessory nerve.     

Uptake, intra-axonal transport and fate of horseradish peroxidase in embryonic spinal neurons of the chick

Horseradish peroxidase (HRP) was injected in ovo into the ventral muscle mass of the hind limb of 5- to 7-day-old chick embryos or into the gastrocnemius muscle of 8- to 18-day embryos and localized histochemically. HRP is extensively incorporated via endocytosis into axonal growth cones or presynaptic terminals in the proximity of the injection site. Much of the tracer is taken up in vesicles and small vacuoles. Most of these are smooth-surfaced and only a few are bristle-coated. A small amount of the tracer is also incorporated into the axon terminal through the openings between the axolemma and an intricate membrane channel. The majority of the tracer-laden vesicles and vacuoles rapidly fuse with one another to become large vacuoles, some of which are transformed into multivesicular bodies (MVBs). In axon shafts, many labeled vacuoles and MVBs are transferred to tubule-like organelles, which appear to be the primary carrier for transporting the tracer back to the cell bodies in the lumbar spinal cord. HRP arrives in the sensory ganglia about 0.5-1 hour earlier than in the motoneurons of the lateral motor column. The maximal rate of the retrograde axoplasmic transport is about 3.5 mm/hour. After arriving in the cell bodies, HRP is transferred from tubule-like organelles to discrete vacuoles of various sizes and appearance. Lysosomal dense bodies and HRP-labeled vacuoles can be distinguished ultrastructurally. A fusion of HRP-labeled vacuoles with lysosomal dense bodies or Golgi vesicles was occasionally observed and the density of HRP-labeled vacuoles diminished after 2 to 3 days. Most of the HRP-labeled organelles were found to contain acid phosphatase activity. Therefore, the complete disappearance of HRP by 4 days postinjection is most likely related to lysosomal degradation. Neuronal cell bodies diffusely labeled with HRP were only observed prior to day 6. After day 6, despite various attempts to injure the peripheral axons, only granularly labeled cell bodies were found. This difference may imply that "mature" neurons have a more efficient mechanism for the sequestration of "free" HRP in the cytoplasmic matrix into membrane-bounded organelles. A mature-like retrograde transport mechanism appears to exist at the earliest stages of axonal growth in vivo.     

Decreased horseradish peroxidase labeling in deafferented spinal motoneurons of the rat

It has been suggested^29,50 that the incorporation and retrograde transport of horseradish peroxidase (HRP) were linked to the level of neuronal activity. Therefore one could postulate that the motor impairment resulting from dorsal rhizotomy affects the HRP labeling of spinal motoneurons in the absence of morphological damage to the motor system. This hypothesis was tested in the adult rat by sectioning bilaterally the L₃-L₅ dorsal roots. 2–18 months after surgery, the L₄ radicular nerve was immersed in a solution of HRP. Labeled motoneurons were counted together with the motor axons of the L₄ ventral root and results were compared with values obtained in paired controls. Deafferentation resulted in a crippling deficit of lower movements with disuse atrophy of muscle fibers but had no effect on the fiber population of the sciatic nerve and the L₄ ventral root. Whereas in normal animals the L₄ HRP-labeled motoneurons represented 71.9–98.3% (average 85.4) of the motor axonal counts, in animals studied 4, 12 and 18 months after dorsal rhizotomy, the number of motoneurons containing HRP granules constituted only 20.1–55.7% (average 46.2) of the number of motor axons and many of the labeled cells were faintly stained. These findings, which may reflect either a decreased retrograde transport of HRP in deafferented motoneurons or an increased turnover of the enzyme in the cell body, call attention to the possibility that the degree of activity in neuronal pathways influences HRP labeling.     

Are cat lingual muscles dually innervated by the hypoglossal nerve and facial nerve?

The origins of the somatic motor nerve innervating the cat lingual muscles were studied using the horseradish peroxidase (HRP) method. Following HRP injection into the lingual muscles, labeled neurons were found not only in the hypoglossal nucleus but also in the facial nucleus, ipsilateral to the injection side. HRP-labeled neurons in the facial nucleus were principally observed in the ventromedial and ventrolateral divisions of the nucleus. This study suggests that cat lingual muscles are innervated by both hypoglossal motoneurons and some of the motoneurons in the ventromedial and ventrolateral divisions of the facial nucleus.     

Anatomical regeneration and behavioral recovery following crush injury of the trigeminal root in lamprey

In normal larval lamprey, bilateral application of horseradish peroxidase (HRP) to the dorsal part of the anterior oral hood labeled subpopulations of trigeminal components on both sides of the brain; peripherally projecting motoneurons, medullary dorsal cells (sensory), and spinal dorsal cells (sensory), as well as centrally projecting afferents in the trigeminal descending tracts. Following unilateral crush injury of the right trigeminal root, HRP labeling of sensory and motor trigeminal components on the right side gradually increased with increasing recovery time, between 2 weeks and 12 weeks postcrush (PC). Axons of trigeminal motoneurons appeared to exhibit robust regeneration, whereas restoration of projections in the descending trigeminal tract ipsilateral to the injury was incomplete. Control experiments indicated that motor and sensory axons from the intact side of the oral hood did not sprout across the midline to the denervated side. Several results suggested that regenerated trigeminal sensory fibers made synapses with brain neurons that have direct or indirect inputs to reticulospinal (RS) neurons. Following a unilateral crush injury of the right trigeminal root, escape behavior in response to stimulation of the right side of the oral hood gradually returned to normal. Muscle recordings at various recovery times confirmed that anatomical regeneration of trigeminal sensory axons was functional. In addition, at 8 or 12 weeks PC, brief stimulation of the oral hood ipsilateral or contralateral to the crush injury elicited synaptic responses in RS neurons on either side of the brain, similar to that in normal animals. In the lamprey, compensatory mechanisms probably allow recovery of behavioral function despite incomplete regeneration of trigeminal sensory axons within the central nervous system. J. Comp. Neurol. 396:322–337, 1998. © 1998 Wiley-Liss, Inc.     

Central distribution of afferent and efferent components of the chorda tympani in the cat as revealed by the horseradish peroxidase method

Central distribution of afferent and efferent components of the chorda tympani (CT) in the cat was examined by using the anterograde and retrograde tracing techniques of horseradish peroxidase (HRP). HRP was applied to the CT in the tympanic cavity. HRP-labeled CT fibers were traced to the brain stem along the ventral surface of the vestibular nerve. The afferent CT fibers were divided into ascending and descending components. The rostrally directed ascending fibers ended within and around the dorsomedial portions of the principal sensory trigeminal nucleus. The descending fibers entered the solitary tract to run caudally as far as the levels slightly rostral to the obex, giving terminals to the solitary nucleus. A cluster of HRP-labeled neurons were seen ipsilaterally in the lateral reticular formation medial to the spinal trigeminal nucleus; it was observed from the caudalmost levels of the exiting root of the facial nerve to the caudal levels of the facial nucleus. HRP-labeled axons arising from the HRP-labeled neurons firstly ran dorsomedially and then medially under the genu of the facial nerve to form a small genu at the region medial to the genu of the facial nerve. Subsequently the labeled axons ran laterally and ventrolaterally to join other CT fibers at the dorsomedial aspect of the spinal trigeminal tract.     

Central distribution of primary afferent fibers in the Arnold's nerve (the auricular branch of the vagus nerve): A transganglionic HRP study in the cat

Central projections of the Arnold's nerve (the auricular branch of the vagus nerve; ABV) of the cat were examined by the transganglionic HRP method. After applying HRP to the central cut end of the ABV, HRP-labeled neuronal somata were seen in the superior ganglion of the vagus nerve. Main terminal labeling was seen ipsilaterally in the solitary nucleus, in the lateral portions of the ventral division of the principal sensory trigeminal nucleus, in the marginal regions of the interpolar subnucleus of the spinal trigeminal nucleus, in the marginal and magnocellular zones of the caudal subnucleus of the spinal trigeminal nucleus, in the ventrolateral portions of the cuneate nucleus, and in the dorsal horn of the C1–C3 cord segments. In the solitary nucleus, labeled terminals were seen in the interstitial, dorsal, dorsolateral and commissural subnuclei; some of these terminals may be connected monosynaptically with solitary nucleus neurons which send their axons to the somatomotor and/or visceromotor centers in the brainstem and spinal cord.     

Selective reinnervation of somatotopically appropriate muscles after facial nerve transection and regeneration in the neonatal rat

The organization of the facial motor nucleus (FMN) has been examined after transection and regeneration of the facial nerve (FN) in neonatal and adult rats. In one series of experiments, horseradish peroxidase (HRP) was applied bilaterally to the superior or inferior buccal ramus 5 months after neonatal FN transection. In another series of experiments, wheat germ agglutinin-horseradish peroxidase conjugate was injected in selected vibrissae follicular muscles on both sides in animals surviving 5 months after FN transection at the neonatal or adult stage. The number and distribution of HRP-labeled cell bodies in the FMN after regeneration was compared with the contralateral side. On the uninjured side, labeled neurons were somatotopically organized. Ipsilateral to nerve injury the number of labeled cells was markedly reduced after neonatal nerve transection, but somatotopy was preserved. However, after nerve lesion at the adult stage, no significant loss of motoneurons occurred, but motor nucleus somatotopy was not maintained. Two alternative principal explanations are proposed for the re-establishment of the normal somatotopy after neonatal injury: that regenerating axons grow in a random fashion but inappropriate connections are subsequently eliminated or that regenerating axons of surviving neurons immediately follow a pathway leading to the appropriate muscle.     

Brainstem projections of sensory and motor components of the vagus nerve in the rat

The sensory and motor connections of the cervical vagus nerves and of its inferior ganglion (nodose ganglion) have been traced in the medulla and upper cervical spinal cord of 16 male Wistar rats by using horseradish peroxidase (HRP) neurohistochemistry. The use of tetramethyl benzidine (TMB) as the substrate for HRP permitted the visualization of transganglionic and retrograde transport in sensory nerve terminals and perikarya, respectively. The vagus nerve in the rat enters the medulla in numerous fascicles with points of entry covering the entire lateral aspect of the medulla extending from level +4 to - 6 mm rostrocaudal to the obex. Fascicles of vagal sensory fibers enter the dorsolateral aspect of the medulla and travel to the tractus solitarius (TS) which was labeled for over 8.8 mm in the medulla. The caudal extent of the TS receiving vagal projections was found in lamina V of the cervical spinal cord (C1 to C2). Sensory terminal fields could be visualized bilaterally in the nucleus of the tractus solitarius (nTS), area postrema (ap) and dorsal motor nucleus of the vagus nerve (dmnX). The ipsilateral projection to the nTS and the dmnX was heavier than that found on the contralateral side. The area postrema was intensely labeled on both sides. Motor fibers from HRP-labeled perikarya in the dmnX travel ventromedially in a distinct fascicle and subsequently subdivide into a number of small fiber bundles that traverse the medullary reticular formation in the form of a fine network of HRP-labeled fibers. As these fibers from the dmnX approach the ventrolateral aspect of the medulla they are joined by axons from the nucleus ambiguus (nA), nucleus retroambigualis (nRA) and the retro facial nucleus (nRF). These latter fibers form hairpin loops in the middle of the reticular formation to accompany the axons from the dmnX exiting from the medulla in a ventrolateral location. HRP-labeled perikarya, in contrast to transganglionically transported HRP in sensory terminals in the nTS, were visualized on one side only, thus indicating that motor control via the vagus nerve is exerted only by motor neurons located ipsilaterally. Sensory information on the other hand, diverges to many nuclear subgroups located on both sides of the medulla.     

An autoradiographic and HRP study of vestibulocollic pathways in the pigeon

This study examines the connections underlying the vestibulocollic system in the adult pigeon by using retrogradely transported horseradish peroxidase (HRP) to identify neck muscle motoneurons in one set of animals, and transneural anterograde transport of tritiated proline-fucose to delineate the descending medial (MVST) and lateral (LVST) vestibulospinal tracts in a second set of animals. Correlations of location and distribution of HRP-labeled motoneurons and autoradiographically labeled fiber tracts and terminal fields were performed between the two sets of experiments. The right biventer cervicis and complexus neck muscles were subdivided into rostral and caudal halves in ten animals and HRP injected into only half of one of the two muscles in each experiment. Following a 16–48-hour survival, the brain was fixed by intracarotid catheterization and perfusion and the HRP in the brain sections reacted with the tetramethylbenzidine (TMB) blue reaction process. Three groups of HRP-labeled motoneurons were identified in the ipilateral ventral horn of the upper cervical spinal cord: a ventromedial and ventrolateral group within lamina VIII innervating the biventer cervicis and the more rostral part of the complexus muscle, and a dorsolateral group of motoneurons within lamina VII innervating the caudal part of the complexus muscle. The dorsolateral motoneurons with their HRP-labeled axons leaving the cord through the dorsal root are homologous to the spinal accessory nucleus of mammals. Labeled motoneurons were also noted in the ipsilateral medulla adjacent to the medial longitudinal fasiculus (ELM) in a location previously identified as the hy-poglossal nucleus. Additional experiments were performed in which HRP was injected directly into the base of the tongue. The resultant HRP-labeled hypoglossal motoneurons were separate and dorsolateral to the collic motoneurons. Descending vestibulospinal projections from one vestibular labyrinth were identified autoradiographicalry (ARG) by transneural anterograde transport of ³H-proline-fucose injected into the left labyrinthine endolytine endolymph in five animals. Heavily labeled MVST fibers were observed crossing the midline of the brain to enter and descend in the contralateral ELM. Labeled MVST fibers were noted to leave the contralateral FLM and surround the previously identified collie motoneurons in the medulla with intense terminal fields suggestive of synaptic contact. Labeled MVST fibers in the contralateral ventral funiculus of the cord were also noted to innervate the HRP-identified ventromedial and ventrolateral cervical motoneurons, but not the dorsolateral motoneurons in lamina VII. Ipsilateral (left) descending MVST and LVST fibers were less heavily labeled at all levels in the medulla and upper cervical cord. Labeled ipsilateral (left) vestibulospinal fibers were also observed to leave the lateralmost aspect of the left ventrolateral funiculus in the upper cervical cord to terminate among left ventrolateral motoneurons. Our findings are compared and contrasted with previous studies of vestibulocollic pathways.     

Bilateral innervation of the superior oblique muscle by the trochlear nucleus

Trochlear motoneurons and their axons were labeled by applying horseradish peroxidase (HRP) solution to the transected trochlear nerve stump in the orbit of cats and rabbits. Although almost all labeled neurons were on the contralateral trochlear nucleus about 5% of them and their axons were on the ipsilateral side. These findings confirmed that the superior oblique muscle was innervated partially by a small number of ipsilateral trochlear nucleus.     

Muscle sensory neurons in the spinal ganglia in the rat determined by the retrograde transport of horseradish peroxidase

The method of retrograde transport of horseradish peroxidase (HRP) was used to identify muscle sensory neurons in the spinal ganglia in the rat. Experiments were conducted on 25 albino rats. Injections of 0.06 to 0.08 ml 2 to 20% Sigma type VIHRP were made unilaterally into anterior tibial muscle. Cells of origin of muscle receptors and motor endings in the same area where HRP was administered were demonstrated. The labeled cells, medium to large, were found in fourth and fifth lumbar ganglia ipsilateral to the site of injection. Simultaneously, labeled neurons were also found in the ipsilateral ventral horn of the same cord segments as the labeled sensory ganglia.     

Topographical arrangement of thalamic neurons projecting to the orbital gyrus in the cat

Distribution of thalamic neurons projecting to the orbital gyrus in the cat was examined by the horseradish peroxidase (HRP) method. After injection of HRP within the cortex of the orbital gyrus, thalamic neurons labeled with HRP were observed ipsilaterally within the pars parvicellularis of the nucleus ventralis posteromedialis (VPMpc) and the caudoventral portions of the nucleus centralis medialis: HRP-labeled neurons in the VPMpc were numerous and those in the nucleus centralis medialis were moderate in number. A few HRP-labeled neurons were seen occasionally in the nucleus paracentralis, nucleus centralis lateralis, nucleus submedius, and nucleus medialis. The VPMpc neurons labeled with HRP injected into the rostral portions of the orbital gyrus were seen chiefly in the dorsal aspects of the VPMpc, whereas the VPMpc neurons labeled with the enzyme injected into the caudal portions of the orbital gyrus were distributed mainly in the ventral aspects of the VPMpc: the region of distribution of the VPMpc neurons projecting onto the rostral portions of the orbital gyrus extended somewhat more rostrally than that of the VPMpc neurons projecting onto the caudal portions of the orbital gyrus. Functional significance of the VPMpc neurons that send their axons to the orbital gyrus is discussed in terms of the relay neurons of the central gustatory pathways.     

Response properties of high-threshold cutaneous cold receptors in the primate

Cutaneous high-threshold cold receptors (HCRs) in the monkey were identified as sensitive only to cold temperatures below 27°C and not responsive to mechanical or heat noxious stimulation. Some HCRs had axons conducting in the low A-δ range while others had C fibers. The response properties of HCRs were contrasted with those of mechanothermal nociceptors, the latter believed to contribute to the sense of cold pain. HCRs with Aδ fibers may contribute to the sense of innocuous cold below temperatures to which low-threshold cold receptors are maximally responsive.     

Cells of origin of propriospinal connections to cat lumbosacral gray as determined with horseradish peroxidase

Propriospinal cells projecting to the lumbosacral spinal cord of cat were identified using the technique of retrograde transport of horseradish peroxidase (HRP). An injection technique was used to preferentially label cells via their terminals rather than through damaged axons of passage. Large volumes (1 to 5 μl) of 35% HRP were slowly ejected from a micropipet penetrating the cord dorsum midway between the dorsal root entry zone and the midline, while the pipet was slowly withdrawn from a depth of 1500 to 2000 μm below the cord surface. This technique resulted in diffusion of HRP throughout the gray matter on the side of the injection and, usually, sperad to the contralateral gray matter. HRP-labeled cells were observed from C1 to S3 after injections in segments L3-S3. Significant differences were seen in the rostrocaudal distributions of labeled cells after injections at different lumbosacral levels: specifically, L6-L7 injections resulted in less labeling of cells at cervical and high thoracic levels than injections that included segments L3-L5 or S1-S3.     

Synaptic connections between trigemino-parabrachial projection neurons and γ-aminobutyric acid- and glycine-immunoreactive terminals in the rat

The synaptic connections between γ-aminobutyric acid (GABA)- and glycine-immunoreactive terminals and neurons projecting to the lateral parabrachial region were examined by a combination of retrograde tracing and immunohistochemical staining in the rat medullary dorsal horn. After injection of horseradish peroxidase (HRP) into the right lateral parabrachial region, HRP retrogradely labeled neurons were observed bilaterally in laminae I, II and III of the medullary dorsal horn with an ipsilateral predominance. GABA- and glycine-like immunoreactive terminals were found in laminae I, II and III. Some of these GABA- and glycine-like immunoreactive terminals were observed chiefly to make symmetric synapses with HRP-labeled neuronal cell bodies and dendritic processes. The present results indicate that neurons in the medullary dorsal horn projecting to the lateral parabrachial region might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons, which might be significantly involved in the regulation of the noxious information transmission.