Nuclear Yellow Research Articles

Demonstration of the potential for chronically injured neurons to regenerate axons into intraspinal peripheral nerve grafts

Experiments were carried out to determine if neurons damaged by injury to the spinal cord retain the ability to regenerate their axonal process for a prolonged period of time after the initial response to injury and if peripheral nerve (PN) grafts could support the regrowth of these processes. True blue (TB) was injected into one side of the adult rat lumbar spinal cord to label neurons with axons coursing through this region. Seven days later spinal cord tissue surrounding the injection sites was removed by aspiration to create a hemisection cavity 3–4 mm in length. Four weeks later scar tissue lining the lesion cavity was removed prior to grafting 1 cm segments of autologous tibial nerve to the rostral and the caudal surfaces of the cavity wall. The distal end of each graft was ligated and left unapposed to spinal cord tissue. Four weeks later the distal end of each PN graft was exposed to nuclear yellow (NY) to retrogradely label neurons that had grown an axon into the graft. Neurons containing both TB and NY were deemed capable of axonal regeneration while in a chronically injured state. Double-labeled ( TB NY ) neurons were found in the ipsilateral spinal cord in laminae IV through X, excluding IX, and in Laminae VI and VII contralateral to the lesion. Most neurons were located within 10 mm of the lesion, with the majority caudal to the lesion. Nearly 50% (range 24–74%) of lumbar dorsal root ganglion neurons containing TB also were labeled with NY. These double-labeled, regenerating ganglion neurons had a mean cell area of 947 μm 2, which was significantly lower than that of the total ganglion cell population. This indicated that primarily small neurons were involved in the regenerative response elicited by the present experimental paradigm. No double-labeled neurons were found in the cervical spinal cord or within regions of the brain known to project to the spinal cord. These results indicate that certain neurons associated with a chronic spinal cord injury have the potential to regenerate their axonal process for at least 4 weeks after sustaining a direct injury.

Retrograde neuronal labeling of motoneurons in the rat by fluorescent tracers, and quantitative analysis of oxidative enzyme activity in labeled neurons

Extensor digitorum longus motoneurons in the rat spinal cord were identified by retrograde labeling with two fluorescent tracers, Fast blue (FB) and Nuclear yellow (NY). Labeled motoneurons had a blue fluorescent cytoplasm at 360 nm excitation wavelength with FB, and a golden-yellow fluorescent nucleus with NY on the cryostat section. Labeled motoneurons were further examined for succinate dehydrogenase activity on the same section used for identification of the motoneurons. This study demonstrates that fluorescent dyes are useful for neuroanatomical studies by the retrograde axonal transport method, and that quantitative analysis of metabolic activity in labeled motoneurons is also possible.

Skin-visceral divergent projection of cholecystokinin — containing dorsal root ganglion neurons: A tri-labelling study with fluorescent tracers and immunohistochemistry

Skin-visceral divergent projections of cholecystokinin (CCK)-containing dorsal root ganglion neurons were studied by combined technique of fluorescent double-labelling and immunohistochemistry. Fast blue (FB) and nuclear yellow (NY) were injected into the coeliac ganglion and the cutaneous branches of left 9th-11th intercostal nerves, respectively. Three kinds of neurons labelled with fluorescein were observed in T9-11 dorsal root ganglia: FB-labelled neurons with blue-fluorescent cytoplasm; NY-labelled neurons with yellow-fluorescent nucleus and double-labelled neurons with blue cytoplasm and yellow nucleus. The double-labelled neurons were found to account for 2.8% of total labelled neurons. The sections containing neurons labelled with fluorescein were stained by CCK-immunohistochemical procedure. Four kinds of neurons could be identified: NY-neurons with CCK-immunoreactivity (NY+CCK); FB-neurons with CCK-immunoreactivity (FB+CCK); NY+FB neurons with CCK-immunoreactivity (NY+FB+CCK); and neurons only CCK-positive. NY+FB+CCK tri-labelled neurons accounted for approximately 11.5% of NY+FB double-labelled neurons, and for 0.4% of all CCK-positive neurons. The findings clearly indicated that the peripheral processes of some sensory dorsal root ganglion neurons divergently project to both skin and visceral structure and contain CCK.

Location of the motoneurons supplying the rabbit pharyngeal constrictor muscles and the peripheral course of their axons: A study using the retrograde HRP or fluorescent labeling technique

The location of the motoneurons supplying the rabbit pharyngeal constrictor muscles (the superior and the middle constrictors, and the thyropharyngeus and the cricopharyngeus; the last two collectively compose the inferior constrictor) was investigated with intramuscular injection of HRP or the fluorescent tracer nuclear yellow into the individual muscles. Moreover, the peripheral course of their axons was investigated by injection of HRP into all of the pharyngeal constrictors in conjunction with intracranial severing of either the vagus or the glossopharyngeal nerves. The pharyngeal constrictor motoneurons were ipsilaterally located within a subdivision of the nucleus ambiguus which is formed by a compact arrangement of the smallest neurons of the nucleus and situated in the rostral half of the nucleus. We named that subdivision the compact cell group (CoG). Axons of the pharyngeal constrictor motoneurons traversed the vagal rootlets. The rostrocaudal extent of the pharyngeal constrictor motoneurons covered almost the entire length of CoG at a level from about 500 to 2,900 microns rostral to the obex, with their number being most numerous in the middle one-third level of the CoG. Although the motoneurons of the superior constrictor, those of the middle constrictor, and those of the thyropharyngeous and the cricopharyngeus overlapped considerably in location, they tended to be arranged rostrocaudally in that order. At the middle one-third level of the CoG, where the CoG is subdivided into dorsomedial and ventrolateral subgroups of neurons, the superior and the middle constrictor motoneurons were confined to the medial portion of the dorsomedial subgroup, while the inferior constrictor motoneurons were distributed throughout its entirety.

Arrangement of neurons in the medullary reticular formation and raphe nuclei projecting to thoracic, lumbar and sacral segments of the spinal cord in the cat

The distribution of neurons in the medullary reticular formation and raphe nuclei projecting to thoracic, lumbar and sacral spinal segments was studied, using the technique of retrograde transport of horseradish peroxidase (HRP), alone or in combination with nuclear yellow (NY). Retrogradely labeled cells were observed in the lateral tegmental field (FTL), paramedian reticular nucleus, magnocellular reticular nucleus (Mc), in the gigantocellular nucleus (Gc), lateral reticular nucleus (LR), lateral paragigantocellular nucleus (PGL), rostral ventrolateral medullary reticular formation (RVR), as well as in the medullary raphe nuclei following the injection of the tracer substance(s) into various levels of the spinal cord. The FTL, the ventral portion of the paramedian reticular nucleus (PRv), Mc, LR, PGL and the raphe nuclei were found to project to thoracic, lumbar and sacral spinal segments. This projection was bilateral; the contralaterally projecting fibers crossed the midline at or near their termination site. The dorsal portion of the paramedian reticular nucleus (PRd), Gc and the RVR projected mainly to thoracic segments. This projection was unilateral. Experiments in which the HRP-injection was combined with lesion of the spinal cord showed that some descending raphe-spinal axons coursed presumably alongside the central canal. Experiments with two tracer substances suggested that some reticulo- and raphe-spinal neurons had axon collaterals terminating both in thoracic and sacral spinal segments.

Morphological relationships among extensor digitorum longus, tibialis anterior, and semitendinosus motor nuclei of the cat: An investigation employing the retrograde transport of multiple fluorescent tracers

To determine the morphological relationships among extensor digitorum longus (EDL), tibialis anterior (TA), and semitendinosus (St) motor nuclei in the spinal cord of the cat, these nuclei were retrogradely labeled with three different fluorescent tracers. The fluorochromes--bisbenzimide, nuclear yellow, and propidium iodide--were applied by intramuscular injection or soaking the muscle nerve. The positions of the labeled motor nuclei were bilaterally symmetrical. The EDL and TA motoneurons were located in close proximity to one another, in the lateral regions of lamina IX in spinal segments L6 and L7. Although the boundaries of each nucleus were tightly opposed, the EDL and TA motor nuclei overlapped minimally, with the somata of EDL motoneurons positioned dorsal to those of TA. The St motor nucleus was located ventromedial to that of EDL and extended from the caudal portion of L6 through S1. Supplemental studies of the reflex effects evoked in EDL, TA, and St muscles by cutaneous nerve stimulation provided physiological observations that may be related to these anatomical results.

Axotomized, adult basal forebrain neurons can innervate fetal frontal cortex grafts: A double fluorescent tracer study in the rat

The ability of axonal regeneration of identified adult basal forebrain (BFB) neurons was examined after homotopic grafting of fetal neocortical tissue to a lesion cavity in the frontal neocortex. Using a four step experimental procedure, adult rats first received an injection of the fluorescent dye Fluoro-Gold (FG) into the sensorimotor cortex in order to label those neurons with projections to the area by retrograde axonal transport. After one week the injection area was removed by aspiration, leaving a cavity in the neocortex. One week later a block of fetal (E14) frontal cortical tissue was placed in the cavity. The animals were then allowed to survive for 6 weeks before a second fluorescent tracer, Nuclear Yellow (NY), was injected into the transplant. The animals were sacrificed 24 h later and analyzed by fluorescence microscopy. Both single labeled, FG and NY containing neurons and double labeled neurons containing both tracers were found in the BFB. The results demonstrate that adult BFB neurons can reestablish cortical projections into fetal cortical grafts (double labeled neurons), and they suggest that other BFB neurons, not initially innervating the lesioned cortical area, have sprouted into the transplant (NY labeled neurons).

The somato-visceral divergent projections of peripheral processes of substance p-containing spinal ganglionic neurons-tri-labeling study of combining fluorescein tracing with immunocytochemistry

The chemical nature of the spinal ganglionic neuron with peripheral processes projecting divergently to the somatic and visceral areas, has been identified by means of tri-labeling method of combining fluorescein tracing with immunocytochemistry. 10 rats were used. First, 2 microliters of 2% fast blue(FB) were injected into the left coeliac ganglion. Two days later, 2% nuclear yellow (NY) was injected into left 9-11 intercostal nerves, 1 microliter for each. On the 4th day, the animal was perfused with 10% formalin in 0.1 mol phosphate buffer. The left T9-11 spinal ganglia were removed and cut into sections by cryostat. The sections were observed under fluorescence microscope and photographed. The results showed that there were three kinds of neurons in the spinal ganglia: 1) single FB labeled cells with blue fluorescent cytoplasm accounted for 38.8% of total cells; 2) single NY labeled cells with yellow fluorescent nuclei accounted for 52.7%; 3) FB and NY double-labeled cells, mostly small or medium in size, accounted for 8.5%. Then, the sections containing double-labeled cells were further processed by substance P-demonstrating PAP immunocytochemical staining. The photographs with immunostaining and fluorescein labelings in the same section were compared. We found that the labeling ratio of SP/NY was 1.4%, SP/FB was 7%, and SP/NY + FB was 28.8%. The present study detected not only the convergence of somato-visceral sensation in the spinal ganglia but also the chemical nature of these neurons containing substance P (SP) for the first time. In addition, these results may provide a morphological basis for the mechanisms of referred pain and somato-visceral reflection.

Segregation and overlap of callosal and association neurons in frontal and parietal cortices of primates: a spectral and coherency analysis

The spatial relations between selected classes of association and callosal neurons were studied in the frontal and parietal lobes of the macaque monkey using retrogradely transported fluorescent dyes. Fast blue and nuclear yellow were injected in the left frontal (areas 4 and 6) and right posterior parietal (area 5) cortices, respectively. These injections led to the retrograde labeling, in the right frontal cortex, of callosal neurons projecting homotopically and association neurons projecting to ipsilateral area 5; in the left superior parietal lobule, of callosal neurons projecting to contralateral area 5 and association neurons projecting to the ipsilateral frontal lobe. In both frontal and parietal cortices, callosal and association neurons were located in layers III and V-VI; a few neurons were also found in layer II. The contribution of layers V-VI to the callosum was significantly higher in areas 4 and 6 than in area 5. Only a small number of neurons (less than 1%) were double labeled. Spectral analyses were used to characterize the spatial periodicities of the distributions of callosal and association neurons. In areas 4, 6, and 5, both association and callosal spectra were dominated by a strong elevation in the range of low spatial frequencies, corresponding to periodicities in cell density with a peak-to-peak distance of about 8 mm. This indicated an arrangement of these corticocortical cells in the form of bands. The latter displayed various shapes and orientations and were composed of more discrete assemblies of cell clusters of about 400-1000 microns width. Their presence was revealed in the power spectra by a small elevation in the range of high spatial frequencies. The coherency analysis assessed the degree of linear relationships for each spatial frequency, and therefore the degree of similarity, between callosal and association cell distributions, together with their phase relations. Little coherency was found in areas 4 and 6 between bands of callosal and association neurons, which suggests that the 2 cell populations are differently and independently distributed in the tangential domain, with no simple phase relations. The overall mean coherency was higher in area 5 than in the frontal cortex: callosal and association bands were more similar in shape, with more extensive zones of overlap. These data indicate that callosal and association neurons share common principles of spatial organization despite the great regional variability of their interrelations in the tangential cortical domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Anatomy of the auditory thalamocortical system of the guinea pig.

We investigated the projection from the medial geniculate body (MG) to the tonotopic fields (the anterior field A, the dorsocaudal field DC, the small field S) and to the nontonotopic ventrocaudal belt in the auditory cortex of the guinea pig. The auditory fields were first delimited in electrophysiological experiments with microelectrode mapping techniques. Then, small quantities of horseradish peroxidase (HRP) and/or fluorescent retrograde tracers were injected into the sites of interest, and the thalamus was checked for labeled cells. The anterior field A receives its main thalamic input from the ventral nucleus of the MG (MGv). The projection is topographically organized. Roughly, the caudal part of the MGv innervates the rostral part of field A and vice versa. After injection of tracer into low or medium best-frequency sites in A, we also found a topographic gradient along the isofrequency contours: the dorsal (ventral) part of a cortical isofrequency strip receives afferents from the rostral (caudal) portions of the corresponding thalamic isofrequency band. However, it is not so obvious whether such a gradient exists also in the high-frequency part of the projection. A second, weaker projection to field A originates in a magnocellular nucleus that is situated caudomedially in the MG and was therefore named the caudomedial nucleus. The dorsocaudal field DC receives input from the same nuclei as the anterior field, but the location of the labeled cells in the MGv is different. This was demonstrated by injection of different tracers into sites with like best frequencies in fields A and DC, respectively. After injection of HRP into the 1-2-kHz isofrequency strip in field A and injection of Nuclear Yellow (NY) into the 1-2-kHz site in field DC, the labeled cells in the MGv form one continuous array that runs from caudal to rostral over the whole extent of the MGv. The anterior part of this array consists of NY-labeled cells; i.e., it projects to field DC. The caudal part is formed by HRP-labeled cells; i.e., it innervates field A. These findings indicate that there is only one continuous tonotopic map in the MGv. This map is split when projected onto the cortex so that two adjacent tonotopic fields (A and DC) result. The cortical maps are rotated relative to the thalamic map in that rostral portions of the MGv project to caudal parts of the tonotopic cortex and vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence of intracerebellar collateralization of nucleocortical cell processes in a prosimian primate (Galago): a fluorescence retrograde study.

The distribution of single and/or double-labeled neurons in the cerebellar nuclei was investigated in a prosimian primate (Galago) by means of fast blue (FB) and nuclear yellow (NY) as retrograde tracers. Injections were made into spatially separate regions of cerebellar cortex on one side (zones C1-C3 in lobules IV and V and in the paramedian lobule) and into the same zones of lobules IV and V on both sides. Following unilateral injections single- and double-labeled somata were seen in the anterior (NIA) and posterior (NIP) interposed cerebellar nuclei on the same side. Single-labeled cells were, in general, more concentrated in NIA from the lobule IV-V injection and in NIP from the PML placement; double-labeled somata were about evenly distributed between NIA and NIP. Cell labeling was sparse in the contralateral NIP. Large and small somata were filled with FB, NY, or FB + NY ipsilateral to the injection sites while the majority of labeled neurons on the contralateral side had small oval- or fusiform-shaped somata. Subsequent to bilateral injections cells labeled with each tracer were concentrated in the NIA, moderate in number in the NIP, both on the ipsilateral side, and sparse in the contralateral NIP. The double labeling of cells in the present study indicates that these neurons project to spatially separated, yet possibly functionally related, areas of ipsilateral cortex. Since larger and small somata were double labeled it is possible that those cells with cerebellofugal processes to extracerebellar targets may simultaneously relay information to divergent cortical sites. In contrast, the few labeled cells seen in the contralateral NIP have small oval and fusiform somata. These neurons may be specifically involved in cerebelloolivary and olivocerebellar feedback loops.

Thalamic branched and unbranched axons to feline polysensory cortex

Thalamic afferents to polysensory neocortex of the suprasylvian, anterior lateral, and pericruciate gyri were demonstrated by retrograde axonal transport of wheat germ agglutinin lectin-bound horseradish peroxidase (HRP) and nuclear yellow (NY). Marker injections were placed in areas identified as responsive to auditory stimuli through the use of click-evoked potentials. Both HRP and NY labeled neurons were found in close proximity or in overlapping regions of the rostral intralaminar nuclei and the adjacent ventral lateral nucleus of the thalamus. While thalamic neurons projecting to different polysensory neocortical areas were often in overlapping fields, double labeling was rare. These results indicate that similar auditory responses seen in different cortical polysensory areas are mediated by mainly separate but topographically related populations of thalamocortical neurons. Similarities in auditory response patterns seen across widely separated areas of polysensory neocortex may be due to common inputs relayed through overlapping or adjacent areas of the thalamus.

ウサギ頚部食道筋支配運動神経細胞の中枢局在

Central location of the cervical esophageal motoneurons was investigated in the rabbit, using the intramuscular injection of retrograde labeling substances: horseradish peroxidase or nuclear yellow. The nucleus ambiguus of the rabbit comprises four subnuclei, CoG, SGm, SG1, and DiG, of which only the ipsilateral CoG was pertinent to the esophageal motoneurons. Labeled neurons were found in the rostral two-thirds of the CoG at levels 1. 2-2. 9 mm rostral to the obex: at the most rostral level, they overlapped in position with the most caudal portion of the facial motor nucleus for about 100, um rostrocaudally. The mean total number of them was 172, and they were maximum in number about a level 2. 5 mm rostral to the obex. In a transverse plane of the brain stem, labeled neurons, although a few in number, were found in the lateral portion of CoG at the most caudal level of their appearance zone. More rostrally, at the mid-level of CoG, in which the CoG was divided into two subgroups, ventrolateral and dorsomedial, labeled neurons increased in number and occupied the entire portion of the former subgroup. Furthermore rostrally, at the rostral level of CoG in which no further division was found, they occupied caudally the dorsal half of CoG and rostrally its entire portion.

Organization of efferent projections of the subthalamic nucleus in the squirrel monkey as revealed by retrograde labeling methods

The cellular origin and degree of collateralization of the subthalamostriatal, subthamonigral and subthalamopallidal projections in the squirrel monkey ( Saimiri sciureus) were studied using lectin-conjugated horseradish peroxidase (WGA-HRP), Nuclear yellow (NY) and Fast blue (FB) as retrograde tracers. In a first experimental group, WGA-HRP was injected in the left putamen and the right caudate nucleus. Followingl these injections numerous retrogradely labeled neurons occured in the dorsolateral two-thirds of the subthalamic nucleus on the putamen-injected side, whereas a smaller numebr of positive cells were found in the ventromedial third of the same nucleus on the caudate-injected side. In a second experimental group NY was injected in the putamen whereas FB was delivered in the substantia nigra on the same side of the brain. After putaminonigral injections subthalamic cells containing the tracer injected in the putamen (about 75–80% of all retrogradely labeled neurons) occured in the dorsolateral two-thirds of the nucleus, whereas those containing the tracer injected in the substantia nigra (about 20–25% of all positive subthalamic cells) were confined to the ventromedial third of the structure. Approximately 5–10% of all subthalamic positive neurons were double-labeled following putaminonigral injections. In a third experimental group, NY was injected in the caudate nucleus and FB in the substantia nigra on the same side. After such injections cells retrogradely labeled with NY or FB were present in about equal number and appeared closely intermingled in the ventromedial third of the subthalamic nucleus. Less than 10% of all positive subthalamic neurons were double-labeled following caudatonigral injections. In a fourth experimental group, NY was delivered in the globus pallidus and FB in the substantia nigra on the same side. In these animal cells containing the tracer delivered in the pallidum were about 4 times more numerous than those labeled with the tracer injected in the substantia nigra, and approximately 10–20% of all positive subthalamic neurons were double-labeled following pallidonigral injections. Most of these double-labeled cells occured in the zone where the two populations of single-labeled cells overlapped. Finally, in a last experimental group, NY was injected in the pedunculopontine nucleus on one side and in the substantia nigra on the other. Following these injections cells containing the tracer delivered in the pedunculopontine nucleus were found to be 5–6 times less numerous than those labeled after substantia nigra injections. These findings suggest that in regard to its efferent projections, the primate subthalamic nucleus is organized according to a complex pattern consisting of: (1) a large (80%) dorsolateral ‘sensorimotor’ zone where most neurons project to the lenticular nucleus and terminate in the globus pallidus and/or the putamen. (2) a small (20%) ventromedial ‘associative’ zone neurons give to either ascending projections to caudate nucleus or descending projections to the substantia nigra, and (3) a smaller (10%) overlapping zone whose neurons send axon collaterals to both the lenticular nucleus and the substantia nigra.

Thalamic projections to fields A, AI, P, and VP in the cat auditory cortex.

Thalamocortical projections to four tonotopic fields (A, AI, P, and VP) of the cat auditory cortex were studied by using combined microelectrode mapping and retrograde axonal transport techniques. Horseradish peroxidase (HRP) or HRP combined with either tritiated bovine serum albumin or nuclear yellow was injected into identified best-frequency sites of one or two different fields in the same brain. Arrays of labeled neurons were related to thalamic nuclei defined on the basis of their cytoarchitecture and physiology. In some cases, patterns of labeling were directly compared with thalamic best-frequency maps obtained in the same brain. We compared only patterns of labeling resulting from injections into similar parts of the frequency representation in different fields to insure that observed differences in patterns of labeling did not simply reflect differences in the frequency representation at the injection sites. The thalamic projection to the four fields is divided among seven nuclei, three tonotopic nuclei (ventral nucleus, V; lateral part of the posterior group of thalamic nuclei, Po; and dorsal cap nucleus, d) and four nontonotopic nuclei (caudodorsal nucleus, cd; ventrolateral nucleus, vl; and small, Ms; and medium-large, Mg, cell regions of the medial division). Projections to each field differ, and each field receives inputs from tonotopic and nontonotopic nuclei. Field A receives its major inputs from Po and Mg, and a minor input from V. Field AI receives its major inputs from V, Po, and Mg, although Po and Mg have heavier projections to field A. Field P receives its major inputs from V, d, and vl; and minor inputs from cd, Ms, Mg, and Po. Field VP receives major inputs from V, vl, and cd; and minor inputs from d, Ms, and Mg. There are segregated territories in V and Po in which most neurons projects to one cortical field (major projection), and a smaller proportion projects to one or more other fields (minor projections). Field VP receives a major projection from the caudal pole of V. Field P receives a major projection from the caudal half of V, and from a thin band along the dorsal border of rostral V. Field AI receives a major projection from most of the rostral one-half of V, and smaller areas in Po and the caudal half of V exclusive of its caudal pole. Field A receives a major projection from most of Po.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibers supplying the laryngeal musculature in the cranial root of the rabbit accessory nerve: Nucleus of origin, peripheral course, and innervated muscles

The location of the rabbit laryngeal motoneurons whose axons traverse the cranial root of the accessory nerve was studied with injection of HRP or nuclear yellow into the laryngeal muscles in combination with the intracranial severing of either the rootlets of the vagus nerve or those of the cranial root. The motoneurons were located in the diffuse cell group that forms a subnucleus occupying the caudal two-thirds of the nucleus ambiguus and sending fibers to the inferior laryngeal nerve. The caudal one-third of the diffuse cell group supplying the lateral cricoarytenoid muscle, was occupied only by these motoneurons, whereas in its rostral two-thirds, they were intermingled with motoneurons having axons that traversed the vagal rootlets. The thyroarytenoid and posterior cricoarytenoid motoneurons are present in the rostral two-thirds of the diffuse cell group; axons of the former traversed the rootlets of the cranial root, and of the latter traversed the vagal rootlets. On the other hand, the medial scattered cell group, located in the rostral one-third of the nucleus ambiguus and sending fibers to the cricothyroid muscle via the superior laryngeal nerve, contained only motoneurons with axons traversing the vagal rootlets. The above findings clarified that fibers of the cranial root enter the inferior laryngeal nerve after joining the vagus, and then reach the adductor muscles for the vocal fold, with their neurons of origin in a caudal portion of the nucleus ambiguus. The vagal rootlet fibers, with their neurons of origin situated more rostrally in the nucleus, innervate the tensor and abductor muscles via the superior and inferior laryngeal nerve, respectively.

The organization of nucleus tegmenti pedunculopontinus neurons projecting to basal ganglia and thalamus: a retrograde fluorescent double labeling study in the rat

The organization of nucleus tegmenti pedunculopontinus (PPN) projections to the basal ganglia and thalamus was studied in the rat by using retrograde transport of fluorescent dyes. Fast blue was injected into the substantia nigra (SN) while Nuclear yellow was delivered to one of the following nuclei: globus pallidus (GP), entopeduncular nucleus, subthalamic nucleus (STN) or parafascicular nucleus of the thalamus. Retrogradely labeled cells were observed throughout the PPN without topographical arrangement. The cells labeled from the SN outnumbered those labeled from other structures. In all cases the majority of cells were single labeled and only a few cells double labeled from SN-GP or SN-STN were found. Labeled cells were either fusiform or multipolar in shape. These data suggest that distinct PPN cells project to their basal ganglia and thalamic targets without a prominent branched organization.

Localization of rabbit laryngeal motoneurons in the nucleus ambiguus

The localization of rabbit laryngeal motoneurons in nucleus ambiguus was investigated using injection of a fluorescent labeling substance, i.e., nuclear yellow, into the individual laryngeal muscles. The nucleus ambiguus of the rabbit comprises four subnuclei, CoG, SGm, SGl, and DiG. The CoG is a group of compactly arranged neurons, and is situated in the rostral one-half of the nucleus. The SG, situated in its rostral one-third, is scattered around the CoG, with a subdivision into SGm and SGl. These subdivisions are medial and lateral to the CoG, respectively. The DiG is formed by diffusely arranged neurons, and is located in the caudal two-thirds of the nucleus. All labeled motoneurons were found in the ipsilateral nucleus ambiguus. The motoneurons supplying the cricothyroid muscle, which is innervated by the superior laryngeal nerve, were present in the SGm, with a clear rostralward segregation from the other motoneurons. The motoneurons supplying the muscles innervated by the inferior laryngeal nerve were located in the DiG, where they displayed a rostrocaudal myotopical arrangement in the order posterior cricoarytenoid, thyroarytenoid, and lateral cricoarytenoid. The posterior cricoarytenoid motoneurons were intermingled with the thyroarytenoid motoneurons in the rostral two-thirds of the DiG, and the former tended to be concentrated more rostrally than the latter. The lateral cricoarytenoid motoneurons were confined to the most caudal one-third of the DiG.

Intrinsically directed pruning as a mechanism regulating the elimination of transient collateral pathways

In neonatal cats, neurons in frontoparietal areas of the cerebral cortex have axons which branch, some collaterals project transiently to the cerebellum, whereas others project by way of the pyramidal tract to the brainstem and spinal cord and persist into the adult. If cerebrocerebellar collaterals are eliminated simply because they are exuberant, then experimentally removing the collaterals in the pyramidal tract should cause the normally ephemeral projections to the cerebellum to persist. To test this hypothesis, the pyramidal tract was cut unilaterally at the pontomedullary junction in 5–9-postnatal-day-old (PND) cats, and 35–68 days later the frontoparietal cortex ipsilateral to the pyramidotomy was injected with tritiated amino acids. From the end of the lesioned pyramidal tract, labeled axons were traced into pathways that descended aberrantly into the caudal medulla and spinal cord, but there was never any transported label in the cerebellum. In a second series of experiments, the fluorescent dye Fast blue (FB) was injected into the spinal cord (2–5 PND) prior to cutting the contralateral pyramidal tract (9–12 PND) to determine if the pyramidotomy caused the axotomized cortical neurons to die. There were no neurons labeled with FB in the frontoparietal cortex on the side of the pyramidotomy, but many retrogradely labeled neurons were present contralaterally in the cortex, suggesting that the pyramidotomy caused the death of all axotomized cortical neurons. In a final set of experiments, FB was injected into the spinal cord and the cerebellar cortex was ablated (2–3 PND) prior to cutting the pyramidal tract (9–72 PND). Cerebellar decortication results in the persistence of cerebrocerebral projections to the partially deafferented deep nuclei, therefore injections of Nuclear yellow (NY) or Diamidino yellow (DY) were made later (32–86 PND) into the cerebellar nuclei on the side of the decortication to determine if these projections persist in pyramidotomized cats. After pyramidotomies at 9 PND, there were no neurons labeled with fluorescent dyes in the ipsilateral frontoparietal cortex, indicating that the cerebrocerebellar collaterals, even under experimental conditions which normally cause them to persist, could not sustain the axotomized cortical neurons. Pyramidotomies at 24 PND or later did not cause all axotomized neurons to die since neurons labeled with FB were present in the ipsilateral cortex. These findings suggest that during development of corticosubcortical pathways there is a hierarchical ordering of axon collaterials. The collaterals in the pyramidal tract must be considered the cortical neuron's primary collateral since their experimental elimination always results in the death of the parent cell. Cerebrocerebellar collaterals, on the other hand, are secondary and expendable since they are normally eliminated without causing the parent neuron to die.

Double-labeling study of axonal branching within the lateral reticulocerebellar projection in the rat.

Collateral axonal branching to the cerebellum from the lateral reticular nucleus (LRN) was studied in the rat by using the fluorescent double-labeling technique. Following injection of Fast Blue (FB) into the cerebellar cortex, followed 3 days later by injection of Nuclear Yellow (NY) into a different region of the cortex, single- and double-labeled cells were found within the LRN. Most LRN-cerebellar projections were bilateral with ipsilateral preponderance, except for the projection to the paramedian lobule, which was completely ipsilateral. The dorsolateral area of the magnocellular division of the LRN contained cells whose axons branch to terminate in the rostral anterior lobe and the caudal part of the ipsilateral paramedian lobule (hindlimb areas of the cerebellar cortex), while the medial area of the LRN contained cells that supply, via collateral axonal branching, the caudal area of the contralateral anterior lobe and the rostral part of the ipsilateral paramedian lobule (forelimb areas of the cerebellum). Branched LRN-cerebellar axons projected to both hemispheres and to both sides of the caudal anterior lobe. No axonal branching was evident in the LRN-cerebellar projection to the rostral anterior lobe. The projection to the anterior and posterior lobe vermis also contained collateral axonal branching.