Retrograde Horseradish Peroxidase Labeling Research Articles

Recombinant ciliary neurotrophic factor promotes nerve regeneration and induces gene expression in silicon tube-bridged transected sciatic nerves in adult rats

Sciatic nerves in adult male rats were transected and reunited via a silicone chamber. This was followed by a focal injection of recombinant ciliary neurotrophic factor (CNTF). To evaluate the effect of this therapeutic approach and to explore its possible mechanisms, nerve regeneration was traced by horseradish peroxidase retrograde labeling. Functional recovery was evaluated by functional assessment of the hind feet and the expression of a number of proteins was detected using immunohistochemistry. The results showed that a single administration of CNTF could promote regeneration of motor axons, with improved functional recovery in adult rats. Growth associated protein (GAP)-43, S100, CD68 and major histocompatibility complex class II immunoreactivity in the regenerative and distal nerves suggested that CNTF could promote axon regeneration, Schwann cell migration, monocyte infiltration and activation. CNTF might also indirectly promote axonal regeneration by further activating the JAK-STAT3 pathway and subsequently upregulating phosphotyrosine, GAP-43 and S100 expression to enhance proliferation, growth and migration of Schwann cells. CNTF has suggested important targets for pharmacological intervention in peripheral nerve disease and injury.

Vesicular acetylcholine transporter–immunoreactive axon terminals enriched in the pontine nuclei of the mouse

Information to the cerebellum enters via many afferent sources collectively known as precerebellar nuclei. We investigated the distribution of cholinergic terminal-like structures in the mouse precerebellar nuclei by immunohistochemistry for vesicular acetylcholine transporter (VAChT). VAChT is involved in acetylcholine transport into synaptic vesicles and is regarded as a reliable marker for cholinergic terminals and preterminal axons. In adult male mice, brains were perfusion-fixed. Polyclonal antibodies for VAChT, immunoglobulin G-peroxidase and diaminobenzidine were used for immunostaining. In the mouse brain, immunoreactivity was seen in almost all major cholinergic cell groups including brainstem motoneurons. In precerebellar nuclei, the signal could be detected as diffusely beaded terminal-like structures. It was seen heaviest in the pontine nuclei and moderate in the pontine reticulotegmental nucleus; however, it was seen less in the medial solitary nucleus, red nucleus, lateral reticular nucleus, inferior olivary nucleus, external cuneate nucleus and vestibular nuclear complex. In particular, VAChT-immunoreactive varicose fibers were so dense in the pontine nuclei that detailed distribution was studied using three-dimensional reconstruction of the pontine nuclei. VAChT-like immunoreactivity clustered predominantly in the medial and ventral regions suggesting a unique regional difference of the cholinergic input. Electron microscopic observation in the pontine nuclei disclosed ultrastructural features of VAChT-immunoreactive varicosities. The labeled bouton makes a symmetrical synapse with unlabeled dendrites and contains pleomorphic synaptic vesicles. To clarify the neurons of origin of VAChT-immunoreactive terminals, VAChT immunostaining combined with wheat germ agglutinin–conjugated horseradish peroxidase retrograde labeling was conducted by injecting a retrograde tracer into the right pontine nuclei. Double-labeled neurons were seen bilaterally in the laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus. It is assumed that mesopontine cholinergic neurons negatively regulate neocortico-ponto-cerebellar projections at the level of pontine nuclei.

Nerve Regeneration through a Healthy Nerve Trunk: A New and Hopeful Conduit for Bridging Nerve Defects

Considering a healthy nerve trunk as the hypothetically ideal conduit, a new experimental model using an intact nerve for bridging a nerve defect was contemplated. Thirty rats were used. In group I (double coaptation), a segment was removed from the peroneal nerve. Both the proximal and distal stumps were repaired end-to-side to the tibial nerve. In group II (only distal coaptation), only the distal nerve stump was repaired. In group III (control), the transected segment was immediately repaired primarily in its original orientation as a nerve graft. A walking track analysis was conducted periodically for 28 months. The horseradish peroxidase retrograde labeling technique was used for tracking the origin of the axons presented in the distal stump of the peroneal nerve in group I, and morphologic studies were also carried out for all the groups. Functional assessment revealed that the difference between group I and group II was significant. The horseradish peroxidase labeling test suggested that the nerve fibers in the distal stump of the peroneal nerve were mostly from its original proximal stump passed by the way of the tibial nerve bridge. This study suggested that the axons of the proximal stump of a sectioned nerve can sprout into another intact nerve trunk by the way of an end-to-side repair site, regenerate, and advance in its epineurium distally for a distance and pass into its original distal stump if it was repaired end-to-side. It was thought that the technique could be used in clinical cases with short nerve defects as an alternative method to grafts and conduits.

Sensory reinnervation of a musculocutaneous flap: an experimental rabbit study

Sensory neurotisation of a muscle (sensory nerve transfer to the motor nerve of a muscle) produces muscle sensibility, but not skin sensibility. How to achieve sensation of a musculocutaneous flap remains a challenge to reconstructive microsurgeons. The purpose of our study was to determine if multiple nerve grafts which were placed vertically between the neuromuscular entrance zone of a muscle and a target area of dermis on the overlying skin could improve sensation. Thirty-six gracilis musculocutaneous flaps (18 rabbits) were raised and divided into three groups: group 1 consisted of 12 sensory neurotised gracilis musculocutaneous flaps with five nerve grafts each; group 2 consisted of another 12 sensory neurotised gracilis flaps with 10 nerve grafts each; and the control group consisted of 12 sensory neurotised gracilis musculocutaneous flaps without any nerve grafts. All nerve grafts spanned the distance between the neuromuscular entrance zone of the gracilis muscle and a specified 3 cm diameter area of the skin island. The saphenous nerve (sensory) was coapted to the obturator nerve (motor nerve of the gracilis) in an effort to achieve improved sensation of the skin island in the musculocutaneous flaps. After 6 months, the flaps were individually evaluated using cortical somatosensory evoked potentials (CSSEP) using normal, painful, cold and hot stimuli. One unoperated rabbit was studied as the baseline CSEEP for comparison. Retrograde horseradish peroxidase (HRP) labelling was then performed to evaluate the possibility of newly established neural pathways. Results of the CSSEP testing revealed that flaps possessing 10 nerve grafts (group 2) demonstrated better sensation when compared to flaps possessing five nerve grafts (group 1) or no nerve grafts (control group). Furthermore, retrograde HRP labelling proved that a new neural pathway had been established from the skin island to the dorsal root ganglia of S1 and S2 via the interposed nerve grafts and the sensory neurotised gracilis muscle in groups 1 and 2 rabbits. The control group did not display any sensory regeneration.

Efficient reinnervation of hindlimb muscles by thoracic motor neurons after nerve cross-anastomosis in rats.

Peripheral motor axons can regenerate through motor endoneurial tubes of foreign nerves to reinnervate different target muscles. This regenerative capacity has been brought to clinical applications for restorative surgery after nerve or root injury. In this study the authors explore the extent to which nerve cross-anastomosis between lower intercostal nerves and lumbar ventral roots would be effective in inducing reinnervation of paralyzed hindlimb muscles after spinal cord hemisection at the thoracolumbar boundary in rats. The proximal extremities of sectioned intercostal nerves T10-12 were surgically connected to the distal extremities of sectioned ipsilateral lumbar ventral roots L3-5, respectively. Motor activity reappeared 2 months postsurgery; however, locomotion was not restored and inappropriate motor patterns persisted at 9 months postsurgery. At that time, data from electrophysiological and histological studies and horseradish peroxidase retrograde labeling demonstrated efficient regrowth of thoracic motor neuron axons that reached hindlimb muscles. They also revealed a persistent maturation defect of regrown fibers, as shown by size heterogeneity and presumable extensive axonal branching. These features are consistent with reduced neural activity subsequent to continuing inappropriate motor patterns. These results indicate that cross-anastomosis of intercostal nerves with lumbar ventral roots allows efficient reinnervation of paralyzed hindlimb muscles after spinal cord hemisection in rats. Stimulating the reorganization of the neuronal circuitry in the central nervous system by locomotion training or other methods would presumably result in both functional and anatomical improvements. This experimental setting provides a convenient animal model to investigate these processes.

Regrowth of the Rostral Spinal Axons into the Caudal Ventral Roots through a Collagen Tube Implanted into Hemisected Adult Rat Spinal Cord

OBJECTIVE A collagen tube was used to guide axonal regrowth from the spinal cord to the periphery to contribute to improvement of paralysis after lower thoracic spinal cord injury. METHODS The spinal cords of adult male Sprague-Dawley rats were lesioned by removing the left hemicord from T12 to 5 mm below this level and additionally sectioning all left lumbar ventral roots. In experimental animals (n = 9), a collagen tube was inserted into this gap, spanning the rostral hemisected cord to the caudal sectioned lumbar ventral roots (gap, 7 mm). In control animals (n = 6), no treatment was performed. RESULTS Six months after surgery, the return of some tension and resistance of the paralyzed hindlimb muscles was observed in all experimental rats except the untreated controls. Nine months postoperatively, muscle action potentials were recorded from the target muscles of the experimental animals while electrostimulating the tissue continuity within the collagen tube. Horseradish peroxidase retrograde labeling showed that the neurons in the rostral cord near the implantation site regrew into the reconnected lumbar ventral roots. Histological examination indicated numerous myelinated axons in the reconnected root pathways and newly formed endplates in the target muscles. No axonal regeneration was found in the control rats. CONCLUSION These results indicate that the rostral spinal axons can regrow into the caudal sectioned and reconnected ventral roots through a collagen tube, thus innervating the denervated peripheral targets in adult rats after spinal cord injury. This surgical repair model also provides a means for testing the use of trophic factors that may further promote axonal regeneration.

Regrowth of the rostral spinal axons into the caudal ventral roots through a collagen tube implanted into hemisected adult rat spinal cord.

A collagen tube was used to guide axonal regrowth from the spinal cord to the periphery to contribute to improvement of paralysis after lower thoracic spinal cord injury. The spinal cords of adult male Sprague-Dawley rats were lesioned by removing the left hemicord from T12 to 5 mm below this level and additionally sectioning all left lumbar ventral roots. In experimental animals (n = 9), a collagen tube was inserted into this gap, spanning the rostral hemisected cord to the caudal sectioned lumbar ventral roots (gap, 7 mm). In control animals (n = 6), no treatment was performed. Six months after surgery, the return of some tension and resistance of the paralyzed hindlimb muscles was observed in all experimental rats except the untreated controls. Nine months postoperatively, muscle action potentials were recorded from the target muscles of the experimental animals while electrostimulating the tissue continuity within the collagen tube. Horseradish peroxidase retrograde labeling showed that the neurons in the rostral cord near the implantation site regrew into the reconnected lumbar ventral roots. Histological examination indicated numerous myelinated axons in the reconnected root pathways and newly formed endplates in the target muscles. No axonal regeneration was found in the control rats. These results indicate that the rostral spinal axons can regrow into the caudal sectioned and reconnected ventral roots through a collagen tube, thus innervating the denervated peripheral targets in adult rats after spinal cord injury. This surgical repair model also provides a means for testing the use of trophic factors that may further promote axonal regeneration.

Innervation of the caudal denervated ventral roots and their target muscles by the rostral spinal motoneurons after implanting a nerve autograft in spinal cord-injured adult marmosets.

The authors conducted a study to determine the effects of using a nerve autograft (NAG) to promote and guide axonal regrowth from the rostral spinal cord to the caudal lumbar ventral nerve roots to restore hindlimb motor function in adult marmosets after lower thoracic cord injury. Nine animals underwent a left-sided hemisection of the spinal cord at T-12 via left-sided T9-L3 hemilaminectomy, with section of all ipsilateral lumbrosacral ventral nerve roots. In the experimental group (five animals), an NAG obtained from the right peroneal nerve was anastomosed with the sectioned and electrophysiologically selected lumbar ventral roots (left L-3 and L-4) controlling the left quadriceps muscle and then implanted into the left ventrolateral T-10 cord. In the control group (four animals), the sectioned/selected lumbar ventral roots were only ligated. After surgery, all marmosets immediately suffered from complete paralysis of their left hindlimb. Five months later, some clinical signs of reinnervation such as tension and resistance began to appear in the paralyzed quadriceps of all experimental animals that received autografts. Nine months postoperatively, three of the five experimental marmosets could maintain their lesioned hindlimb in hip flexion. Muscle action potentials and motor evoked potentials were recorded from the target quadriceps in all experimental marmosets, but these potentials were absent in the control animals. Horseradish peroxidase retrograde labeling from the distal sectioned/reconnected lumbar ventral roots traced 234+/-178 labeled neurons in the ipsilateral T8-10 ventral horn, mainly close to the NAG tip. Histological analysis showed numerous regenerating axons in this denervated/reconnected nerve root pathway, as well as newly formed motor endplates in the denervated/reinnervated quadriceps. No axonal regeneration was detected in the control animals. These data indicate that the rostral spinal neurons can regrow into the caudal ventral roots through an NAG, thereby innervating the target muscle in adult marmosets after spinal cord injury.

Acellular nerve graft re-seeded by Schwann cells migrating from the nerve stump can stimulate spinal motoneurons for functional reinnervation of the rat muscle

The acellular nerve graft was utilised to restore a functional reinnervation of the biceps brachii muscle from the motoneuron pool of the cervical spinal cord. The musculocutaneous nerve stump was sutured to an acellular nerve graft, the opposite end of which was inserted into the cervical spinal cord cranial to the avulsed C5 ventral root. The acellular nerve graft was repopulated by Schwann cells heavily immunostained for NGFr within 90 days. The Schwann cells migrating from the nerve stump reached the spinal cord grey matter, where they stimulated the motoneurons to send axonal sprouts. The functional reinnervation of the biceps brachii muscle was assessed by means of the behavioural (grooming) test and EMG, the presence of myelinated and unmyelinated axons was demonstrated by light and electron microscopy. The axonal reconnection of the musculocutaneous nerve stump was verified by horseradish peroxidase retrograde labelling of the spinal motoneurons. Moreover, the motoneurons on the operated side of the C5 spinal segment displayed increased immunostaining for GAP-43 in comparison to the contralateral side, whereas the pattern of AChE histochemical reaction was similar on both the operated and contralateral side, of the C5 segment 150 days after acellular graft implantation. The regenerated axons bridged a 4-cm long originally acellular nerve graft to reach and reinnervate the biceps brachii muscle. The reinnervation of the neuromuscular junctions was morphologically determined by immunofluorescence for neurofilaments. The number of myelinated axons in the acellular nerve graft was significantly higher than those growing over the cellular graft, but their diameter was smaller. The results of experiments presented here demonstrate functional recovery of the biceps muscle reinnervation through the acellular nerve graft repopulated by migrating Schwann cells. The process of reinnervation by acellular nerve graft is however delayed and worse in comparison with the cellular graft.

Reinnervation of denervated lumbar ventral roots and their target muscle by thoracic spinal motoneurons via an implanted nerve autograft in adult rats after spinal cord injury

Intraspinally implanting a nerve autograft (NAG) to promote axonal regeneration toward periphery was investigated as a surgical treatment for spinal cord injury in adult rats. Fifteen animals underwent a left hemisection of the spinal cord at T12 level and an intradural section of all ipsilateral lumbar ventral roots. In repaired animals (n = 9), the electrophysiologically selected left L3 and L4 lumbar ventral roots supplying the quadriceps muscle were anastomosed to a NAG. The NAG was taken from the right peroneal nerve and then ventrolaterally implanted into the cord at a level 7 mm rostral to the hemisection. In the control group (n = 6), sectioned lumbar ventral roots were left unrepaired. Nine months later, the animals were assessed with clinical, electrophysiological, and histological examinations. Muscle action potential and motor evoked potential were obtained from the denervated/reinnervated quadriceps in all repaired animals, with a mean amplitude of 918.3 ± 328.9 μV and 215.8 ± 39.7 μV, respectively. Horseradish peroxidase retrograde labeling from the denervated/repaired lumbar ventral roots, performed in five repaired animals, showed that the mean of labeled neurons, ipsilaterally located in the thoracic ventral horn near the implantation site, was 145.8 ± 111.7. Histological analysis showed numerous myelinated axons in the NAG and denervated/repaired lumbar ventral roots of all repaired animals. The study of neuromuscular junctions furthermore confirmed numerous newly formed endplates appearing in the denervated/reinnervated quadriceps. These changes were absent in the control animals. These data indicate that the rostral thoracic spinal motoneurons can innervate the caudal denervated/repaired lumbar ventral roots and the target quadriceps via an implanted NAG, thereby inducing some functional recovery in adult rats after lower thoracic spinal cord injury. J. Neurosci. Res. 56:506–517, 1999. © 1999 Wiley-Liss, Inc.

Reinnervation of denervated lumbar ventral roots and their target muscle by thoracic spinal motoneurons via an implanted nerve autograft in adult rats after spinal cord injury.

Intraspinally implanting a nerve autograft (NAG) to promote axonal regeneration toward periphery was investigated as a surgical treatment for spinal cord injury in adult rats. Fifteen animals underwent a left hemisection of the spinal cord at T12 level and an intradural section of all ipsilateral lumbar ventral roots. In repaired animals (n = 9), the electrophysiologically selected left L3 and L4 lumbar ventral roots supplying the quadriceps muscle were anastomosed to a NAG. The NAG was taken from the right peroneal nerve and then ventrolaterally implanted into the cord at a level 7 mm rostral to the hemisection. In the control group (n = 6), sectioned lumbar ventral roots were left unrepaired. Nine months later, the animals were assessed with clinical, electrophysiological, and histological examinations. Muscle action potential and motor evoked potential were obtained from the denervated/reinnervated quadriceps in all repaired animals, with a mean amplitude of 918.3+/-328.9 microV and 215.8+/-39.7 microV, respectively. Horseradish peroxidase retrograde labeling from the denervated/repaired lumbar ventral roots, performed in five repaired animals, showed that the mean of labeled neurons, ipsilaterally located in the thoracic ventral horn near the implantation site, was 145.8+/-111.7. Histological analysis showed numerous myelinated axons in the NAG and denervated/repaired lumbar ventral roots of all repaired animals. The study of neuromuscular junctions furthermore confirmed numerous newly formed endplates appearing in the denervated/reinnervated quadriceps. These changes were absent in the control animals. These data indicate that the rostral thoracic spinal motoneurons can innervate the caudal denervated/repaired lumbar ventral roots and the target quadriceps via an implanted NAG, thereby inducing some functional recovery in adult rats after lower thoracic spinal cord injury.

Abducens internuclear and ascending tract of Deiters inputs to medial rectus motoneurons in the cat oculomotor nucleus: Synaptic organization

Abducens internuclear and ascending tract of Deiters (ATD) inputs to medial rectus motoneurons in the oculomotor nucleus are important for conjugate horizontal movements. In the present study, the organization of these separate populations of neurons and their synaptic connections with medial rectus motoneurons in the cat oculomotor nucleus have been examined by light and electron microscopy by using retrograde and anterograde axonal tracers. Consistent with the patterns of retrograde horseradish peroxidase labeling, the abducens internuclear projection is predominantly, if not exclusively, contralateral, whereas the ATD projection is exclusively ipsilateral, as demonstrated by anterograde autoradiographic and biocytin labeling. Both populations of synaptic endings contain spheroidal synaptic vesicles and establish synaptic contacts with modest postsynaptic densifications. In addition, ATD synaptic endings frequently are associated with subjunctional dense bodies and subsurface cisternae. The two populations of excitatory inputs differ, however, in their soma-dendritic distribution. The majority of abducens internuclear synaptic endings contact distal dendrites, whereas the majority of ATD synaptic endings contact proximal dendrites or somata. Abducens internuclear synaptic endings furthermore have a higher density of mitochondria than ATD synaptic endings. The more proximal location of ATD synaptic endings is consistent with the faster rise time and earlier reversal to polarizing currents of ATD excitatory postsynaptic potentials in comparison to those evoked by the abducens internuclear pathway as determined electrophysiologically. Given the differences in the physiologic signals conveyed by the abducens internuclear (eye velocity and eye position) and ATD (head velocity) pathways, the findings in this study suggest that the soma-dendritic stratification of the two inputs to medial rectus motoneurons may provide a means for the separate control of visuomotor and vestibular functions, respectively. J. Comp. Neurol. 405:141–159, 1999. © 1999 Wiley-Liss, Inc.

Abducens internuclear and ascending tract of Deiters inputs to medial rectus motoneurons in the cat oculomotor nucleus: Synaptic organization

Abducens internuclear and ascending tract of Deiters (ATD) inputs to medial rectus motoneurons in the oculomotor nucleus are important for conjugate horizontal movements. In the present study, the organization of these separate populations of neurons and their synaptic connections with medial rectus motoneurons in the cat oculomotor nucleus have been examined by light and electron microscopy by using retrograde and anterograde axonal tracers. Consistent with the patterns of retrograde horseradish peroxidase labeling, the abducens internuclear projection is predominantly, if not exclusively, contralateral, whereas the ATD projection is exclusively ipsilateral, as demonstrated by anterograde autoradiographic and biocytin labeling. Both populations of synaptic endings contain spheroidal synaptic vesicles and establish synaptic contacts with modest postsynaptic densifications. In addition, ATD synaptic endings frequently are associated with subjunctional dense bodies and subsurface cisternae. The two populations of excitatory inputs differ, however, in their soma-dendritic distribution. The majority of abducens internuclear synaptic endings contact distal dendrites, whereas the majority of ATD synaptic endings contact proximal dendrites or somata. Abducens internuclear synaptic endings furthermore have a higher density of mitochondria than ATD synaptic endings. The more proximal location of ATD synaptic endings is consistent with the faster rise time and earlier reversal to polarizing currents of ATD excitatory postsynaptic potentials in comparison to those evoked by the abducens internuclear pathway as determined electrophysiologically. Given the differences in the physiologic signals conveyed by the abducens internuclear (eye velocity and eye position) and ATD (head velocity) pathways, the findings in this study suggest that the soma-dendritic stratification of the two inputs to medial rectus motoneurons may provide a means for the separate control of visuomotor and vestibular functions, respectively.

Motor versus sensory neuron regeneration through collagen tubules.

Differences in regeneration of sensory and motor nerves were studied in rats to determine the effects of entubulation with collagen conduits. The rat sciatic nerve was repaired either with a 10-mm saline-filled gap or with a no-gap end-to-end repair cuffed within collagen tubules. These repairs were compared with the standard epineurial repairs. The populations of regenerated motor and sensory neurons in the peroneal nerves of all repairs were compared against the populations of normal peroneal neurons using horseradish peroxidase retrograde labeling. The epineurial repair resulted in regeneration of 65 percent (409 +/- 150) of motor neurons and 79 percent (2127 +/- 516) of sensory neurons (n = 6). The no-gap end-to-end repair in a collagen tubule resulted in regeneration of 53 percent (338 +/- 203) of motor and 70 percent (1893 +/- 794) of sensory neurons (n = 7). In the 10-mm gap repair, only 6.2 percent (39 +/- 18) of motor neurons but 63 percent (1710 +/- 557) of sensory neurons regenerated (n = 5). These results show that collagen entubulation supports nerve regeneration in end-to-end nerve repairs comparably to standard epineurial suture repairs. With the 10-mm gap repairs in collagen tubules, sensory neurons regenerated consistently better than motor neurons in the same environment. Therefore, intrinsic differences exist between motor and sensory neuron regeneration in the same nerve.

Assessment of axons from the different types of neurons regenerating through sites of repair after nerve repair

The percentage of axons from different types of neurons regenerating through the site of repair was assessed by retrograde horseradish peroxidase labelling in 30 male SD rats. These rats were randomly divided into groups and underwent epineurial, perineurial suturing and 4 mm nerve graft for transected peroneal nerves in lower limbs on one side. The contralateral nerves were not injured and served as controls. The number of labelled neurons in the experimental side divided by those in the control gave a percentage of regeneration. Four to 9 weeks after nerve repair, axonal regeneration through the repair site was assessed by number, location and diameter. Results revealed that the percentage of motor axons crossing the nerve repair site was the same as the sensory axons. The percentage of neurons with axons innervating muscle spindles was statistically lower that those innervating the other end organs. Perineurial repair produced a higher percentage of motor axons across the repair than epineurial repair or nerve graft.

A critical period for the specification of motor pools in the chick lumbosacral spinal cord.

When 3-4 segments of the chick lumbosacral neural tube are reversed in the anterior-posterior axis at stage 15 (embryonic day 2.5), the spinal cord develops with a reversed organization of motoneurons projecting to individual muscles in the limb (C. Lance-Jones and L. Landmesser (1980) J. Physiol. 302, 581-602). This finding indicated that motoneuron precursors or components of their local environment were specified with respect to target by stage 15. To identify the timing of this event, we have now assessed motoneuron projections after equivalent neural tube reversals at earlier stages of development. Lumbosacral neural tube segments 1-3 (+/- one segment cranial or caudal) were reversed in the anterior-posterior axis at stages 13 and 14 (embryonic day 2). The locations of motoneurons innervating two thigh muscles, the sartorius and femorotibialis, were mapped via retrograde horseradish peroxidase labeling at stages 35-36 (embryonic days 9-10). In a sample of embryos, counts were made of the total number of motoneurons in the lateral motor columns of reversed segments. The majority of motoneurons projecting to the sartorius and femorotibialis were in a normal position within the spinal cord. Segmental differences in motor column size were also similar to normal. These observations indicate that positional cues external to the LS neural tube can affect motoneuron commitment and number at stages 13-14. Since these observations stand in contrast to results following stage 15 reversals, we conclude that regional differences related to motoneuron target identity are normally specified or stabilized within the anterior LS neural tube between stages 14 and 15. To examine the role of the notochord in this process, neural tube reversals were performed at stages 13-14 as described above, except that the underlying notochord was also reversed. Projections to the sartorius and femorotibialis muscles did not differ significantly from those in embryos with neural tube reversals alone, indicating that the notochord is not the source of cues for target identity at stages 13-14.

Regeneration of Respiratory Pathways within Spinal Peripheral Nerve Grafts

Central respiratory neurons exhibit normal activity after axonal regeneration within blind-ended peripheral nerve grafts (PNGs) inserted near the corresponding cell bodies in the medullary respiratory centers. Part of these medullary respiratory neurons project toward the spinal cord and contribute to descending respiratory pathways that control respiratory motoneurons. The present work investigates to what extent cervical respiratory pathways could be directed out of the central nervous system within PNGs inserted distant to the medullary respiratory nuclei. In adult rats (n=13), autologous segments of the peroneal nerve were implanted into the ventrolateral part of the C2 spinal cord at the level of the descending respiratory pathways. Two to four months after grafting, electrophysiological recording of teased graft filaments (n=562) revealed the presence of regenerated nerve fibers with unitary impulse traffic (n=164) in all tested PNGs (n=6). Respiratory discharges (n=52) corresponded to efferent and afferent activity. Efferent respiratory discharges (n=32) originated from central respiratory neurons which remained functional and preserved afferent connections. Retrograde horseradish peroxidase labeling applied to the distal cut end of PNGs (n=7) revealed stained (42/1997) neurons in areas where respiratory cells have been described. Afferent respiratory discharges (n=20) were synchronized with lung inflation but their origin (stretch pulmonary receptors and/or respiratory muscle receptors) was not determined. On the basis of additional data from light and electron microscopy of PNGs, comparison was made between anatomical, retrograde labeling, and electrophysiological data. The main conclusion is that spinal PNGs appear to be able to promote axonal regeneration of functional respiratory efferent and afferent pathways.

Projections of the lateral terminal accessory optic nucleus of the common marmoset (Callithrix jacchus)

The connections of the lateral terminal nucleus (LTN) of the accessory optic system (AOS) of the marmoset monkey were studied with anterograde 3H-amino acid light autoradiography and horseradish peroxidase retrograde labeling techniques. Results show a first and largest LTN projection to the pretectal and AOS nuclei including the ipsilateral nucleus of the optic tract, dorsal terminal nucleus, and interstitial nucleus of the superior fasciculus (posterior fibers); smaller contralateral projections are to the olivary pretectal nucleus, dorsal terminal nucleus, and LTN. A second, major bundle produces moderate-to-heavy labeling in all ipsilateral, accessory oculomotor nuclei (nucleus of posterior commissure, interstitial nucleus of Cajal, nucleus of Darkschewitsch) and nucleus of Bechterew; some of the fibers are distributed above the caudal oculomotor complex within the supraoculomotor periaqueductal gray. A third projection is ipsilateral to the pontine and mesencephalic reticular formations, nucleus reticularis tegmenti pontis and basilar pontine complex (dorsolateral nucleus only), dorsal parts of the medial terminal accessory optic nucleus, ventral tegmental area of Tsai, and rostral interstitial nucleus of the medial longitudinal fasciculus. Lastly, there are two long descending bundles: (1) one travels within the medial longitudinal fasciculus to terminate in the dorsal cap (ipsilateral >> contralateral) and medial accessory olive (ipsilateral only) of the inferior olivary complex. (2) The second soon splits, sending axons within the ipsilateral and contralateral brachium conjunctivum and is distributed to the superior and medial vestibular nuclei. The present findings are in general agreement with the documented connections of LTN with brainstem oculomotor centers in other species. In addition, there are unique connections in marmoset monkey that may have developed to serve the more complex oculomotor behavior of nonhuman primates.

On the Agonistic Display of the Siamese Fighting Fish

The agonistic display of the Siamese fighting fish is characterized by tonic extension of the gill opercula, which is mediated by a single abductor, the opercular dilator muscle. This muscle consists of three bellies, which have different origins and biochemical properties. As part of an effort to define in detail the neural substrate for agonistic display, the distribution, number, and morphology of opercular dilator motoneurons in the trigeminal motor nucleus were examined by retrograde horseradish peroxidase labelling and Golgi-impregnation. The trigeminal motor nucleus consists of three subnuclei: rostral, caudomedial and caudolateral. Neuron numbers among these three subnuclei are relatively constant among animals, and between males and females. Innervation of the opercular dilator muscle originates predominantly from the caudolateral subnucleus; a smaller contingent of motoneurons originates from the caudomedial subnucleus. Labelling of the caudolateral subnucleus is usually intense, whereas caudomedial neurons are weakly labelled. These observations suggest that neurons in the caudomedial subnucleus provide either sparse and widespread innervation, or innervate only a small, circumscribed area of the opercular dilator muscle complex, whereas caudolateral neurons probably provide extensive and dense innervations. The dendritic morphology of trigeminal motoneurons is independent of their targets and of the subnuclei in which they are located. All neurons observed are similar in shape. Primary dendrites of neurons in the rostral and caudomedial subnuclei are long, whereas those in the caudolateral subnucleus are short or absent. Regardless of the position of the somata, dendrites arborize only in the lateral half of the reticular formation in the vicinity of the descending tectobulbal tract, the descending trigeminal tract, and the ascending secondary gustatory tract. The morphological differences suggest that inputs to terminal dendrites have stronger effects on neurons in the caudolateral than those in the other two subnuclei.

Topographical relations between ipsilateral cortical afferents and callosal neurons in the second somatic sensory area of cats

Experiments were carried out on the second somatic sensory area (SII) of cats to study 1) the laminar distribution of axon terminals from the ipsilateral first somatic sensory cortex (SI); and 2) the topographical relations between their terminal field and the callosal neurons projecting to the contralateral homotopic cortex. To label simultaneously in SII both ipsilateral cortical afferents and callosal cells, cats were given iontophoretic injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in the forepaw zone of ipsilateral SI, and pressure injections of horseradish peroxidase (HRP) in the same zone of contralateral SII. The possibility that ipsilateral cortical axon terminals synapse callosal neurons was investigated with the electron microscope by combining lesion-induced degeneration with retrograde HRP labelling. Fibers and terminations immunolabelled with PHA-L from ipsilateral SI were distributed in SII in a typical patchy pattern and were mostly concentrated in supragranular layers. Labelled fibers formed a very dense plexus in layer III and ramified densely also in layers I and II. Labelled axon terminals were both en passant and single-stalked boutons. Counts of 8,303 PHA-L-labelled terminals of either type showed that 82.40% were in supragranular layers. The highest concentration was in layer III (43.99%), followed by layers II (30.32%) and I (8.09%). The remaining terminals were distributed among layers IV (6.96%), V (4.93%), and VI (5.68%). The same region of SII containing anterogradely labelled axons and terminals also contained numerous neurons retrogradely labelled with HRP from contralateral SII. Callosal projection neurons were pyramidal, dwelt mainly in layer III, and were distributed tangentially in periodic patches. Patches of anterograde and retrograde labelling either interdigitated or overlapped both areally and laminarly. In the zones of overlap, numerous PHA-L-labelled axon terminals were seen in close apposition to HRP-labelled pyramidal cell dendrites. Combined HRP-electron microscopic degeneration experiments showed that in SII axon terminals from ipsilateral SI form asymmetric synapses with HRP-labelled dendrites and dendritic spines pertaining to callosal projection neurons. These results are discussed in relation to the layering and function of the SI to SII projection, and to the evidence that SII neurons projecting to the homotopic area of the contralateral hemisphere have direct access to the sensory information transmitted from ipsilateral SI.