Axonal regeneration of descending and ascending spinal projection neurons in spinal cord-transected larval lamprey

The distributions of descending and ascending spinal projection neurons (i.e., spinal neurons with moderate to long axons) were compared in normal larval lamprey and in animals that had recovered for 8 weeks following a complete spinal cord transection at 50% body length (BL, normalized distance from the anterior head). In normal animals, application of HRP to the spinal cord at 60% BL (40% BL) labeled an average of 713.8 +/- 143.2 descending spinal projection neurons (718.4 +/- 108.0 ascending spinal projection neurons) along the rostral (caudal) spinal cord, most of which were unidentified neurons. Some of these neurons project for at least approximately 50-60 spinal cord segments (approximately 36-47 mm in animals with an average length of approximately 90 mm used in the present study). At 8 weeks posttransection, the numbers of HRP-labeled descending or ascending spinal neurons that extended their axons through the transection were about 40% of those in similar areas of the spinal cord in normal animals. Thus, in larval lamprey, axonal regeneration of descending and ascending spinal projection neurons is incomplete, similar to that found for descending brain neurons. The majority of restored projections were from unidentified spinal neurons that have not been documented previously. In contrast to results from several other lower vertebrates, in the lamprey ascending spinal neurons exhibited substantial axonal regeneration. Identified descending spinal neurons, such as lateral interneurons and crossed contralateral interneurons, and identified ascending spinal neurons, such as giant interneurons and edge cells, regenerated their axons at least 9 mm beyond the transection site in animals with an average length of approximately 90 mm, which is appreciably farther than previously reported. In contrast, most dorsal cells, which are centrally located sensory neurons, exhibited very little axonal regeneration.     

Conditioning lesions enhance axonal regeneration of descending brain neurons in spinal-cord-transected larval lamprey

In larval lamprey, with increasing recovery times after a transection of the rostral spinal cord, there is a gradual recovery of locomotor behavior, and descending brain neurons regenerate their axons for progressively greater distances below the transection site. In the present study, spinal cord "conditioning lesions" (i.e., transections) were performed in the spinal cord at 30% body length (BL; normalized distance from the head) or 50% BL. After various "lesion delay times" (D), a more proximal spinal cord "test lesion" (i.e., transection) was performed at 10% BL, and then, after various recovery times (R), horseradish peroxidase was applied to the spinal cord at 20% BL to determine the extent of axonal regeneration of descending brain neurons. Conditioning lesions at 30% BL, lesion delay times of 2 weeks, and recovery times of 4 weeks (D-R = 2-4 group) resulted in a significant enhancement of axonal regeneration for the total numbers of descending brain neurons as well as neurons in certain brain cell groups compared to control animals without conditioning lesions. Experiments with hemiconditioning lesions, which reduce interanimal variability, confirmed that conditioning lesions do significantly enhance axonal regeneration and indicate that axotomy rather than diffusible factors released at the injury site is primarily involved in this enhancement. Results from the present study suggest that conditioning lesions "prime" descending brain neurons via cell body responses and enhance subsequent axonal regeneration, probably by reducing the initial delay and/or increasing the initial rate of axonal outgrowth.     

Biophysical properties of descending brain neurons in larval lamprey

In the brains of larval lamprey, biophysical properties of reticulospinal (RS) neurons were determined by applying depolarizing and hyperpolarizing current pulses under current clamp conditions. In response to above threshold depolarizing current pulses, almost all RS neurons produced an initial relatively high spiking frequency (F_i) followed by a variable decay to a steady-state firing frequency (F_ss). Spike-frequency adaptation (SFA), defined as [(F_i−F_ss)/F_i]×100%, was minimal at the lowest currents and more pronounced with larger applied current pulses. Some RS neurons, particularly those in the posterior rhombencephalic reticular nucleus (PRRN), either adapted very quickly, and stopped firing, or fired in short bursts during a constant depolarizing current pulse. Several types of RS neurons, including some Muller cells and unidentified neurons in the middle rhombencephalic reticular nucleus (MRRN), displayed delayed excitation (DE) in which spiking in response to a depolarizing current pulse was delayed if preceded by a hyperpolarizing prepulse. Very few neurons fired action potentials following a hyperpolarizing pulse, such as in the case of post-inhibitory rebound (PIR), and no neurons were found that displayed plateau potentials. The possible contributions of these properties to the descending activation of spinal locomotor networks is discussed.     

Regeneration of descending projections in Xenopus laevis tadpole spinal cord demonstrated by retrograde double labeling

Xenopus laevis tadpoles functionally recover from spinal cord transection. Because this recovery requires the tadpole to metamorphose, it may result from compensatory changes initiated by de novo growth of axons involved in limb dominant locomotion rather than from regeneration of cut axons. To determine whether axonal regrowth contributes to functional recovery, sequential retrograde double labeling with two fluorescent dextran amines was used to identify neurons with regenerated axons. Rhodamine dextran amine was applied to hemisected spinal cords of prometamorphic tadpoles between the 4th and 5th vertebrae. After metamorphosis, in animals that had recovered movement, fluorescein dextran amine was applied to the lumbar spinal cord. Two weeks later, the CNS of these animals was examined for the presence of double-labeled neurons, i.e., those whose axons had regenerated. Double-labeled neurons were found in the reticular, raphe, and solitary tract nuclei, and in the interstitial nucleus of the medial longitudinal fasciculus. Because Xenopus expresses all the known mammalian molecular inhibitors of CNS axon regeneration, the determination that these phylogenetically conserved populations of neurons are indeed capable of axon regeneration should facilitate molecular studies of successful recovery from spinal cord trauma.     

Extensive injury of descending neurons demonstrated by retrograde labeling in a virus-induced murine model of chronic inflammatory demyelination

Persistent Theiler's virus infection of SJL/J mice was used as a model to quantitatively assess the extent of descending neuron injury by chronic inflammatory demyelination of the spinal cord. By 9 months postinfection, inflammatory demyelinating lesions were present throughout the spinal cord, affecting up to 31% of the cross-sectional area of the ventrolateral columns. Axon dropout was evident in the lesions by electron microscopy and by quantitation of axons in normal-appearing white matter. Axon number in the ventrolateral columns at L1/L2 was reduced by 23% and total axon area was reduced by 37%, compared with uninfected mice. The most informative data on descending neuron injury, however, was a reduction in retrograde. Fluoro-Gold labeling. Labeling from T11/T12 of rubrospinal, reticulospinal/raphespinal, and vestibulospinal neurons was reduced by 60%, 70%, and 93%, respectively. Retrograde responses to axonal injury were observed, consisting of atrophied cell bodies, indented nuclei, and abundant lipofuscin, but cell body dropout was minimal. The number of cell bodies of vestibulospinal neurons was reduced by only 35%, whereas the number of cell bodies of rubrospinal neurons was unchanged. These results demonstrate that chronic inflammatory demyelination can severely injure axons and emphasize the need to design neuroprotective therapies in human multiple sclerosis.     

Axonal regeneration in lamprey spinal cord

Spinal cords of sea lamprey larvae were transected at one of two levels: (a) rostral, at the last gill, or (b) caudal, at the cloaca. Following various recovery times, regeneration of the posteriorly projecting giant reticulospinal axons (RAs) was demonstrated by intra-axonal injection of horseradish peroxidase (HRP). Regeneration of axons of anteriorly projecting dorsal cells (DCs) and giant interneurons (GIs) was demonstrated by intrasomatic HRP injection into cells located just below the transection scar. After 40 days of recovery, 55% of proximally transected RAs (rostral cut) regenerated at least as far as the center of the scar, whereas only 15% of distally transected RAs (caudal cut) did so. Maximum distance of regeneration was 5.3 mm beyond the scar for proximally transected RAs but only 38 u for distally transected RAs. Proximally transected RAs also branched more profusely than distally transected ones. These data (when combined with others in the literature) suggest that the regenerative capacity of RAs may decrease with distance of axotomy from the cell body. Distance of regeneration and degree of branching of proximally transected RAs peaked between 40 and 100 days. Thereafter, there appeared to be a tendency toward neurite retraction. Of axotomized GIs, 76% regenerated anteriorly at least as far as the center of a caudal transection scar (GIs are located only in the caudal part of the cord). The maximum distance of regeneration was 1.3 mm beyond the scar. Of DC axons, 56% regenerated anteriorly at least as far as the transection site. The maximum distance was 1.1 mm beyond the scar. DCs located just below a caudal transection regenerated at least as well as those located below a rostral transection. Axonal regeneration was also demonstrated for a few lateral cells, edge cells, and crossed caudally projecting interneurons.     

Functional regeneration of descending brainstem command pathways for locomotion demonstrated in the in vitro lamprey CNS

The lamprey brainstem contains a ‘command system’ which descends into the spinal cord to activate motor networks and initiate locomotion. In the present study, partial lesions were made in the rostral spinal cord in order to spare various tracts and determine which tracts carry the descending command signal to the spinal cord. Sparing the medial areas of the rostral spinal cord usually blocked both sensory-evoked and spontaneous locomotion, while sparing the lateral regions of the rostral spinal cord did not abolish voluntary locomotor activity. Either the ventrolateral or dorsolateral spinal tracts could support the initiation of locomotion. Brainstem structures rostral to the mesencephalon were not necessary for the initiation of locomotor behavior. The data indicate that the lateral spinal tracts contain a significant part of the descending command pathway for locomotion. In contrast, the medial spinal tracts were neither necessary nor usually sufficient to support locomotor behavior, suggesting that the larger reticulospinal Muller cells, which project in these tracts, do not contribute significantly to the initiation of locomotion.     

Axonal regeneration in the adult lamprey spinal cord

Larval sea lampreys recover from complete spinal transection by a process involving directionally specific axonal regeneration. In order to determine whether this is also true of adults, 14 adult lampreys were transected at the level of the 5th gill and allowed to recover for 10 weeks. Müller and Mauthner cells and their giant reticulospinal axons (GRAs) were impaled with microelectrodes and injected with horseradish peroxidase (HRP). The tissue was processed for HRP histochemistry and wholemounts of brain and spinal cord were prepared. All animals recovered coordinated swimming; 61 of 121 (50%) neurites emanating from 30 axons regenerated caudal to the scar into the distal stump. Of the neurites which had grown beyond the scar, 92% were correctly oriented, i.e., caudalward and ipsilateral to the parent axon. Retransection in two additional animals eliminated the recovered swimming. Thus, behavioral recovery in adult sea lampreys is accompanied by directionally specific axonal regeneration.     

Octavolateral neurons projecting to the middle and posterior rhombencephalic reticular nuclei of larval lamprey: A retrograde horseradish peroxidase labeling study

The octavolateral area of lampreys, which receives primary fibers from the octaval and lateral line nerves, is involved in the premotor organization of body movements through secondary projections to the reticular formation. Here, the typology of neurons of the three octavolateral nuclei (ventral, medial, and dorsal) that putatively project to the middle and posterior rhombencephalic reticular nuclei were studied by retrograde transport of horseradish peroxidase (HRP) applied to these reticular nuclei. Several types of neurons were labeled in the ventral nucleus, both ipsilateral and contralateral to the site of HRP application. Some of these neurons showed a rather simple morphology (octavomotor neurons, monopolar cells), but most had more- or less-branched dendrites that were associated with one, or several, fields of terminal fibers in the octavolateral area. Unlike those of the ventral nucleus, labeled neurons of the medial nucleus were homogeneous in appearance (mostly pear-shaped). The dorsal nucleus was scarcely developed in larvae, as judged from the very simple and small labeled cells. The presence of terminal or “en-passant” boutons of secondary octavolateral fibers in the reticular area and the commissural nature of these fibers were also investigated by means of application of HRP or indocarbocyanine dye to the octavolateral nuclei. In addition, neurons of other alar plate nuclei that were labeled by the HRP application to the reticular nuclei (trigeminal descending root nucleus and solitary nucleus) were also characterized. The functional significance of these results is discussed. J. Comp. Neurol. 384:396–408, 1997. © 1997 Wiley-Liss, Inc.     

Axonal branching of canine sympathetic postganglionic cardiopulmonary neurons. A retrograde fluorescent labeling study

In 11 dogs fluorescent retrograde tracers were injected into physiologically identified left-sided sympathetic cardiopulmonary nerves. When two different ipsilateral cardiopulmonary nerves were injected, labeled cells from each injected nerve had overlapping distributions in the middle cervical and stellate ganglia. Most retrogradely labeled neurons were located in the middle cervical ganglion and cranial pole of the stellate ganglion. Following the injection of two different tracers into two different nerves, some neurons in the middle cervical ganglion were retrogradely labeled with two tracers. Double-labeled neurons were rarely found in the stellate ganglion. There were areas within the ganglia in which labeled neurons projected predominantly to one cardiopulmonary nerve. In the thoracic autonomic nervous system Fast Blue was transported most effectively. Bisbenzimide was not transported as well as Fast Blue and Nuclear Yellow was very poorly transported in cardiopulmonary nerves. The results demonstrate that some efferent postganglionic sympathetic neurons project axons into at least two different cardiopulmonary nerves and that an anatomical substrate for axo-axonal reflexes exists in the thoracic sympathetic nervous system.     

Combined retrograde labeling and calcium imaging in spinal cord and brainstem neurons of the lamprey

Neurons in the brainstem and spinal cord of the lamprey were retrogradely labeled with Calcium Green-dextran, an indicator dye that increases its fluorescence when intracellular calcium levels increase. Optical signals could be recorded from these labeled neurons during spinal cord stimulation, nerve stimulation, or spontaneous activity, up to 4 days after dye application and for distances of 5–14 mm away from the application site. Optical signals were enhanced by 4-AP, a potassium channel blocker, and blocked by cadmium, a calcium channel blocker. Taken together, the results suggest that the optical signals recorded from labeled neurons were due to calcium influx during electrical activity. Thus, retrograde labeling with calcium indicator dyes may provide a general purpose method for simultaneously monitoring the activity-related changes of intracellular calcium in anatomically identified groups of neurons in the lamprey nervous system.     

Bilaterally diverging axon collaterals and contralateral projections from rat locus coeruleus neurons,demonstrated by fluorescent retrograde double labeling and norepinephrine metabolism

Summary Evans Blue (EB) and a mixture of 4-6-diamidino-2-phenylindol 2 HCl and primuline (DAPI-Pr), fluorescing at different wave-lengths were injected into the rat hippocampus, frontal cortex or lateral part of the thalamus. After unilateral injection either of the two substances was retrogradely transported not only to ipsilateral but also to contralateral locus coeruleus (LC) neurons. Moreover after simultaneous injections of EB and DAPI-Pr respectively into the opposite brain structures of individual animals double-labeled neurons were observed in the bilateral LC.Unilateral electrical stimulation of the LC induced significant decreases of norepinephrine and increases of 3-methoxy-4-hydroxyphenylethyleneglycol in both the ipsi- and contralateral frontal cortex and whole forebrain, respectively. These ipsi- and contralateral alterations of the amine and its metabolite correlated highly significantly.These results indicate that several LC neurons have both contralateral and bilateral projections to the brain areas mentioned above.     

Descending brain neurons in larval lamprey: Spinal projection patterns and initiation of locomotion

In larval lamprey, partial lesions were made in the rostral spinal cord to determine which spinal tracts are important for descending activation of locomotion and to identify descending brain neurons that project in these tracts. In whole animals and in vitro brain/spinal cord preparations, brain-initiated spinal locomotor activity was present when the lateral or intermediate spinal tracts were spared but usually was abolished when the medial tracts were spared. We previously showed that descending brain neurons are located in eleven cell groups, including reticulospinal (RS) neurons in the mesenecephalic reticular nucleus (MRN) as well as the anterior (ARRN), middle (MRRN), and posterior (PRRN) rhombencephalic reticular nuclei. Other descending brain neurons are located in the diencephalic (Di) as well as the anterolateral (ALV), dorsolateral (DLV), and posterolateral (PLV) vagal groups. In the present study, the Mauthner and auxillary Mauthner cells, most neurons in the Di, ALV, DLV, and PLV cell groups, and some neurons in the ARRN and PRRN had crossed descending axons. The majority of neurons projecting in medial spinal tracts included large identified Müller cells and neurons in the Di, MRN, ALV, and DLV. Axons of individual descending brain neurons usually did not switch spinal tracts, have branches in multiple tracts, or cross the midline within the rostral cord. Most neurons that projected in the lateral/intermediate spinal tracts were in the ARRN, MRRN, and PRRN. Thus, output neurons of the locomotor command system are distributed in several reticular nuclei, whose neurons project in relatively wide areas of the cord.     

Medullary neurons with projections to lamina X of the rat as demonstrated by retrograde labeling after HRP microelectrophoresis

Brainstem neurons were retrogradely labeled with microelectrophoresis of HRP or WGA-HRP into lamina X of the cervical or lumbar cord of rats. The results reveal that lamina X of the lumbar cord receives bulbar projections originating mainly within the nucleus raphe magnus and the nucleus reticularis paragigantocellularis (including the medial or alpha-ventral part and lateral part) and that lamina X of the cervical cord receives projections from similar but more extensive regions in the lower brainstem. These findings provide a neuroanatomical substrate for medullary descending modulation of nociceptive transmission in lamina X.     

Axonal branching in the projections from precerebellar nuclei to the lobulus simplex of the rat's cerebellum investigated by retrograde fluorescent double labeling

The projections to lobulus simplex and Crus I of the cerebellum from various brainstem nuclei have been examined in adult rats by using the retrograde fluorescent double labeling technique. True blue was injected into the lobulus simplex on one side and nuclear yellow on the other and the brainstem was examined for labeled neurons. The lateral reticular nucleus, pontine tegmental reticular nucleus, and nucleus praepositus hypoglossi were similar to the equivalent nuclei in other species but all contained double- as well as single-labeled neurons and it was concluded that these nuclei have neurons whose axons branch to both sides of the cerebellum. More neurons in the rostral part of the lateral reticular nucleus were bilaterally projecting than in the caudal and the significance of this in relation to its afferents is considered. The individual neurons in the pontine nuclei, inferior olivary nucleus, and cuneate nuclei only appear to project to one side and the recent evidence for axonal branching of pontine neurons in the cat is discussed.     

Electrophysiologic evidence of regeneration of lamprey spinal neurons

Morphologic evidence has shown that the anteriorly projecting axons of giant interneurons (GIs) can regenerate after spinal transection in larval sea lampreys (19). In the present study, we showed that the regenerating neurites of GIs were electrically excitable. We also showed evidence for regeneration of descending afferent connections to GIs. Spinal cords were transected at the level of the cloaca. After at least 70 days recovery, GIs located 1.5 to 17.0 mm below the scar were impaled with microelectrodes. Stimulating electrodes were placed at various distances above the scar. Six of 13 GIs located 4 to 17 mm below the scar could be activated antidromically. For 1 GI, the rostralmost point of stimulation which elicited these responses was 13.5 mm above the scar. For the others, the range was 0.5 to 4.5 mm. Estimated average conduction velocity in regenerated neurites was 0.50 m/s compared with 1.94 m/s for the parent axon. Twelve GIs could be orthodromically activated by fixed-latency EPSPs. The most rostral point of stimulation that could elicit such responses was 0.5 to 8.5 mm above the scar. There was an inverse relationship between the farthest distance of stimulation and the distance of the GI from the scar. These findings are consistent with the hypothesis that regeneration of axons across a spinal transection is limited to neurons whose cell bodies are situated within 1 to 2 cm from the transection, and that regenerating neurites grow only a few millimeters beyond the scar.     

Collaterals of rubrospinal neurons to the cerebellum in rat. A retrograde fluorescent double labeling study

In a previous study the collateralization of the rubrospinal tract in the spinal cord of rat, cat and monkey was studied by means of the fluorescent retrograde double labeling technique. In the present study the existence of rubrospinal collaterals to the cerebellar interpositus nucleus (NI) has been studied using the same technique. In rat 'True Blue' (TB) was injected in the cerebellar NI and 'Nuclear Yellow' (NY) was injected ipsilaterally in white and gray matter of C5-C8 spinal segments. In some cases a new fluorescent retrograde tracer was used instead of NY, i.e. 'Diamidino Yellow' (DY), which produces retrograde labeling similar to NY but which migrates only very slowly out of the retrogradely labeled neurons. In these experiments only very few single TB-labeled rubrocerebellar neurons occurred, but many (+/- 90%) of the TB-fluorescent rubrocerebellar neurons were TB-NY or TB-DY double-labeled from the spinal cord. At least 37% of the NY and DY-fluorescent rubrospinal neurons were NY-TB and DY-TB double-labeled from the cerebellum. These findings indicate that, in rat, almost all rubrocerebellar fibers represent collaterals of rubrospinal neurons, and that at least 37% of the rubrospinal neurons give rise to such cerebellar collaterals.     

The abducens motor and internuclear neurons in the rabbit: retrograde horseradish peroxidase and double fluorescent labeling

Horseradish peroxidase and the fluorochromes Fast blue and propidium iodide were injected into the lateral rectus and retractor bulbi muscles and/or the oculomotor nucleus of the rabbit to determine the locations and basic morphology of motoneurons and internuclear neurons in the abducens nucleus. The 1000–1100 motoneurons found were distributed throughout the nucleus except in the rostral and caudal tips, but were most densely clustered in the dorsomedial area, especially in the middle third of the nucleus, where 60% of these cells were found. The rostral and caudal tips were composed of internuclear neurons, 25% of which lay in the rostral third of the nucleus, 35% in the middle third and 40% in the caudal third. In the middle third, interneurons occupied the ventral and lateral areas of the nucleus (where they mingled with motoneurons); in the rostral and caudal thirds they were more widely distributed. At the level of the caudal half of the nucleus it was impossible to distinguish clearly between the most lateral abducens interneurons and the most rostromedial labeled vestibular neurons. The abducens interneurons of the rabbit (320–380) thus differ in interesting respects from those described previously in either lateral eyed or frontal eyed mammals.     

Further evidence for excitatory amino acid transmission in lamprey reticulospinal neurons: selective retrograde labeling with (3H)D-aspartate

The distribution of radiolabeled neurons in the brain stem of Lampetra fluviatilis was studied following unilateral injections of (3H)D-aspartate in the rostral spinal cord. After survival periods of 1-3 days, labeled perikarya were present within and nearby the posterior, middle, and anterior rhombencephalic reticular nuclei and in the mesencephalic reticular nucleus. The highest number of (3H)D-aspartate labeled cell bodies were present in the posterior rhombencephalic reticular nucleus. The labeled reticulospinal neurons were distributed mainly ipsilateral to the injection site and included the giant Müller cells as well as medium-sized and small neurons. Contralateral labeling occurred in cell bodies scattered along the lateral margin of the rhombencephalic reticular formation, the most rostral of these contralaterally projecting neurons being the Mauthner cell. The (3H)D-aspartate labeling correlates with previous electrophysiological studies showing that lamprey reticulospinal neurons utilize excitatory amino acid transmission.