Neuronal activity of the external oculomotor nucleus during saccadic eye movement in the waking cat

The activity of antidromically identified abducens nucleus motoneurons and inter-nuclear neurons has been recorded during saccadic eye movements in the alert cat. The activity of these neurons has been demonstrated to be the sum of a velocity component proportional to eye velocity plus a position component proportional to instantaneous eye position during the movement. Results are discussed in relation to proposed models about the generation of saccadic eye movements.     

Computer simulation of the neural discharge carried by the abducens nerve during eye fixation in the cat

The neural signal carried by the abducens nerve during eye fixations was simulated. The neural discharge was defined by the number of spikes carried by the abducens nerve within each ms. Calculations were based on real neurophysiological data. The computed neural signal showed frequency histograms and variance/mean ratios typical of Poisson distribution. A peak was obtained in the power spectral density function of the simulated neural signal. This peak appeared in the frequency range corresponding to the firing rate of single motoneurons for each eye position. It remained as a broad spectral peak after filtering by a second-order differential equation simulating the ocular mechanics. The obtained spectra are similar to the described power spectral density function of eye position recordings. Present results add evidence of a possible neural basis for ocular tremor.     

Mapping and quantitative analysis of gephyrin cytoplasmic trafficking pathways in motoneurons, using an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure

In the present study, an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure was used to map and quantify the pathways involved in the trafficking and subcellular targeting of gephyrin in identified abducens motoneurons. Gephyrin is a scaffolding protein, which plays a crucial role in the clustering of the GABA_A and glycine receptors to the cytoskeleton. TEMCI associated several accurate tools: (i) nanogold immunodetection of gephyrin in motoneurons identified on the basis of their immunoreactivity to Choline Acetyl Transferase, (ii) low magnification color scale coding of gephyrin densities on series of ultrathin sections of motoneurons, which gave a map of the cytoplasmic distribution of the protein, (iii) statistical analysis of the subcellular distribution of the immunolabeling. The color map of gephyrin densities in the cell bodies reflected the distribution of inhibitory synapses over the membrane. The TEMCI analysis of motoneurons with various patterns of synaptic covering made it possible to visualize for the first time the cytoplasmic transport pathway of gephyrin towards its target at synaptic contact. A high magnification quantitative analysis, including the study of 109 inhibitory synapses, showed that most gephyrin-associated immunogold particles (67%) were located in the subsynaptic regions facing the active zones, and the second most densely occupied regions were the perisynaptic regions (19.5% of immunogold particles). A consistent proportion of the gephyrin (11.5%), significantly higher than densities present in the rest of the cytoplasm (2%), was detected in the extrasynaptic submembrane region.     

The dorsomedial nuclear group of cranial nerves in the frog.

The dorsomedial motor nuclei were demonstrated by the cobalt-labeling technique applied to the so-called somatic motor cranial nerves. The motoneurons constituting these nuclei are oval-shaped and smaller than the motoneurons in the ventrolateral motor nuclei. They give rise to ventral and dorsal dendrite groups which have extensive arborization areas. A dorsolateral cell group in the rostral three quarters of the oculomotorius nucleus innervates ipsilateral eye muscles (m.obl.inf., m.rect.inf., m.rect.med.) and a ventromedial cell group innervates the contralateral m. rectus superior. Ipsilateral axons originate from ventral dendrites, contralateral axons emerge from the medial aspect of cell bodies, or from dorsal dendrites, and form a "knee" as they turn around the nucleus on their way to join the ipsilateral axons. A few labeled small cells found dorsal and lateral to the main nucleus in the central gray matter are regarded as representing the nucleus of Edinger-Westphal. The trochlearis nucleus is continuous with the ventromedial cell group of the oculomotorius nucleus. The axons originate in dorsal dendrites, run dorsally along the border of the gray matter and pierce the velum medullare on the contralateral side. A compact dendritic bundle of oculomotorius neurons traverse the nucleus, and side branches appear to be in close apposition to the trochlearis neurons. A dorsomedial and a ventrolateral cell group becomes labeled via the abducens nerve. The former supplies the m. rectus lateralis, while the latter corresponds to the accessorius abducens nucleus which innervates the mm. rectractores. Neurons in this latter nucleus are large and multipolar, resembling the neurons in the ventrolateral motor nuclei. Their axons originate from dorsal dendrites and form a "knee" around the dorsomedial aspect of the abducens nucleus. Cobalt applied to the hypoglossus nerve reaches a dorsomedial cell group (the nucleus proper), spinal motoneurons and sympathetic preganglionic neurons. Of the dorsomedial motor cells, the hypoglossus neurons are the largest, and a branch of their ventral dendrites terminates on the contralateral side. Some functional and developmental biological aspects of the morphological findings, such as the crossing axons and the peculiar morphology of the accessory abducens nucleus, are discussed.     

Postsynaptic potentials in cat abducens motoneurons evoked by stimulation of cortical eye fields.

Intracellular recordings were carried out on abducens motoneurons of encephale isole cats in order to analyse synaptic influences of cortical areas engaged in control of saccadic eye movements. It was found that, in addition to the "frontal eye field" (FEF), eye movements containing a contraversive component may be triggered by electrical stimulation of the 1st and the 2nd sensorimotor areas (SM). Correspondingly, sustained postsynaptic responses (EPSPs) and rhythmic firing of abducens motoneurons could be reliably induced by prolonged stimulus trains. In this respect, the efficiencies of FEF and SM were about the same. They appeared to be higher than the efficiency of excitatory pyramidal actions on spinal motoneurons as reported by others. EPSPs elicited from both regions by short stimuli were, on the major part, polysynaptic. Quite complex multineuronal chains appeared to be stronger engaged in the transmission of FEF effects. EPSPs of SM origin contained a disynaptic fraction which could not be reliably identified in FEF responses. Recipocal innervation of abducens nuclei on both sides was found to be reflected in the asymmetry of excitatory and inhibitory influences from two hemispheres: EPSPs predominated in responses to contralateral, IPSPs and mixed PSPs - to ipsilateral stimulation.     

Anatomic and physiological characteristics of the ferret lateral rectus muscle and abducens nucleus.

The ferret has become a popular model for physiological and neurodevelopmental research in the visual system. We believed it important, therefore, to study extraocular whole muscle as well as single motor unit physiology in the ferret. Using extracellular stimulation, 62 individual motor units in the ferret abducens nucleus were evaluated for their contractile characteristics. Of these motor units, 56 innervated the lateral rectus (LR) muscle alone, while 6 were split between the LR and retractor bulbi (RB) muscle slips. In addition to individual motor units, the whole LR muscle was evaluated for twitch, tetanic peak force, and fatigue. The abducens nucleus motor units showed a twitch contraction time of 15.4 ms, a mean twitch tension of 30.2 mg, and an average fusion frequency of 154 Hz. Single-unit fatigue index averaged 0.634. Whole muscle twitch contraction time was 16.7 ms with a mean twitch tension of 3.32 g. The average fatigue index of whole muscle was 0.408. The abducens nucleus was examined with horseradish peroxidase conjugated with the subunit B of cholera toxin histochemistry and found to contain an average of 183 motoneurons. Samples of LR were found to contain an average of 4,687 fibers, indicating an LR innervation ratio of 25.6:1. Compared with cat and squirrel monkeys, the ferret LR motor units contract more slowly yet more powerfully. The functional visual requirements of the ferret may explain these fundamental differences.     

Development of midbrain and anterior hindbrain ocular motoneurons in normal and Wnt-1 knockout mice

The effect of homozygotic Wnt-1^?/? mutations on the development of ocular motoneurons was examined with the lipophilic dye DiI and compared to control and phenotypic wild-type mouse embryos. A piece of DiI-soaked filter paper was inserted into the orbit, the midbrain, or rhombomere 5 of the hindbrain in six paraformaldehydefixed litters (10.5, 12.5, and 14.5 days postcoitum) containing Wnt-1, Wnt^+/?, and Wnt-1^+/+ individuals and three control litters. We labeled all ocular motoneurons retrogradely and all relevant nerves anterogradely in all control and phenotypic wild-type animals. In all phenotypically identified Wnt-1^?/? mutants we could always label the abducens nerve and motoneurons and the optic fibers to the thalamus, but we were unable to lable oculomotor or trochlear nerves or motoneurons. In addition to Wnt-1 knockout mutants, we also labeled mice from the WZT9B transgenic line carrying a lacZ reporter gene driven by the Wnt-1 gene enhancer. In these embryos we tested for co-localization of Wnt-1 expression in biotinylated dextran amine-labeled ocular motoneurons using a newly developed technique. In younger embryos we obtained evidence for co-localization of the β-galactosidase reaction product derived from lacZ gene activity in some retrogradely filled oculomotor motoneurons and adjacent to other oculomotor and the trochlear motoneurons. Acetylcholine esterase, a marker of early differentiating cholinergic neurons, showed a similar topology with respect to the lacZ reaction product. Thus, at least some future oculomotor motoneurons express Wnt-1, whereas others and the trochlear motoneurons caudal to the ventral midbrain expression of Wnt-1 may be exposed to the short range diffusion of the Wnt-1 gene product. Thus, the Wnt-1^?/? mutation precludes formation or survival of midbrain and anterior hindbrain neurons, including oculomotor and trochlear motoneurons. © 1995 John Wiley & Sons, Inc.     

Effects of the steroid hormone, 20-hydroxyecdysone, on the growth of neurites by identified insect motoneurons in vitro.

During metamorphosis in the hawkmoth, Manduca sexta, identified larval leg motoneurons survive the degeneration of their larval targets to innervate new muscles of the adult legs. The dendrites and axon terminals of these motoneurons regress at the end of the larval stage and then regrow during adult development. Previous studies have implicated the insect steroid, 20-hydroxyecdysone (20-HE), in similar examples of dendritic reorganization during metamorphosis. The present studies were undertaken to test whether 20-HE acts directly on the leg motoneurons to regulate dendritic growth. Larval leg motoneurons were labeled with a fluorescent dye to permit their identification in culture following the dissociation of thoracic ganglia at later stages of development. Leg motoneurons isolated from early pupal stage animals (just before the normal onset of dendritic regrowth) survived in vitro and grew processes regardless of whether 20-HE was added to the culture medium. The extent of process outgrowth, however, as measured by the total length of all processes and the number of branches, was significantly greater for motoneurons maintained in the presence of 20-HE. The enhancement could be blocked by the addition of a juvenile hormone analog. By contrast, larval leg motoneurons that were isolated just before the normal period of dendritic regression did not show enhanced growth of neurites in the presence of 20-HE. The results suggest that 20-HE acts directly on the leg motoneurons to regulate the growth of processes during metamorphosis.     

Comparison of identified leg motoneuron structure and function between larval and adult Manduca sexta

Persistent leg motoneurons of the moth Manduca sexta were investigated in larval and adult animals to compare their dendritic structures, intrinsic electrical properties and pattern of target innervation. The study focused on two identified motoneurons of the prothoracic leg. Despite the complete remodeling of leg muscles, the motoneurons innervated pretarsal flexor muscles in both larval and adult legs. Similarly, although the central dendrites regress and regrow, the branching pattern was similar with the exception of a prominent midline branch that was not present in the adult stage. The intrinsic electrical properties of the motoneurons differed between larval and adult stages. Larval motoneurons had significantly higher membrane input resistances and more depolarized resting membrane potentials than did motoneurons in pharate adults or adults. In all stages, one motoneuron had a low maximal firing frequency, whereas the second motoneuron, which innervated the other half of the muscle, had a high maximum firing frequency. Although the two motoneurons continued to innervate the same halves of the target muscle, their relative effects on muscular contraction were reversed during metamorphosis along with concomitant changes in intrinsic properties. Pretarsal flexor motoneurons in pharate adults (just prior to emergence) displayed properties similar to those in emerged adults. Accepted: 8 January 2000     

Intrinsic properties guide proximal abducens and oculomotor nerve outgrowth in avian embryos

Proper movement of the vertebrate eye requires the formation of precisely patterned axonal connections linking cranial somatic motoneurons, located at defined positions in the ventral midbrain and hindbrain, with extraocular muscles. The aim of this research was to assess the relative contributions of intrinsic, population-specific properties and extrinsic, outgrowth site-specific cues during the early stages of abducens and oculomotor nerve development in avian embryos. This was accomplished by surgically transposing midbrain and caudal hindbrain segments, which had been pre-labeled by electroporation with an EGFP construct. Graft-derived EGFP+ oculomotor axons entering a hindbrain microenvironment often mimicked an abducens initial pathway and coursed cranially. Similarly, some EGFP+ abducens axons entering a midbrain microenvironment mimicked an oculomotor initial pathway and coursed ventrally. Many but not all of these axons subsequently projected to extraocular muscles that they would not normally innervate. Strikingly, EGFP+ axons also took initial paths atypical for their new location. Upon exiting from a hindbrain position, most EGFP+ oculomotor axons actually coursed ventrally and joined host branchiomotor nerves, whose neurons share molecular features with oculomotor neurons. Similarly, upon exiting from a midbrain position, some EGFP+ abducens axons turned caudally, elongated parallel to the brainstem, and contacted the lateral rectus muscle, their originally correct target. These data reveal an interplay between intrinsic properties that are unique to oculomotor and abducens populations and shared ability to recognize and respond to extrinsic directional cues. The former play a prominent role in initial pathway choices, whereas the latter appear more instructive during subsequent directional choices.     

The sizes of motoneurons supplying hindlimb muscles in the mouse

Motoneurons supplying identified muscle groups in the mouse spinal cord were labelled by retrograde transport of horseradish peroxidase. The size of motoneurons was estimated by measuring perimeter and cross-sectional area at the level of the nucleolus for the following seven major muscle groups: quadriceps femoris, adductors and gracilis, gluteal musculature, hamstring muscles, posterior crural musculature, anterolateral crural musculature and intrinsic musculature of the foot. The qualitative observation of two size ranges of motoneuron was supported by the measurements. Frequency distribution histograms of motoneuronal cross sectional area were bimodal for all motoneuronal groups except for the foot musculature. The population parameters and proportions for the six bimodal histograms were estimated by the method of maximum likelihood. It was found that the mean area of the small neuron component, which were presumed to be gamma motoneurons, was similar for the six bimodal systems. In contrast to this the mean area of the large neuron component, presumed to be alpha motoneurons, was found to be different for the six bimodal systems; motoneurons supplying more proximal muscles showed a larger mean area than those supplying distal muscles. The mean area of both components was unaffected by survival time and this was interpreted as indicating that changes in survival time did not label greater numbers of small or large motoneurons. The proportion of motoneurons in the small neuron component was found to vary from 9 to 27%.     

Perineal muscles and motoneurons are sexually monomorphic in the naked mole-rat (Heterocephalus glaber)

Naked mole-rats are eusocial mammals that live in colonies with a single breeding female and one to three breeding males. All other members of the colony, known as subordinates, are nonreproductive and exhibit few sex differences in behavior or genital anatomy. This raises questions about the degree of sexual differentiation in subordinate naked mole-rats. The striated perineal muscles associated with the phallus [the bulbocavernosus (BC), ischiocavernosus (IC), and levator ani (LA) muscles], and their innervating motoneurons, are sexually dimorphic in all rodents examined to date. We therefore asked whether perineal muscles and motoneurons were also sexually dimorphic in subordinate naked mole-rats. Muscles similar to the LA and IC of other rodents were found in naked mole-rats of both sexes. No clear BC muscle was identified, although a large striated muscle associated with the urethra in male and female naked mole-rats may be homologous to the BC of other rodents. There were no sex differences in the volumes of the LA, IC, or the urethral muscles. Motoneurons innervating the perineal muscles were identified by retrograde labeling with cholera-toxin-conjugated horseradish peroxidase. All perineal motoneurons were found in a single cluster in the ventrolateral lateral horn, in a position similar to that of Onuf's nucleus of carnivores and primates. There was no sex difference in the size or number of motoneurons in Onuf's nucleus of naked mole-rats. Thus, unlike findings in any other mammal, neither the perineal muscles nor the perineal motoneurons appear to be sexually differentiated in subordinate naked mole-rats.     

Activity of premotor vestibular neurons in the alert cat

The activity of medial vestibular nucleus neurons projecting to the contralateral abducens nucleus (premotor vestibular neurons) has been recorded during spontaneous and vestibular induced eye movements in the alert cat. Recorded neurons were identified by their antidromic activation from the abducens nucleus and by the post-synaptic field potential induced in this nucleus. The activity of identified medial vestibular neurons increased significantly with horizontal eye position and velocity toward the contralateral side, and decreased abruptly during ipsilateral saccades. The activity of these neurons was also related to head velocity toward the ipsilateral side. The functional role and origin of eye position and velocity signals present in these vestibular neurons are discussed.     

Respecification of larval proleg motoneurons during metamorphosis of the tobacco hornworm, Manduca sexta: segmental dependence and hormonal regulation

The principal locomotory appendages of the Manduca sexta caterpillar, the prolegs, are present on the third through sixth abdominal segments (anal prolegs located on the terminal segment were not included in this study). Previous studies have characterized some of the proleg retractor muscles and their motoneurons. In the present study we identified additional proleg motoneurons and their putative homologs in the non-proleg-bearing segments. One of the motoneurons present in the proleg-bearing segments is absent in the non-proleg-bearing segments. At pupation the prolegs are lost, their muscles degenerate, and some of their motoneurons regress structurally. Subsequently, subsets of the proleg motoneurons and their homologs in other segments die in a segment-specific pattern. This is the first report of segment-specific motoneurons, and of segment-specific death of identified motoneurons, in Manduca. During adult development the surviving proleg motoneurons innervate the tergosternal muscle (TSM) and grow bilateral dendritic arbors. Dendritic growth is completed by about the 12th of the 18 days of adult development. Following adult emergence all but one of the respecified proleg motoneurons dies. The hormonal dependence of dendritic outgrowth was tested by isolating abdomens to eliminate the ecdysteroid-secreting glands in the thorax. Between the second and fifth days after pupation the motoneurons became progressively more competent to undergo dendritic outgrowth following abdomen isolation. The extent of dendritic outgrowth paralleled the degree of morphological development attained by isolated abdomens. It is concluded that ecdysteroids are required for motoneuron outgrowth, but our findings suggest that, unless an abdominal source of ecdysteroids exists in pupae, a relatively small exposure may be sufficient.     

Extensor motor neurons of the crayfish abdomen

Summary The somata of five deep extensor motoneurons of the third abdominal ganglion of the crayfish(Procambarus clarkii) were located and identified. The positions of these somata within the ganglion and their distal distribution to muscles have been mapped and were constant. The soma of the extensor inhibitor was noted to touch the soma of the flexor inhibitor. Three of the excitatory neurons were clustered near their exit route.Sensory and cord routes of activation of the extensor motoneurons were also found and were constant from preparation to preparation. Sub-threshold recording showed that these motoneurons exhibited radically different types of post-synaptic response to stimuli at different sites in the nervous system. No interaction between extensor motoneurons or between the extensor and flexor motoneurons was observed.     

Columnar arrangement of lumbar motoneurons innervating a pair of antagonistically acting leg muscles in the rat

Various problems concerning the physiology of muscular units depend on the exact localization of motoneurons innervating antagonistically acting muscles. The present communication is focussed on the distribution of motoneurons innervating the gastrocnemius (GC) and tibialis anterior (TA) muscles. After injection of horseradish peroxidase (HRP) into these muscles and a survival time ensuring sufficient retrograde transport, the number of motoneurons, their segmental distribution, the mean area covered the labeled cells and the mean diameter of their somata were determined. After injections into the GC-muscle, 129 +/- 6 labeled perikarya were found, and following injections into the TA-muscle, 120 +/- 9 motoneurons were marked with HRP. The motoneurons of both muscles were distributed in spinal cord segments L4-5-6; however, the GC-neurons accumulated in segments L5-6 (approximately 94%) and the TA-neurons in L4-5 (approximately 95%). Although the motoneurons innervating both muscles were located in a rather similar area of the ventral column, i.e. its dorsolateral portion as judged from transverse sections, the GC-motoneurons were situated ventrolaterally to the TA-motoneurons. The measurement of the area of the somata and the mean soma diameter did not reveal any conspicuous differences between both pools of motoneurons. An unimodal distribution pattern of these parameters suggests a broad overlap in the size of alpha-, beta-, and gamma-motoneurons.     

Perineal muscles and motoneurons are sexually monomorphic in the naked mole‐rat (Heterocephalus glaber)

Naked mole‐rats are eusocial mammals that live in colonies with a single breeding female and one to three breeding males. All other members of the colony, known as subordinates, are nonreproductive and exhibit few sex differences in behavior or genital anatomy. This raises questions about the degree of sexual differentiation in subordinate naked mole‐rats. The striated perineal muscles associated with the phallus [the bulbocavernosus (BC), ischiocavernosus (IC), and levator ani (LA) muscles], and their innervating motoneurons, are sexually dimorphic in all rodents examined to date. We therefore asked whether perineal muscles and motoneurons were also sexually dimorphic in subordinate naked mole‐rats. Muscles similar to the LA and IC of other rodents were found in naked mole‐rats of both sexes. No clear BC muscle was identified, although a large striated muscle associated with the urethra in male and female naked mole‐rats may be homologous to the BC of other rodents. There were no sex differences in the volumes of the LA, IC, or the urethral muscles. Motoneurons innervating the perineal muscles were identified by retrograde labeling with cholera‐toxin‐conjugated horseradish peroxidase. All perineal motoneurons were found in a single cluster in the ventrolateral lateral horn, in a position similar to that of Onuf's nucleus of carnivores and primates. There was no sex difference in the size or number of motoneurons in Onuf's nucleus of naked mole‐rats. Thus, unlike findings in any other mammal, neither the perineal muscles nor the perineal motoneurons appear to be sexually differentiated in subordinate naked mole‐rats. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 33–42, 2002     

Sexual differentiation in the CNS of the moth,Manduca sexta. I. Sex and segment-specificity in production,differentiation, and survival of the imaginal midline neurons

We analyzed the development of several sets of postembryonic sex-specific motoneurons in Manduca sexta which belong to a group of homologous lineage of neurons called the imaginal midline neurons (IMNs). Adult female oviduct motoneurons and male sperm duct motoneurons are IMNs that show similar anatomical features and differentiate during metamorphosis, despite appearing in different segments: A7 for oviduct neurons, A9 for sperm duct neurons. These cells are born at the same time and, initially, similar sets are found in A7 and A9 ganglia of larvae of both sexes. The dimorphic adult pattern is generated by sex-specific production and cell death. A7 IMNs differentiate in both sexes through early pupal stages, whereupon they disappear in the male and become the oviduct motoneurons in the female. A9 IMNs are overproduced in the male, and subsequent cell death reduces male cell number and eliminates the small complement of female cells; the surviving male cells develop into the sperm duct motoneurons. Similar IMN arrays are generated in nongenital ganglia, but show non-sex-specific fates. This suggests that both the sex of these cells and their segment of residence play major roles in their subsequent differentiation. 1994 John Wiley & Sons, Inc.     

Changes in the activity of cells in the lateral geniculate nucleus by stimulation of the abducens nucleus area in rabbits

These investigations are aimed at studying the influence of the electrical stimulation of the VIth nucleus (abducens nucleus) on responses of lateral geniculate cells in rabbits. The animals were prepared in the usual fashion for single cell recordings at the lateral geniculate nucleus (LGN). Results show that: Electrical stimulation of the VIth nucleus always produced excitatory discharges whose latency varied from 30 to 400 ms. Interestingly, an electrical pulse applied to the abducens nucleus was capable of enhancing the light-evoked responses without altering the spontaneous rate of firing. It thus seems that the ascending influence of the VIth nucleus manifests itself when it coincides with light responses. Most cells which were sensitive to electrical activation of the abducens nucleus had their receptive field located peripherally (greater than 50 degrees). Histological reconstructions of recording electrode tracts suggest that cells which responded to electrical stimulation were located in a narrow band lying dorsally relative to the LGN. This area can be paralleled with the perigeniculate area observed in other mammals, although not identified in rabbits. It is suggested that these extraretinal impulses which reach the LGN and emerge from an area surrounding the VIth nucleus are associated with corollary discharges.     

Pathfinding by zebrafish motoneurons in the absence of normal pioneer axons.

Individually identified primary motoneurons of the zebrafish embryo pioneer cell-specific peripheral motor nerves. Later, the growth cones of secondary motoneurons extend along pathways pioneered by primary motor axons. To learn whether primary motor axons are required for pathway navigation by secondary motoneurons, we ablated primary motoneurons and examined subsequent pathfinding by the growth cones of secondary motoneurons. We found that ablation of the primary motoneuron that pioneers the ventral nerve delayed ventral nerve formation, but a normal-appearing nerve eventually formed. Therefore, the secondary motoneurons that extend axons in the ventral nerve were able to pioneer that pathway in the absence of the pathway-specific primary motoneuron. In contrast, in the absence of the primary motoneuron that normally pioneers the dorsal nerve, secondary motoneurons did not pioneer a nerve in the normal location, instead they formed dorsal nerves in an atypical position. This difference in the ability of these two groups of motoneurons to pioneer their normal pathways suggests that the guidance rules followed by their growth cones may be very different. Furthermore, the observation that the atypical dorsal nerves formed in a consistent incorrect location suggests that the growth cones of the secondary motoneurons that extend dorsally make hierarchical pathway choices.