Development of the human abducens nucleus: a morphometric study.

BACKGROUND: The abducens nucleus directly innervates the lateral rectus muscle and plays a role in controlling conjugate horizontal eye movements. Although the neuronal cytoarchitecture of the abducens nucleus has been extensively investigated in various species of vertebrates, few studies have been undertaken in humans, especially in fetuses or neonates. DESIGN/SUBJECTS: We examined 12 human brains from preterm infants aged 20-43 postmenstrual weeks to document the histology and morphometry of the abducens nucleus. The brain was processed into celloidin-embedded serial sections stained with the Klüver-Barrera and other conventional methods. RESULTS: The nucleus was identified as a mass of cells as early as 20 weeks. Its neurons were clearly distinguished from glial cells due to droplet-like, clear nuclei containing prominent nucleoli and surrounded by a basophilic perikaryon. Neurons of various sizes and shapes were intermingled within the nucleus, although larger neurons were located towards the center of the nucleus. Immature granular or reticular Nissl bodies were seen at 20-21 weeks. Tigroid, coarse Nissl bodies appeared around 28-29 weeks in larger neurons, although in smaller neurons Nissl bodies were dispersed or concentrated peripherally. Morphometric results were: (1) the nuclear volume exponentially increased with age between 20 and 43 weeks; (2) the histograms of neuronal profile areas showed a non-normal distribution trailing toward the right and widening with age; (3) the geometric average of neuronal profile areas increased linearly with age. CONCLUSION: Our study suggests that the human abducens nucleus enlarges more quickly toward the end of gestation, and comprises heterogeneous groups of neurons.

Carboxypeptidase A6 in zebrafish development and implications for VIth cranial nerve pathfinding.

Carboxypeptidase A6 (CPA6) is an extracellular protease that cleaves carboxy-terminal hydrophobic amino acids and has been implicated in the defective innervation of the lateral rectus muscle by the VIth cranial nerve in Duane syndrome. In order to investigate the role of CPA6 in development, in particular its potential role in axon guidance, the zebrafish ortholog was identified and cloned. Zebrafish CPA6 was secreted and interacted with the extracellular matrix where it had a neutral pH optimum and specificity for C-terminal hydrophobic amino acids. Transient mRNA expression was found in newly formed somites, pectoral fin buds, the stomodeum and a conspicuous condensation posterior to the eye. Markers showed this tissue was not myogenic in nature. Rather, the CPA6 localization overlapped with a chondrogenic site which subsequently forms the walls of a myodome surrounding the lateral rectus muscle. No other zebrafish CPA gene exhibited a similar expression profile. Morpholino-mediated knockdown of CPA6 combined with retrograde labeling and horizontal eye movement analyses demonstrated that deficiency of CPA6 alone did not affect either VIth nerve development or function in the zebrafish. We suggest that mutations in other genes and/or enhancer elements, together with defective CPA6 expression, may be required for altered VIth nerve pathfinding. If mutations in CPA6 contribute to Duane syndrome, our results also suggest that Duane syndrome can be a chondrogenic rather than a myogenic or neurogenic developmental disorder.

Abducens internuclear neurons depend on their target motoneurons for survival during early postnatal development.

The highly specific projection of abducens internuclear neurons onto medial rectus motoneurons in the oculomotor nucleus is a good model to evaluate the dependence on target cells for survival during development and in the adult. Thus, the procedure we chose to selectively deprive abducens internuclear neurons of their natural target was the enucleation of postnatal day 1 rats to induce the death of medial rectus motoneurons. Two months later, we evaluated both the extent of reduction in target size, by immunocytochemistry against choline acetyltransferase (ChAT) and Nissl counting, and the percentage of abducens internuclear neurons surviving target loss, by calretinin immunostaining and horseradish peroxidase (HRP) retrograde tracing. Firstly, axotomized oculomotor motoneurons died in a high percentage ( approximately 80%) as visualized 2 months after lesion. In addition, we showed a transient (1 month) and reversible down-regulation of ChAT expression in extraocular motoneurons induced by injury. Secondly, 2 months after enucleation, 61.6% and 60.5% of the population of abducens internuclear neurons appeared stained by retrograde tracing and calretinin immunoreaction, respectively, indicating a significant extent of cell death after target loss (38.4% or 39.5%). By contrast, in the adult rat, neither extraocular motoneurons died in response to axotomy nor abducens internuclear neurons died due to the loss of their target motoneurons induced by the retrograde transport of toxic ricin injected in the medial rectus muscle. These results indicate that, during development, abducens internuclear neurons depend on their target motoneurons for survival, and that they lose this dependence with maturation.

Abducens length and vulnerability?

BACKGROUND: Several sources have attributed the vulnerability of the abducens nerve to its long intracranial course. However, other anatomic factors likely contribute to the apparent vulnerability of the abducens nerve to mass lesions and trauma. METHODS: The authors performed a two-part anatomic study of the abducens nerve. In the first part of the study, they compared the length of the abducens with another cranial nerve, the trochlear, at the autopsy of 26 pediatric patients. In the second part of the study, the authors used an endoscopic exposure of these two cranial nerves in a preserved human cadaver head. RESULTS: The abducens nerve was consistently approximately one-third the length of the trochlear nerve at all ages that they studied. The endoscopic views revealed the structural and vascular relationships of the abducens nerve in situ. CONCLUSIONS: The authors conclude from these findings and the literature that abducens nerve vulnerability results from factors other than its intracranial length.

A-, T-, and H-type currents shape intrinsic firing of developing rat abducens motoneurons.

During postnatal development, profound changes take place in the excitability of nerve cells, including modification in the distribution and properties of receptor-operated channels and changes in the density and nature of voltage-gated channels. We studied here the firing properties of abducens motoneurons (aMns) in transverse brainstem slices from postnatal day (P) 1-13 rats. Recordings were made from aMNs in the whole-cell configuration of the patch-clamp technique. Two main types of aMn could be distinguished according to their firing profile during prolonged depolarizations. Both types were identified as aMns by their fluorescence following retrograde labelling with the lipophilic carbocyanine DiI in the rectus lateralis muscle. The first type (BaMns) exhibited a burst of action potentials (APs) followed by an adaptation of discharge and were encountered in approximately 70 % of aMns. Their discharge profile resembled that of adult aMns and was encountered in all aMns after P9. BaMns exhibited a hyperpolarization-induced rebound potential that was blocked by low concentrations of Ni2+ or by Ca2+-free external solution. This current had the properties of the T-type current. Action potentials of BaMns showed a complex afterhyperpolarization (AHP). An inward rectification was evidenced following hyperpolarization and was blocked by external application of caesium or ZD7288, indicating the presence of the hyperpolarization-activated cationic current (IH). Blocking the IH current almost doubled the input resistance of BaMns. The second class of aMns (DaMns) displayed a delayed excitation that was mediated by A-type K+ currents and was observed only between P4 and P9. DaMns exhibited immature characteristics: an action potential with a simple AHP, a linear current-voltage relation and a large input resistance. The number of aMns remained unchanged when both types were present (P5-P6) and later in development when only BaMns were encountered (P19), suggesting that DaMns mature into BaMns during postnatal development. We conclude that aMns display profound reorganization in their intrinsic excitability during postnatal development.

Intrinsic determinants of synaptic phenotype: an experimental study of abducens internuclear neurons connecting with anomalous targets.

The present experiments investigate the role of postsynaptic neurons in the morphological differentiation of presynaptic terminals that are formed de novo in the adult CNS. Abducens internuclear neurons in the adult cat were chosen as the experimental model. These neurons project onto the contralateral medial rectus motoneurons of the oculomotor nucleus. Abducens internuclear axon terminals were identified by their anterograde labeling with biocytin and analyzed at the electron microscopic level. To promote the formation of new synapses, two different experimental approaches were used. First, after the selective ablation of medial rectus motoneurons with ricin, abducens internuclear neurons reinnervated the neighboring oculomotor internuclear neurons. Second, after axotomy followed by embryonic cerebellar grafting, abducens internuclear axons invaded the implanted tissue and established synaptic connections in both the molecular and granule cell layer. Boutons contacting the oculomotor internuclear neurons developed ultrastructural characteristics that resembled the control synapses on medial rectus motoneurons. In the grafted cerebellar tissue, abducens internuclear axons and terminals did not resemble climbing or mossy fibers but showed similarities with control boutons. However, labeled boutons analyzed in the granule cell layer established a higher number of synaptic contacts than controls. This could reflect a trend towards the mossy fiber phenotype, although labeled boutons significantly differed in every measured parameter with the mossy fiber rosettes found in the graft. We conclude that at least for the abducens internuclear neurons, the ultrastructural differentiation of axon terminals reinnervating novel targets in the adult brain seems to be mainly under intrinsic control, with little influence by postsynaptic cells.

Development and organization of the ocular motor nuclei in the larval sea lamprey, Petromyzon marinus L.: an HRP study.

Retrograde transport of horseradish peroxidase (HRP) after its application into the orbit was used to investigate the development of the different ocular motor nuclei in larvae of the sea lamprey (Petromyzon marinus) and to identify their regions of origin. In the smallest larvae studied (10-19 mm in length), the oculomotor and abducens neurons were ipsilateral to the site of HRP application, whilst trochlear neurons were contralateral. These motoneurons did not have dendritic processes. In larvae more than 19 mm in length, both ipsilateral and contralateral components were found in the oculomotor and trochlear nuclei; dendrites were present, and their length and branching increased with larval age. An adult-like pattern of topographic organization and dendritic arborization was reached in larvae of about 45-60 mm in length. In oculomotor neurons, medial dendrites appear first, then dorsolateral dendrites, and finally ventral dendrites. Similarly, in trochlear neurons ventral and ventrolateral dendrites develop first, followed by dorsal dendrites that course either to the caudal optic tectum or to the terminal fields of the octaval and lateral line nerves in the cerebellar plate. Dorsal and ventral dendrites of the abducens neurons arise at the same time, but dorsal dendrites attain an adult-like morphology earlier. A few motoneurons showed ventricular attachments in larvae longer than 40 mm. The significance of these processes and their possible usefulness as a marker for the regions of origin of the ocular motor nuclei are discussed. Finally, the results presented here indicate that differentiation of the ocular motor nuclei in larval lampreys precedes and is independent of the maturation of the eye at transformation.

Development of the abducens nuclei in the Xenopus laevis.

The development of the main (nVI) and the accessory abducens (nVIa) nuclei was studied with the horseradish peroxidase and cobaltic-lysine labeling techniques in Xenopus laevis tadpoles. In earliest labeling was obtained at stage 39, and neuroblasts of both nuclei formed two separate groups according to their definitive positions in relation to other rhombencephalic structures in this young age of development. Conspicuous morphological differences were observed between the two nuclei: the accessory abducens neuroblasts were twice as big as the abducens neuroblasts and the characteristic nVIa 'knee' was present from this time of the first successful labeling. The two different dendritic arborization patterns, which clearly distinguished the abducens neurons from the accessory abducens neurons, gradually developed in tadpoles. It is suggested that the form and position of abducens and accessory abducens neurons are determined at a prefunctional stage, probably before the beginning of axonal outgrowth, and neurobiotaxis may not play the role attributed previously in the differentiation of these two nuclei.

Development of myelinated nerve fibers in the sixth cranial nerve of the rat: a quantitative electron microscope study.

Myelination was studied quantitatively in the sixth cranial nerves of rats by counting and measuring all myelinated fibers during the first three postnatal weeks. In transverse semithin and thin sections cut serially at a well-defined anatomical site in the midsphenoid region, only a few axons (mean 12) were myelinated at birth. On days 2, 4, and 8, counts of myelinated fibers were respectively 5 times (mean 57), 20 times (mean 230), and 24 times (mean 273) the number seen at birth. During the second postnatal week, the number of myelinated fibers remained constant, whereas growth of axons and their myelin sheaths continued. By 15 days these fibers were large and relatively uniform in size; they had compact, circular myelin sheaths. During the third postnatal week, myelination of previously unmyelinated, smaller axons began. The number of myelinated fibers increased again and the size distribution of myelinated fibers became bimodal. Axon diameters, fiber diameters, and myelin sheath dimensions for all fibers were calculated from measurements made on electron micrographs. The transverse length of the myelin membrane increased exponentially with time. The growth increased rapidly during the formation of the first 20 spiral layers and remained relatively constant during the subsequent enlargement of the compact sheath. The association of axon diameter and myelin sheath thickness was poor at young ages, but it improved progressively with maturation of the sheath. The results show that myelination begins around axons that have a wide range of diameters. Also, the first axons to be myelinated become the large myelinated fibers of the sixth nerve. The small myelinated fibers originate from axons that do not become myelinated until the third postnatal week. Myelination, though differing in onset by 2 weeks, appeared to be similar in both populations as judged by similarity of sheath morphology and growth rates. It is of interest that at the level studied, the sixth nerve also contains a fascicle of unmyelinated cranial sympathetic fibers.

P2, P1, and P0 myelin protein expression in developing rat sixth nerve: a quantitative immunocytochemical study.

Myelination and the expression of myelin proteins P2, P1, and P0 were studied quantitatively in the rat sixth cranial nerve during development. The postnatal development and growth of all myelin sheaths in this nerve have been studied morphometrically in a companion paper. Epon-embedded blocks with closely matched topography in the transverse plane were selected from rats perfused at ages 1-4, 8, 15, and 20 days. From each block, serial semithin sections were cut, etched, and immunostained according to the peroxidase-antiperoxidase method with well-characterized polyclonal antisera that reacted specifically with P0 glycoprotein and the basic proteins P1 and P2. The immunoreactivities of individual myelin sheaths were measured by densitometry. Numbers of compact myelin lamellae, myelin spiral lengths, and axon diameters were determined on electronmicrographs of adjacent thin sections. At birth anti-P0 immunoreactivity was found on sheaths with two and more compact lamellae; neither P1 nor P2 immunoreactivity was observed. On day 2, myelin sheaths with five and eight lamellae were stained respectively by anti-P1 and anti-P2. On day 3 the percentages of myelin sheaths stained were substantially higher: P0 95%, P1 78%, P2 15%. By day 4, anti-P0 and anti-P1 immunoreactivity was present in 95% of myelin sheaths; 35% were stained by anti-P2. For P2, staining intensity and percentage of myelin sheaths stained continued to increase and by day 20, 85% were anti-P2-positive. The density of immunoreactivity was not uniform in all myelin sheaths. At young ages staining varied with all three proteins. The variability decreased as myelin sheaths thickened; it persisted longest for anti-P2. We conclude that the density and distribution of immunoreactivities of P0, P1, and P2 reflect their relative concentrations during myelin sheath development and growth. We attribute lack of detectable anti-P2 immunoreactivity in some small sheaths at 20 days to their early stage of myelination and also to limitations of the method. We infer from our observations that all myelin-forming Schwann cells express P2 basic protein.

The vestibulo-ocular reflex arc in the newborn kitten. An electrophysiologic investigation.

Field potentials and postsynaptic potentials were recorded in the vestibular and abducens nuclei and neurons following vestibular nerve stimulation in anesthetized newborn kittens (within 72 h after birth). Stimulation of the ipsilateral vestibular nerve evoked an initial P wave and an N1 field potential in the vestibular nuclei. No N2 potential was evoked. Latencies of the peak of the P wave, the onset and the peak of the N1 potential were 0.99 +/- 0.16 ms, 1.66 +/- 0.18 ms, and 2.51 +/- 0.23 ms, respectively. Ipsilateral vestibular nerve stimulation evoked monosynaptic excitatory postsynaptic potentials (EPSPs) and polysynaptic inhibitory postsynaptic potentials (IPSPs) in vestibular nuclear neurons. Stimulation of the contralateral vestibular nerve evoked polysynaptic IPSPs in vestibular nuclear neurons. In abducens motoneurons, ipsilateral vestibular nerve stimulation evoked monosynaptic EPSPs and disynaptic IPSPs; contralateral vestibular nerve stimulation produced disynaptic EPSPs. We conclude that short circuit pathways of the vestibulo-vestibular and vestibulo-ocular reflex arc are present in the kitten already at birth.

Morphometric analysis of the effects of exenteration and enucleation on the development of third and sixth cranial nerves in the rat.

The effects on rat cranial nerve growth of removing various amounts of extraocular muscle was studied using morphometric techniques. Growth in the third cranial nerves was found to be severely retarded when most of the muscle tissue was removed. By contrast, removal of the eye alone, leaving extraocular muscles relatively intact, was found to have little or no effect on the subsequent growth of third and sixth cranial nerve fibers and of extraocular muscle fibers. This conclusion could be drawn only through the application of statistical methods which take into account several generally unrecognized facts: frequency distributions of axon circumference and myelin sheath thickness are highly variable from nerve to nerve even in normal rats, which often have more large fibers in left than in right nerves. The bimodal nature of peripheral nerve fiber distributions precludes the use of such parametric tests as the commonly and inappropriately used t-test, but a non-parametric test such as the Kolmogorov-Smirnov test, extensively used in these studies, is inadequate for data comprising several sets of distributions to be compared. The application of the analysis of variance to some of the data and the merits of the procedure are discussed.