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.

Immunohistochemical determination of the sympathetic pathway in the orbit via the cranial nerves in humans.

OBJECT: The present study was undertaken to elucidate the extent and precise distribution of the postganglionic sympathetic fibers in the cranial nerves projecting to the orbit and to reconstruct sympathetic routes in the orbit in humans. For this purpose, the authors made an immunohistochemical determination of the sympathetic fibers by using an antibody against norepinephrine-synthetic enzyme, tyrosine hydroxylase (TH). METHODS: Specimens containing the orbit and the cavernous sinus were obtained from formalin-fixed human cadavers. First, it was confirmed that the superior cervical ganglion contained strongly immunostained TH-positive neuronal cell bodies and fibers. After careful dissection of the cranial nerves projecting to the orbit, different segments of each cranial nerve were processed for immunohistochemical analysis for TH. All of the intraorbital cranial nerves contained TH-positive sympathetic fibers, although the amounts were very different in each cranial nerve. At the proximal site of the common tendinous ring, TH-positive fibers were found mainly in the abducent and trochlear nerves. At the distal site of this ring, TH-positive fibers were lost or markedly reduced in number in the abducent and trochlear nerves and were distributed mostly in the ophthalmic and oculomotor nerves. Among the cranial nerves projecting to the orbit, the ophthalmic nerve and its bifurcated nerves--frontal, lacrimal, and nasociliary--contained numerous TH-positive fibers. CONCLUSIONS: The authors conclude that the postganglionic sympathetic fibers are distributed to all cranial nerves projecting to the orbit and that the ophthalmic nerve provides a major sympathetic route in the orbital cavity in humans.

Organization of choline acetyltransferase-containing structures in the cranial nerve motor nuclei and spinal cord of the monkey.

Cholinergic structures in the cranial nerve motor nuclei and ventral and lateral horns of the spinal cord of the monkey, Macaca fuscata, were investigated immunohistochemically with a monoclonal antibody against monkey choline acetyltransferase (ChAT). ChAT-immunoreactive perikarya and dendrites were present in the oculomotor, trochlear, abducent, trigeminal motor, facial and hypoglossal nuclei, nucleus of Edinger-Westphal, nucleus ambiguus, dorsal nucleus of the vagus, lamina IX of the cervical, thoracic and lumbar spinal cords, and intermediolateral nucleus of the thoracic spinal cord. The neuropil of the trigeminal motor, facial and hypoglossal nuclei, nucleus ambiguus and lamina IX of the cervical, thoracic and lumbar spinal cords contained many ChAT-positive bouton-like structures and they were seemingly in contact with perikarya and dendrites of motoneurons, suggesting that motoneurons in these nuclei are cholinoceptive as well as cholinergic. The oculomotor, trochlear and abducent nuclei, nucleus of Edinger-Westphal, dorsal nucleus of the vagus and intermediolateral nucleus of the thoracic spinal cord contained a small number of ChAT-immunoreactive bouton-like structures, but they did not contact with perikarya and dendrites of ChAT-positive neurons. These observations suggest that the organization of the motor nuclei is complex, at least regarding the cholinoceptivity.

Histochemical localization of acetylcholinesterase in relation to motor neurons and internuclear neurons of the cat abducens nucleus.

Three distinct patterns of AChE localization have been observed in relation to cat abducens motor neurons and internuclear neurons labelled by retrograde transport of horseradish peroxidase. First, AChE was localized predominantly within cisternae of granular endoplasmic reticulum and agranular reticulum of motor neuron somata, dendrites and axons, but was absent from internuclear neurons. AChE was also associated with saccules of the Golgi apparatus in the motor neurons, but was was absent from all other cytoplasmic organelles. Second, AChE was observed on the soma-dendritic and axonal surface membrane of the motor neurons, particularly at sites of apposition of synaptic endings of all morphological types, but was usually absent from the surface membranes of internuclear neurons. Third, AChE was associated both extracellularly and intracellularly with certain synaptic endings that contained spheroidal synaptic vesicles and that contacted both motor neurons and internuclear neurons. A similar pattern of staining of synaptic endings was observed at the neuromuscular junctions in the lateral rectus muscle. Axotomy of the VIth nerve resulted in loss of intracellular AChE associated with the Golgi apparatus and extracellular AChE on the somatic surface membrane of the motor neurons. The patterned localization of AChE contrasted with the localization of butyrylcholinesterase, which was associated predominantly with astrocytes. The findings suggest different roles of AChE as a function of the different patterns of localization.