In Vivo and Ex Vivo Comprehensive Evaluation of Corneal Reinnervation in Eyes Neurotized With Contralateral Supratrochlear and Supraorbital Nerves.

PURPOSE: To use an automated morphometric analysis system of in vivo confocal microscopy (IVCM) images for evaluating reinnervation occurring at the subbasal nerve plexus (SNP) after direct corneal neurotization (DCN) and to further report neurophysiological and histopathological findings. METHODS: Prospective interventional case series including 3 eyes with neurotrophic keratitis that underwent DCN. Deep anterior lamellar keratoplasty was performed 18 months after DCN in patient 1. The following evaluations were performed before and at 3, 6, and 12 months after DCN: clinical evolution of keratitis; corneal sensitivity; IVCM images of the SNP analyzed with "ACCMetrics;" neurophysiological study of corneal reflex. Protein gene product 9.5 immunofluorescence staining assay and transmission electron microscopy were conducted on the neurotized button excised during deep anterior lamellar keratoplasty. RESULTS: Complete healing was obtained in all patients by 3 months postoperatively. Corneal sensitivity was absent preoperatively in all eyes and improved after surgery, reaching an average value of 30 mm 1 year postoperatively. The corneal SNP was not visible at IVCM in any of the cases preoperatively and became visible by 3 months postoperatively, showing IVCM metrics comparable to normal contralateral eyes at 1 year. In all cases, neurophysiological evaluation showed a partial recovery of the electrical activity of the cornea. In patient 1, protein gene product (PGP) 9.5 staining of neurotized cornea showed nerve fascicles at the SNP, whereas transmission electron microscopy showed amyelinic nerve axons and nerve endings. CONCLUSIONS: The corneal SNP exhibited IVCM metrics comparable to the normal contralateral eye 1 year after DCN. Ex vivo histopathological assessment of neurotized corneas confirmed the presence of nerves with normal ultrastructure.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice.

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Motor axon guidance of the mammalian trochlear and phrenic nerves: dependence on the netrin receptor Unc5c and modifier loci.

Netrin signaling is important to guide migrating neurons and axons in many systems. Experiments with vertebrate CNS explants suggested netrin is bifunctional, attracting some axons and repelling others. Netrin1-expressing cells attracted spinal commissural axons and repelled trochlear cranial nerve axons in these experiments. Subsequent genetic studies demonstrated that multiple axon types, including those of the spinal commissural neurons, are attracted to netrin in vivo; however, an in vivo role for netrin signaling in trochlear nerve repulsion has not been observed. Here, we demonstrate that mice with a null mutation in the netrin receptor Unc5c on the inbred C57BL/6J (B6) genetic background have ventral/ipsilateral trochlear nerve misprojections. These misprojections are attenuated on a hybrid B6 x SJL background. In addition, B6.Unc5c(-/-) mice die as neonates of apparent respiratory distress and have incomplete phrenic nerve innervation of the diaphragm muscle. Neither the trochlear nerve misprojections nor the phrenic nerve phenotype was observed in B6 embryos lacking the netrin receptors DCC or Neogenin1, or the ligand netrin1, indicating these signaling molecules are dispensable for guidance of these axons. Like the trochlear nerve, the phrenic nerve phenotype is modified in a B6 x SJL hybrid background. To identify these modifier loci, we performed genome scans of the hybrid Unc5c(-/-) mice and found a major SJL-derived suppressor locus on Chromosome 17. Our results provide the first evidence that genes involved in netrin signaling are necessary for proper mammalian spinal motor axon development and trochlear axon guidance. In addition, they demonstrate the importance of modifier genes in vertebrate axonal guidance.

Reciprocal gene replacements reveal unique functions for Phox2 genes during neural differentiation.

The paralogous paired-like homeobox genes Phox2a and Phox2b are involved in the development of specific neural subtypes in the central and peripheral nervous systems. The different phenotypes of Phox2 knockout mutants, together with their asynchronous onset of expression, prompted us to generate two knock-in mutant mice, in which Phox2a is replaced by the Phox2b coding sequence, and vice versa. Our results indicate that Phox2a and Phox2b are not functionally equivalent, as only Phox2b can fulfill the role of Phox2a in the structures that depend on both genes. Furthermore, we demonstrate unique roles of Phox2 genes in the differentiation of specific motor neurons. Whereas the oculomotor and the trochlear neurons require Phox2a for their proper development, the migration of the facial branchiomotor neurons depends on Phox2b. Therefore, our analysis strongly indicates that biochemical differences between the proteins rather than temporal regulation of their expression account for the specific function of each paralogue.

Monorail/Foxa2 regulates floorplate differentiation and specification of oligodendrocytes, serotonergic raphé neurones and cranial motoneurones.

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphenucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

Development of oculomotor axon projections in the chick embryo.

The pattern of innervation of the extraocular muscles is highly conserved across higher vertebrate species and mediates sophisticated visuomotor processes. Defects in oculomotor development often lead to strabismus, a misalignment of the eyes that can cause partial blindness. Although it has been intensively studied from a clinical perspective, relatively little is known about how the system develops embryonically. We have therefore mapped the development of the oculomotor nerve (OMN) in chick embryos by using confocal microscopy. We show that OMN development follows a series of stereotyped steps that are tightly regulated in space and time. The OMN initially grows past three of its targets to innervate its distal target, the ventral oblique muscle, only later forming branches to the more proximal muscles. We have also investigated spatiotemporal aspects of the unusual contralateral migration of a subpopulation of oculomotor neurons by using molecular markers and have found the semaphorin axon guidance molecules and their receptors, the neuropilins, to be expressed in discrete subnuclei during this migration. Finally, we have created an embryological model of Duane retraction syndrome (DRS), a form of strabismus in which the OMN is believed to innervate aberrantly the lateral rectus, the normal target of the abducens nerve. By ablating rhombomeres 5 and 6 and hence the abducens, we have mimicked a proposed oculomotor deficit occurring in DRS. We find that the absence of the abducens nerve is not sufficient to produce this inappropriate innervation, so other factors are required to explain DRS.

Navigation of trochlear motor axons along the midbrain-hindbrain boundary by neuropilin 2.

Trochlear motor axons project dorsally along the midbrain-hindbrain boundary (MHB) to decussate at the dorsal midline. We report on the roles of neuropilin 2 and its ligands in the molecular mechanisms controlling this trajectory. In chick embryos, neuropilin 2 was expressed in the neuroepithelium of the dorsal isthmus in addition to the trochlear neurons, and Sema3F transcripts were localized along the caudal margin of the midbrain. Misexpression of Sema3F demonstrated that Sema3F displays repulsive activity in vivo that guides the trochlear motor axons along the MHB. An unexpected result was that misexpression of neuropilin 2 canceled the midbrain-evoked repulsion, allowing trochlear motor axons to cross the MHB and invade the tectum. A binding assay with neuropilin 2 ectodomain revealed the existence of neuropilin 2 ligands in the midbrain, which were masked by ectopic neuropilin 2. We therefore propose that neuropilin 2 neutralizes the repulsive activity in order to steer trochlear motor axons towards the dorsal decussation point. Taken together, our results suggest that the interaction of neuropilin 2 with its ligands has crucial roles for establishing trochlear trajectory along the MHB.

Differential expression of Group I metabotropic glutamate receptors in motoneurons at low and high risk for degeneration in ALS.

Glutamate excitotoxicity has been suggested to play a role in amyotrophic lateral sclerosis (ALS), yet it remains unclear why some groups of motoneurons (MNs) are more vulnerable to degeneration than others. Our aim was to compare, in normal adult rats, the expression of Group I metabotropic glutamate receptors (mGluR1 and mGluR5) in MNs normally affected in ALS (XII and spinal MNs) with those which are spared (III and IV MNs). RT-PCR analysis of tissue punches taken from III and XII motor nuclei revealed mRNA for both 'a' and 'b' splice variants of the mGluR1 and mGluR5 receptor subtypes, with expression of the 'a' variant dominant for both receptor subtypes in III and XII nuclei. Immunolabeling for mGluR1a protein was strong in vulnerable (XII and spinal) but negligible in the resistant (III and IV) MNs. Immunoreactivity for mGluR5 was not detected in the cell bodies or proximal dendrites of any MN pool examined. Greater expression of mGluR1a receptor protein within vulnerable MN pools may predispose these neurons to neurodegeneration as seen in ALS.

Morphological identification of nitric oxide sources and targets in the cat oculomotor system.

Nitric oxide (NO) production by specific neurons in the prepositus hypoglossi (PH) nucleus is necessary for the correct performance of eye movements in alert cats. In an attempt to characterize the morphological substrate of this NO function, the distribution of nitrergic neurons and NO-responding neurons has been investigated in different brainstem structures related to eye movements. Nitrergic neurons were stained by either immunohistochemistry for NO synthase I or histochemistry for reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. The NO targets were identified by cyclic guanosine monophosphate (cGMP) immunohistochemistry in animals treated with a NO donor immediately before fixation of the brain. Connectivity between cells of the NO-cGMP pathway was analyzed by injections of the retrograde tracers horseradish peroxidase or fast blue in different structures. The motor nuclei commanding extraocular muscles did not contain elements of the NO-cGMP pathway, except for some scattered nitrergic neurons in the most caudal part of the abducens nucleus. The PH nucleus contained the largest number of nitrergic cell bodies and a rich neuropil, distributed in two groups in medial and lateral positions in the caudal part, and one central group in the rostral part of the nucleus. An abundant cGMP positive neuropil was the only NO-sensitive element in the PH nucleus, where no cGMP-producing neuronal cell bodies were observed. The opposite disposition was found in the marginal zone between the PH and the medial vestibular nuclei, with a large number of NO-sensitive cGMP-producing neurons and almost no nitrergic cells. Both nitrergic and NO-sensitive cell bodies were found in the medial and inferior vestibular nuclei and in the superior colliculus, whereas the lateral geniculate nucleus contained nitrergic neuropil and a large number of NO-sensitive cell bodies. Some of the cGMP-positive neurons in the marginal zone and medial vestibular nucleus projected to the PH nucleus, predominantly to the ipsilateral side. These morphological findings may help to explain the mechanism of action of NO in the oculomotor system.

Differential regulation of alpha- and beta-CGRP mRNAs within oculomotor, trochlear, abducens, and trigeminal motoneurons in response to axotomy.

Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague-Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35S-labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons.

Altered expression of microtubule-associated proteins in cat trochlear motoneurons after peripheral and central lesions of the trochlear nerve.

Neurons lesioned in the peripheral nervous system (PNS) generally regenerate and survive, while neurons lesioned in the central nervous system (CNS) do not regenerate and often die. Investigators have traditionally compared the neuronal responses to PNS and CNS lesions in two separate populations of neurons. In this study, we compared the effects of PNS and CNS lesions on the expression of cytoskeletal proteins in a single neuronal population, the trochlear motoneurons of the cat. The trochlear nerve was lesioned either unilaterally in the PNS or bilaterally in the CNS (within the anterior medullary velum), and animals were allowed to survive 1, 2, or 4 weeks. Brain sections were reacted immunocytochemically using antibodies against microtubule -associated protein-2 (MAP-2) and a phosphorylated isoform of MAP1B, termed MAP1B-P. MAP-2 immunoreactivity (IR) was significantly decreased in the CNS-lesioned trochlear nucleus, compared to the lesioned and the unlesioned trochlear nucleus of PNS-lesioned animals. MAP1B-P IR was significantly increased in PNS- and CNS- lesioned trochlear axons, compared to axons in the unlesioned trochlear nerve of PNS-lesioned animals, and appeared in a small percentage of PNS- and CNS-lesioned cell bodies. These results support the growing body of evidence that MPA-2 can serve as a marker for cells that will eventually die following neuronal insult. The increased immunostaining of MAP1B-P in lesioned axons and its appearance in lesioned cell bodies are characteristic of the immature CNS and may reflect an initial recapitulation of early development, when the levels of this protein are high.

Immunohistochemical localization of neurotransmitters utilized by neurons in the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) that project to the oculomotor and trochlear nuclei in the cat.

The rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) contains excitatory and inhibitory burst neurons that are related to the control of vertical and torsional eye movements. In the present study, light microscopic examination of the immunohistochemical localization of amino acid neurotransmitters demonstrated that the riMLF in the cat contains overlapping populations of neurons that are immunoreactive to the putative inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and the excitatory neurotransmitters glutamate and aspartate. By using a double-labelling paradigm, GABA-, glutamate-, and aspartate-immunoreactive neurons in the riMLF were retrogradely labelled by transport of horseradish peroxidase (HRP) from the oculomotor and trochlear nuclei. Electron microscopy showed that the oculomotor and trochlear nuclei contain synaptic endings that are immunoreactive to GABA, glutamate, or aspartate. Each neurotransmitter-specific population of synaptic endings has distinctive ultrastructural and synaptic features. Synaptic endings in the oculomotor and trochlear nuclei that are anterogradely labelled by transport of biocytin from the riMLF are immunoreactive to GABA, glutamate, or aspartate. Taken together, the findings from these complimentary retrograde and anterograde double-labelling studies provide rather conclusive evidence that GABA is the inhibitory neurotransmitter, and glutamate and aspartate are the excitatory neurotransmitters, utilized by premotor neurons in the riMLF that are related to the control of vertical saccadic eye movements.

Calcitonin gene-related peptide increases following axotomy of trochlear motoneurons.

Neuropeptides have long been considered to act as neurotransmitters or neuromodulators, but they may also contribute to a variety of regulatory and trophic neuronal functions. In the present study, we determined the effects of axotomy on the levels of the neuropeptide calcitonin gene-related peptide (CGRP) in trochlear motoneurons (TMNs) of adult cats. The number of neurons with detectable CGRP immunoreactivity, and the intensity of their CGRP immunoreactivity, increased dramatically 1 week postaxotomy, gradually returned toward normal levels, but remained significantly higher than normal 12 weeks postaxotomy--a time when axonal regeneration and functional reinnervation of the superior oblique muscle should be complete. Our observation that CGRP levels in TMNs increase after axotomy suggests a role for this peptide in the response of motoneurons to injury and in regeneration. In addition, since many axotomized TMNs die, we suggest that the maintenance of high CGRP levels even after regeneration is complete may reflect an increased load on those TMNs that survive.

Neuronal response of the hippocampal formation to injury: blood flow, glucose metabolism, and protein synthesis.

The reaction of the hippocampal formation to entorhinal lesions was studied from the viewpoints of cerebral blood flow ([123I]isopropyl-iodoamphetamine[IMP])-glucose utilization ([14C]2-deoxyglucose), and protein synthesis ([14C]leucine), using single- and double-label autoradiography. Our study showed (i) decreased glucose utilization in the inner part, and increased glucose utilization in the outer part of the molecular layer of the dentate gyrus, starting 3 days after the lesion; (ii) increased uptake of [123I]IMP around the lesion from 1 to 3 days postlesion; and (iii) starting 3 days after the lesion, marked decrease in [14C]leucine incorporation into proteins and cell loss in the dorsal CA1 and dorsal subiculum in about one-half of the rats. These changes were present only in animals with lesions which invaded the ventral hippocampal formation in which axons of CA1 cells travel. By contrast, transsection of the 3rd and 4th cranial nerves resulted, 3 to 9 days after injury, in a striking increase in protein synthesis in the oculomotor and trochlear nuclei. These results raise the possibility that in some neurons the failure of central regeneration may result from the cell's inability to increase its rate of protein synthesis in response to axonal injury.

Radioautographic study of 3H-GABA uptake in the oculomotor nucleus of the cat.

The uptake of tritiated gamma-aminobutyric acid (3H-GABA) in the oculomotor nucleus of the cat was studied, using light and electron microscopic examination of radioautograms after intracerebral in vivo administration of the amino-acid. A glial uptake by oligodendrocytes was seen together with a neuronal uptake of the tracer in a certain type of axon terminals found in synaptic contact with both dendrites and soma, some of them exhibiting all the ultrastructural features of motoneurons. Previous neurochemical, electrophysiological and immunocytochemical studies indicate that GABA might well be the inhibitory neurotransmitter in the vestibuloocular reflex arc. The present results show that a morphological substrate exists for the presumed postsynaptic GABAergic inhibition of ocular motoneurons, at least in the oculomotor nucleus of the cat.

Studies of suspected neurotransmitters in the vestibuloocular pathways.

Isolated fresh cat trochlear and oculomotor nuclei, which contain the axon terminals of inhibitory neurons whose cell bodies are in the superior vestibular nucleus (SVN), actively synthesize and store [3H]GABA, [14C]acetylcholine, [3H]dopamine and [3H]tyramine from labeled precursors of these compounds. Twelve to 14 days following lesions of the ipsilateral superior vestibular nucleus or its efferent pathway to the oculomotor and trochlear nuclei, at a time when there is extensive degeneration of superior vestibular nucleus axon terminals in these nuclei, the synthesis and storage of GABA in the ipsilateral trochlear nucleus is markedly reduced compared to that in the contralateral trochlear nucleus; the synthesis of acetylcholine, dopamine and tyramine is not measurably affected. The oculomotor nuclei, which unlike the trochlear nuclei receive a heavy bilateral projection from the SVN, show no asymmetric decrease after SVN lesions in their ability to synthesize any of the compounds tested. The data support the identity of GABA as an inhibitory transmitter in the superior vestibular nucleus-trochlear nucleus pathway.

Neurofilament and glycogen changes during cold acclimation in the trochlear nucleus of lizards (Sceloporus undulatus).

In lizards (Sceloporus undulatus), long term (13 or 19 weeks) acclimation to an environment of 6 degrees C produces a striking increase in the argyrophilic neurofibrillar network in most large perikarya of the trochlear nucleus. In electron micrographs the cells contain numerous bundles of 10-30 regularly-spaced 90 A neurofilaments. In the cells from warm acclimated animals, a plexus of neurofibrils is seen by light microscopy. The electron micrographs show scattered neurofilaments and fewer, thinner bundles than in the cold. Within the cell bodies of the cold animals, glycogen particles are organized in regional accumulations from which other organelles are excluded except for the bundles of neurofilaments which are distributed throughout the cytoplasm. The aggregations of rough endoplasmic reticulum (RER) are also penetrated by the neurofilament bundles. The increased neurofilamentous network in the cold is not accompanied by obvious changes in the amount or distribution of RER or of microtubules which are present in limited numbers in both conditions. The dendrites of trochlear cells and axon terminals within the nucleus also show a cold induced increase in neurofilaments, as well as in the distinctive accumulations of glycogen particles.