L1 CAM expression is increased surrounding the lesion site in rats with complete spinal cord transection as neonates.

L1 is a cell adhesion molecule associated with axonal outgrowth, fasciculation, and guidance during development and injury. In this study, we examined the long-term effects of spinal cord injury with and without exercise on the re-expression of L1 throughout the rat spinal cord. Spinal cords from control rats were compared to those from rats receiving complete mid-thoracic spinal cord transections at postnatal day 5, daily treadmill step training for up to 8 weeks, or both transection and step training. Three months after spinal cord transection, we observed substantially higher levels of L1 expression by both Western blot analysis and immunocytochemistry in rats with and without step training. Higher expression levels of L1 were seen in the dorsal gray matter and in the dorsal lateral funiculus both above and below the lesion site. In addition, L1 was re-expressed on the descending fibers of the corticospinal tract above the lesion. L1-labeled axons also expressed GAP-43, a protein associated with axon outgrowth and regeneration. Treadmill step training had no effect on L1 expression in either control or transected rats despite the fact that spinal transected rats displayed improved stepping patterns indicative of spinal learning. Thus, spinal cord transection at an early age induced substantial L1 expression on axons near the lesion site, but was not additionally augmented by exercise.

The paralogous Hox genes Hoxa10 and Hoxd10 interact to pattern the mouse hindlimb peripheral nervous system and skeleton.

The most 5' mouse Hoxa and Hoxd genes, which occupy positions 9-13 and which are related to the Drosophila AbdB gene, are all active in patterning developing limbs. Inactivation of individual genes produces alterations in skeletal elements of both forelimb and hindlimb; inactivation of some of these genes also alters hindlimb innervation. Simultaneous inactivation of paralogous or nonparalogous Hoxa and Hoxd genes produces more widespread alterations, suggesting that combinatorial interactions between these genes are required for proper limb patterning. We have examined the effects of simultaneous inactivation of Hoxa10 and Hoxd10 on mouse hindlimb skeletal and nervous system development. These paralogous genes are expressed at lumbar and sacral levels of the developing neural tube and surrounding axial mesoderm as well as in developing forelimb and hindlimb buds. Double-mutant animals demonstrated impaired locomotor behavior and altered development of posterior vertebrae and hindlimb skeletal elements. Alterations in hindlimb innervation were also observed, including truncations and deletions of the tibial and peroneal nerves. Animals carrying fewer mutant alleles show similar, but less extreme phenotypes. These observations suggest that Hoxa10 and Hoxd10 coordinately regulate skeletal development and innervation of the hindlimb.

Laparoscopic ovariectomy and ovariohysterectomy in llamas and alpacas.

The minimally invasive nature of laparoscopic techniques and shorter convalescent periods have made these techniques increasingly popular for use in New World camelids (llamas and alpacas). This article outlines the instruments and steps needed to perform laparoscopic surgery on the female reproductive tract in llamas and alpacas.

Enhanced sensitivity to N-methyl-D-aspartate receptor activation in transgenic and knockin mouse models of Huntington's disease.

We used two mouse models of Huntington's disease (HD) to examine changes in glutamate receptor sensitivity and striatal electrophysiology. One model, a transgenic, consisted of mice expressing exon 1 of the human HD gene and carrying 141-157 CAG repeat sequences (R6/2 line). The second model, a CAG repeat "knockin," consisted of mice with different lengths of CAG repeats (CAG71 and CAG94 repeats). The effects of glutamate receptor activation were examined by visualizing neurons in brain slices with infrared videomicroscopy and differential interference contrast optics to determine changes in somatic area (cell swelling). Striatal and cortical neurons in both models (R6/2 and CAG94) displayed more rapid and increased swelling to N-methyl-D-aspartate (NMDA) than those in controls. This effect was specific as there were no consistent group differences after exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or kainate (KA). Intracellular recordings revealed that resting membrane potentials (RMPs) in the R6/2 transgenics were significantly more depolarized than those in their respective controls. RMPs in CAG94 mice also were more depolarized than those in CAG71 mice or their controls in a subset of striatal neurons. Confirming previous results, R6/2 mice expressed behavioral abnormalities and nuclear inclusions. However, CAG71 and CAG94 knockins did not, suggesting that increased sensitivity to NMDA may occur early in the disease process. These findings imply that NMDA antagonists or compounds that alter sensitivity of NMDA receptors may be useful in the treatment of HD.

Targeted disruption of Hoxd9 and Hoxd10 alters locomotor behavior, vertebral identity, and peripheral nervous system development.

The five most 5' HoxD genes, which are related to the Drosophila Abd-B gene, play an important role in patterning axial and appendicular skeletal elements and the nervous system of developing vertebrate embryos. Three of these genes, Hoxd11, Hoxd12, and Hoxd13, act synergistically to pattern the hindlimb autopod. In this study, we examine the combined effects of two additional 5' HoxD genes, Hoxd9 and Hoxd10. Both of these genes are expressed posteriorly in overlapping domains in the developing neural tube and axial mesoderm as well as in developing limbs. Locomotor behavior in animals carrying a double mutation in these two genes was altered; these alterations included changes in gait, mobility, and adduction. Morphological analysis showed alterations in axial and appendicular skeletal structure, hindlimb peripheral nerve organization and projection, and distal hindlimb musculature. These morphological alterations are likely to provide the substrate for the observed alterations in locomotor behavior. The alterations observed in double-mutant mice are distinct from the phenotypes observed in mice carrying single mutations in either gene, but exhibit most of the features of both individual phenotypes. This suggests that the combined activity of two adjacent Hox genes provides more patterning information than activity of each gene alone. These observations support the idea that adjacent Hox genes with overlapping expression patterns may interact functionally to provide patterning information to the same regions of developing mouse embryos.

Monoclonal antibody against a neuronal antigen impairs formation of the axon scaffold in grasshopper central nervous system.

The pathway a growing axon follows is determined by a number of cues, including differential adhesion to surface molecules on axons and the matrix of the fascicles along which they grow. We have characterized the differential expression of an extracellular antigen and the effects of a monoclonal antibody against this molecule on the development of the grasshopper central nervous system (CNS). The 5C1 monoclonal antibody was generated against ganglion chains of grasshopper embryos; it labels cell bodies of newly differentiated neurons and their axons as they extend. Electron microscopy of embryos at 42% of development reveals that 5C1 labels neuronal filopodia, axons and somata, and areas of glial membrane in apposition to neurite fascicles. After 70% of development, labeling is lost from axon bundles, but remains on cell bodies. 5C1 also cross-reacts with an epitope expressed in Drosophila CNS during embryonic development. Enzymatic digestion suggests that the antigen recognized by the antibody is likely to be a glycolipid. In embryos exposed to 5C1 during early stages of development of the CNS, at the time when the first axons begin to extend, the formation of axon pathways is blocked or greatly delayed. Our results suggest that the 5C1 antigen participates in the formation of the axon scaffold and may play a functional role in the initiation and maintenance of axon outgrowth during early development of the CNS.

Neural tube expression of pituitary adenylate cyclase-activating peptide (PACAP) and receptor: potential role in patterning and neurogenesis.

Neural tube patterning in vertebrates is controlled in part by locally secreted factors that act in a paracrine manner on nearby cells to regulate proliferation and gene expression. We show here by in situ hybridization that genes for the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and one of its high-affinity receptors (PAC1) are widely expressed in the mouse neural tube on embryonic day (E) 10.5. Transcripts for the ligand are present in differentiating neurons in much of the neural tube, whereas the receptor gene is expressed in the underlying ventricular zone, most prominently in the alar region and floor plate. PACAP potently increased cAMP levels more than 20-fold in cultured E10.5 hindbrain neuroepithelial cells, suggesting that PACAP activates protein kinase A (PKA) in the neural tube and might act in the process of patterning. Consistent with this possibility, PACAP down-regulated expression of the sonic hedgehog- and PKA-dependent target gene gli-1 in cultured neuroepithelial cells, concomitant with a decrease in DNA synthesis. PACAP is thus an early inducer of cAMP levels in the embryo and may act in the neural tube during patterning to control cell proliferation and gene expression.

Targeted disruption of Hoxd-10 affects mouse hindlimb development.

Targeted disruption of the Hoxd-10 gene, a 5' member of the mouse HoxD linkage group, produces mice with hindlimb-specific defects in gait and adduction. To determine the underlying causes of this locomotor defect, mutant mice were examined for skeletal, muscular and neural abnormalities. Mutant mice exhibit alterations in the vertebral column and in the bones of the hindlimb. Sacral vertebrae beginning at the level of S2 exhibit homeotic transformations to adopt the morphology of the next most anterior vertebra. In the hindlimb, there is an anterior shift in the position of the patella, an occasional production of an anterior sesamoid bone, and an outward rotation of the lower part of the leg, all of which contribute to the defects in locomotion. No major alterations in hindlimb musculature were observed, but defects in the nervous system were evident. There was a decrease in the number of spinal segments projecting nerve fibers through the sacral plexus to innervate the musculature of the hindlimb. Deletion of a hindlimb nerve was seen in some animals, and a shift was evident in the position of the lumbar lateral motor column. These observations suggest a role for the Hoxd-10 gene in establishing regional identity within the spinal cord and imply that patterning of the spinal cord may have intrinsic components and is not completely imposed by the surrounding mesoderm.

REGA-1 is a GPI-linked member of the immunoglobulin superfamily present on restricted regions of sheath cell processes in grasshopper.

REGA-1 is a glycoprotein localized to sheath cell processes in the developing CNS when NBs are producing progeny and neurons are maturing and extending processes. It is also present on a subset of muscles and on the lumenal surface of the ectoderm in the embryonic appendages when pioneer neurons are growing into the CNS. REGA-1 is associated with the extracellular side of the cell membrane by a glycosyl-phosphatidylinositol linkage. We have identified a cDNA clone encoding REGA-1 using a sequence from purified protein. Sequence analysis defines REGA-1 as a novel member of the immunoglobulin superfamily containing three immunoglobulin domains and one fibronectin type III repeat. Each Ig domain has distinct sequence characteristics that suggest discrete functions. REGA-1 is similar to other Ig superfamily members involved in cell adhesion events and neurite outgrowth.

Loss of Hox-A1 (Hox-1.6) function results in the reorganization of the murine hindbrain.

Targeted disruption of the murine hox-A1 gene results in severe defects in the formation of the hindbrain and associated cranial ganglia and nerves. Carbocyanine dye injections were used to trace afferent and efferent projections to and from the hindbrain in hox-A1-/hox-A1- mutant mice. Defects were observed in the position of efferent neurons in the hindbrain and in their projection patterns. In situ hybridization was used to analyze the transcription pattern of genes expressed within specific rhombomeres. Krox-20, int-2 (fgf-3), and hox-B1 all display aberrant patterns of expression in hox-A1- mutant embryos. The observed morphological and molecular defects suggest that there are changes in the formation of the hindbrain extending from rhombomere 3 through rhombomere 8 including the absence of rhombomere 5. Also, motor neurons identified by their axon projection patterns which would normally be present in the missing rhombomere appear to be respecified to or migrate into adjacent rhombomeres, suggesting a role for hox-A1 in the specification of cell identity and/or cell migration in the hindbrain.

The location and distribution of neural crest-derived Schwann cells in developing peripheral nerves in the chick forelimb.

The location and distribution of neural crest-derived Schwann cells during development of the peripheral nerves of chick forelimbs were examined using chick-quail chimeras. Neural crest cells were labeled by transplantation of the dorsal part of the neural tube from a quail donor to a chick host at levels of the neural tube destined to give rise to brachial innervation. The ventral roots, spinal nerves, and peripheral nerves innervating the chick forelimb were examined for the presence of quail-derived neural crest cells at several stages of embryonic development. These quail cells are likely to be Schwann cells or their precursors. Quail-derived Schwann cells were present in ventral roots and spinal nerves, and were distributed along previously described neural crest migratory pathways or along the peripheral nerve fibers at all stages of development examined. During early stages of wing innervation, quail-derived Schwann cells were not evenly distributed, but were concentrated in the ventral root and at the brachial plexus. The density of neural crest-derived Schwann cells decreased distal to the plexus, and no Schwann cells were ever seen in advance of the growing nerve front. When the characteristic peripheral nerve branching pattern was first formed, Schwann cells were clustered where muscle nerves diverged from common nerve trunks. In still older embryos, neural crest-derived Schwann cells were evenly distributed along the length of the peripheral nerves from the ventral root to the distal nerve terminations within the musculature of the forelimb. These observations indicate that Schwann cells accompany axons into the developing limb, but they do not appear to lead or direct axons to their targets. The transient clustering of neural crest-derived Schwann cells in the ventral root and at places where axon trajectories diverge from one another may reflect a response to some environmental feature within these regions.

The distribution of neural crest-derived Schwann cells from subsets of brachial spinal segments into the peripheral nerves innervating the chick forelimb.

Neural crest cells from brachial levels of the neural tube populate the ventral roots, spinal nerves, and peripheral nerves of the chick forelimb where they give rise to Schwann cells. The distribution of neural crest cells in the developing forelimb was examined using homotopic and heterotopic chick-quail chimeras to label neural crest cells from subsets of the brachial spinal segments. Neural crest cells from particular regions of the spinal cord populated ventral roots and spinal nerves adjacent to or immediately posterior to the graft. Crest cells also populated the brachial plexus in accord with their segmental origins. In the forelimb, neural crest cells populated muscle nerves with anterior brachial spinal segments populating nerves to anterior musculature of the forelimb and posterior brachial spinal segments populating nerves to posterior musculature. Similar patterns were seen following both homotopic and heterotopic transplantation. In both types of grafts, the distribution of neural crest cells largely matched the sensory and motor projection pattern from the same spinal segmental level. This suggests that neural crest-derived Schwann cells from a particular spinal segment may use sensory and motor fibers emerging from the same segmental level as substrates to guide their migration into the periphery.

Developmental expression of REGA-1, a regionally expressed glial antigen in the central nervous system of grasshopper embryos.

Glial cells are a large component of the developing nervous system, appearing before the onset of axon outgrowth in a variety of developing systems. Their time of appearance and their location in conjunction with developing axon pathways may allow them to define the position of axon pathways. Specific glial cells may be utilized as guideposts by growing axons, allowing them to recognize the appropriate pathway, or conversely, glial cells may inhibit axons from growing along an inappropriate pathway. The 7F7 monoclonal antibody labels a subset of glial cells in grasshopper embryos that may play a role in defining the location of selected axonal pathways. This antibody recognizes the REGA-1 molecule, a cell-surface antigen with a molecular weight of 60 kDa, which is regionally expressed on developing glial cells. REGA-1 is expressed around the edges of clusters of glial cells and on lamellae extending from glial cells to line the edges of some axonal pathways. REGA-1 expression is first seen in the neuroblast sheet, surrounding neuroblast 4-1. Slightly later in development, 2 glial cells extend processes that express REGA-1 and demarcate the caudal edge of the anterior commissure. As the animal matures, cell processes expressing REGA-1 line the edges of the longitudinal connective, then expand to surround the central neuropil of the segmental ganglia. REGA-1 expression is also seen in conjunction with axons leaving the segmental ganglia via the segmental nerves and the intersegmental connectives. REGA-1 expression is limited to a subset of glial cells; some known glial cells such as the segment boundary cell do not express REGA-1. Glial cell processes expressing REGA-1 are seen only in association with axons, which suggests that these processes may act as borders or guard rails confining axons to the appropriate regions of the developing CNS. Axons navigating a path through the CNS may be prohibited from growing into inappropriate regions based on their inability to cross the boundaries established by glial cells expressing REGA-1.