High rate of detection of subtelomeric aberration by using combined MLPA and subtelomeric FISH approach in patients with moderate to severe mental retardation.

OBJECTIVES: (1) To evaluate the prevalence of subtelomeric deletion in moderate to severe mental retardation population, (2) to assess the feasibility and cost-effectiveness of combined methodology in routine workup of this sub-population. METHOD: Twenty unrelated patients using strict selection criteria were recruited for the study from the Clinical Genetic Service. Patients were initially screened by Multiplex Ligation-dependent Probe Amplification (MLPA) for subtelomeric imbalance followed by FISH analysis for anatomical integrity. This is then followed by parental subtelomeric FISH analysis. RESULTS: Three subtelomeric deletions were identified. They were Deletion 1p36, Deletion 1q44 and Deletion 10q26; these were previously unidentified by conventional technique. CONCLUSIONS: The prevalence of subtelomeric deletion in our cohort of moderate to severe mental retardation patients is consistent with published findings of around 10%. The figure is on the higher side if more stringent criteria is used. The combination of strict clinical criteria, MLPA and selective subtelomeric FISH was shown to be feasible and cost-effective.

Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA).

OBJECTIVES: To evaluate the efficacy of Multiplex Ligation-dependent Probe Amplification (MLPA) technique in comparison with the traditional multiplex PCR assay in detection of exon deletions and duplications of the DMD gene. DESIGN AND METHODS: The sensitivity and accuracy of MLPA were assessed and compared with the multiplex PCR in a total of 63 subjects including 43 subjects with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) and 20 female carriers. RESULTS: MLPA was able to detect all the known deletions and duplications; it detected four additional mutations that had been missed by multiplex PCR. In addition, the extent of the deletions and duplications could be more accurately defined which in turn facilitated a genotype-phenotype correlation. CONCLUSIONS: MLPA is superior to multiplex PCR. It should be the method of choice for the detection of exon deletions and duplications of the DMD gene in patients with DMD or BMD, as well as in female carriers.

Potential of stem cell based therapy and tissue engineering in the regeneration of the central nervous system.

The insufficiency of self-repair and regeneration of the central nervous system (CNS) leads to difficulty of rehabilitation of the injured brain. In the past few decades, the significant progress in cell therapy and tissue engineering has contributed to the functional recovery of the CNS to a great extent. The present review focuses on the potential role of stem cell based therapy and tissue engineering in the regeneration of the CNS.

Remodeling of motor terminals during metamorphosis of the moth Manduca sexta: expression patterns of two distinct isoforms of Manduca fasciclin II.

During metamorphosis of the moth Manduca sexta, the neuromuscular system of the thoracic legs is reorganized dramatically. Larval leg muscles degenerate at the end of larval life, and new adult leg muscles develop during the ensuing pupal stage. Larval leg motoneurons persist, but undergo substantial remodeling of central and peripheral processes. As part of our on-going investigation of mechanisms underlying the remodeling of motor terminals, we have used antisera generated against Manduca-specific isoforms of the homophilic adhesion molecule fasciclin II (MFas II) to label motor terminals during metamorphosis. Antisera generated against the glycosyl-phosphatidylinositol (GPI) -linked isoform of MFas II (GPI-MFas II) labeled the motor nerves at all stages and seemed to be associated with glial cells ensheathing the peripheral nerves. In addition, the anti-GPI-MFas II antisera labeled regions associated with synaptic boutons at both larval and adult stages. In contrast, antisera generated against a transmembrane isoform of MFas II (TM-MFas II) only labeled specific neuronal processes at discrete intervals during remodeling. Identified leg motoneurons (such as the femoral depressor motoneuron) expressed detectable levels of TM-MFas II in their peripheral processes only during phases of motor-terminal retraction and initial stages of motor-terminal re-growth. Putative modulatory neurons (such as the unpaired median neurons), however, expressed TM-MFas II in their processes during larval stages as well as during remodeling. Use of the isoform-specific anti-MFas II antisera provided a novel method for visualizing remodeling of motor terminals during metamorphosis and helped distinguish different components of the motor nerves and neuromuscular junction.

Development of the labial pit organ glomerulus in the antennal lobe of the moth Manduca sexta: the role of afferent projections in the formation of identifiable olfactory glomeruli.

Iterated neuropil modules called glomeruli are characteristic of primary olfactory centers in both vertebrates and invertebrates. To gain insight into the developmental mechanisms underlying the formation of such structured, organized neuropil, we have examined the development of an identified glomerulus in the olfactory (antennal) lobe of the moth Manduca sexta. The labial pit organ glomerulus (LPOG) receives bilateral sensory projections from the labial pit organs in the labial palps of the mouthparts, while other glomeruli in the antennal lobe receive unilateral projections from the antenna. Here, we chronicle the development of the LPOG under normal and perturbed conditions. Our findings suggest that the sensory axons of the labial pit organ, like those of the antenna, induce and shape growth of interneuronal arborizations, but specific features of interneuronal arborizations such as the relative position of glomerular arborizations within the antennal lobe are independent of both classes of afferent innervation. Labial pit organ axons and antennal axons exhibit a high degree of specificity for their respective target regions, independent of the presence or absence of the other class of afferent axon or the route taken to the antennal lobe. Specification of glomerular position is intrinsic to the antennal lobe rather than a consequence of competition between afferent axons.

Leg proprioceptors of the tobacco hornworm, Manduca sexta: organization of central projections at larval and adult stages.

Organization of the central neuropil of the insect ganglion is characterized in part by a modality-specific layering of afferent projections. This organization has been particularly well described for the central projections of thoracic leg sensory neurons of adult locusts, crickets, and flies. Tactile sensory neurons project into a ventral layer of neuropil, while proprioceptive sensory neurons project into an intermediate layer of neuropil. In order to determine whether a modality-specific layering exists in the CNS of larval Manduca sexta, we have examined the projections of sensory neurons innervating one class of putative proprioceptors, the campaniform sensilla, of the larval metathoracic legs. We find that campaniform sensory neurons of the larval legs have central projection patterns that generally distinguish them from each other and from the tactile sensory neurons. The campaniform projections, however, are not completely segregated from tactile projections in ventral layers of neuropil, as has been described in other insects. By contrast, the projections of campaniform sensory neurons from the adult legs are more extensive and elaborate than their larval counterparts and dramatically different from projections of nearby adult tactile hairs, having extensive arborizations in more dorsal regions of neuropil while those of tactile sensory neurons are restricted to very ventral layers of neuropil. This difference in organization of the afferent projections in larval and adult ganglia may reflect different functions of the leg sensilla and different functions of the legs at the two stages.

Remodeling of the peripheral processes and presynaptic terminals of leg motoneurons during metamorphosis of the hawkmoth, Manduca sexta.

During metamorphosis of the hawkmoth, Manduca sexta, the muscles, cuticular structures, and most sensory neurons of the larval thoracic legs are replaced by new elements in the adult legs. The thoracic leg motoneurons, however, survive the loss of the larval muscles and persist to innervate new targets in the imaginal legs. Here we have used biocytin staining, immunocytochemistry, and confocal microscopy to follow the fates of the peripheral processes and presynaptic terminals of the leg motoneurons. Although the most distal processes of the motor nerves retract following the degeneration of larval leg muscles, the axon terminals always retain close association with the muscle remnants and the anlagen of the new adult muscles. As the imaginal muscles differentiate and enlarge, the motor terminals expand to form adult presynaptic terminals. An antibody to the presynaptic protein, synaptotagmin, revealed its localization to the terminal varicosities in both larval and adult stages but distribution within pre-terminal branches during adult development. Electrophysiological methods revealed that functional neuromuscular transmission first occurs quite early during metamorphosis, before the differentiation of contractile elements in the muscle fibers.

Dendritic reorganization of an identified neuron during metamorphosis of the moth Manduca sexta: the influence of interactions with the periphery.

During metamorphosis of the moth, Manduca sexta, an identified leg motor neuron, the femoral extensor motor neuron (FeExt MN) undergoes dramatic reorganization. Larval dendrites occupy two distinct regions of neuropil, one in the lateral leg neuropil and a second in dorsomedial neuropil. Adult dendrites occupy a greater volume of lateral leg neuropil but do not extend to the dorsomedial region of the ganglion. The adult dendritic morphology is acquired by extreme dendritic regression followed by extensive dendritic growth. Towards the end of larval life, MN dendrites begin to regress, but the most dramatic loss of dendrites occurs in the 3 days following pupation, such that only a few sparse dendrites are retained in the lateral region of leg neuropil. Extensive dendritic growth occurs over the subsequent days such that the MN acquires an adult-like morphology between 12 and 14 days after pupation. This basic process of dendritic remodeling is not dependent upon the presence of the adult leg, suggesting that neither contact with the new target muscle nor inputs from new leg sensory neurons are necessary for triggering dendritic changes. The final distribution of MN dendrites in the adult, however, is altered when the adult leg is absent, suggesting that cues from the adult leg are involved in directing or shaping the growth of MN dendrites to specific regions of neuropil.

Sensory organs of the thoracic legs of the moth Manduca sexta.

The thoracic legs of the moth Manduca sexta acquire a new form and develop a new complement of sensory organs and muscles during metamorphosis from larva to adult. Because of our interest in the reorganization of neural circuitry and the acquisition of new behaviors during metamorphosis, we are characterizing sensory elements of larval and adult legs so that we may determine the contribution of new sensory inputs to the changes in behaviors. Here we describe the sensory structures of adult legs using scanning electron microscopy to view the external sensilla and cobalt staining to examine innervation by underlying sensory neurons. We find that, in contrast to larval legs, the adult legs are covered with a diverse array of sensilla. All three pairs of thoracic legs contain scattered, singly innervated scalelike sensilla. Campaniform sensilla occur singly or in clusters near joints. Hair plates, consisting of numerous singly innervated hairs, are also present near joints. Other more specialized sensilla occur on distal leg segments. These include singly innervated spines, two additional classes of singly innervated hairs, and three classes of multiply innervated sensilla. Internal sensory organs include chordotonal organs, subgenual organs, and multipolar joint receptors.

Neural control of leg movements in a metamorphic insect: persistence of larval leg motor neurons to innervate the adult legs of Manduca sexta.

During metamorphosis of the hawkmoth Manduca sexta, the larval thoracic legs along with their associated sensory organs and muscles degenerate and new adult legs develop. The larval legs are small and relatively simple structures capable of lateral extension and medial flexion allowing them to grasp the substrate as the caterpillar crawls along. By contrast, the adult legs are used for walking with an alternating gait. They are much larger than the larval legs and articulate such that they are capable of movement in several directions. This change in form and function is accompanied by a reorganization of the neural circuits controlling leg movements. In a previous report (Kent and Levine: J. Comp. Neurol. 271:559-576, '88) we described motor neurons innervating the larval prothoracic legs, and here we describe motor neurons innervating the prothoracic legs of the adult. Using a combination of cobalt staining methods and the persistent fluorescent dye Fluoro-Gold, we have found that some, if not all, larval leg motor neurons are retained and innervate the new adult leg muscles. Moreover, we have been able to discover the fate of individual larval leg motor neurons by marking a single larval neuron with Fluoro-Gold and using a second fluorescent dye to double label the same neuron in the adult. Our results suggest that specific larval leg motor neurons innervate corresponding muscles in the adult stage, although their apparent function is significantly different. In addition, the motor neurons undergo significant remodeling of their dendritic branching patterns during metamorphosis, alterations which doubtless contribute to their new roles in adult behavior.

Neural control of leg movements in a metamorphic insect: sensory and motor elements of the larval thoracic legs in Manduca sexta.

During the metamorphosis of the hawkmoth Manduca sexta the larval thoracic legs degenerate to be replaced in the adult by legs of very different form and function. This change must be accompanied by a reorganization of the neural circuits controlling leg movements. As an initial step in the study of this reorganization we describe here the sensory and motor elements of this circuitry in the larval stage of life. Sensory neurons innervating mechanoreceptive hairs on the thoracic surface were stained individually with cobalt. Those innervating hairs on the general thoracic surface project topographically into two ventral regions of the segmental ganglia. Sensory neurons innervating leg sensilla also map topographically to the more ventral of these regions but in addition have arborizations in a midlateral region. The density of branching within this lateral "leg neuropil" is greatest for sensory neurons form sensilla on the more distal leg segments. Leg motor neurons were identified with intracellular recording and cobalt injection techniques. Those innervating muscles controlling distal leg segments have dense dendritic arbors in the lateral "leg neuropil," while motor neurons controlling more proximal segments and muscles of the ventral body wall have extensive arborizations in a dorsomedial region of the ganglion. In general, flexor motor neurons are excited by medial and inhibited by lateral leg sensilla, while the opposite is true of extensors. Distal segment motor neurons respond most strongly to sensory neurons from distal segments, thus suggesting some interaction within the lateral "leg neuropil." Thus, in the larval nervous system a highly ordered array of of sensory and motor elements underlies the specific behavioral responses of the legs to tactile stimulation.

A novel serotonin-immunoreactive neuron in the antennal lobe of the sphinx moth Manduca sexta persists throughout postembryonic life.

A single serotonin-immunoreactive neuron in the antennal lobe (AL) of the brain of the sphinx moth Manduca sexta is present in larval, pupal, and adult stages. This neuron has a neurite that extends to the contralateral AL, where it forms sparse arborizations in each glomerulus. Other neurites from this neuron project into the ipsilateral and contralateral protocerebrum. This cell is morphologically very different from other neurons previously characterized in the adult AL. The neuron maintains the same basic profile in the adult as in the larva, although fine processes such as the arborizations within the AL neuropil appear to be restructured to conform to the larger, more anatomically differentiated regions of the adult brain.

Cephalic sensory pathways in the central nervous system of larval Manduca sexta (Lepidoptera : Sphingidae).

Central projections of neurons innervating sensory structures on the head of larval Manduca sexta were traced by using methods of anterograde cobalt-diffusion. Regions of the deutocerebrum and tritocerebrum in the brain receive input from the antenna, labrum, maxilla, labial palps, hypopharynx and other unidentified regions of the buccal cavity. Antennal, maxillary and labial inputs project to the larval antennal centre (LAC) of the deutocerebrum. Stemmatal neurons and a few antennal neurons project into the protocerebrum. The suboesophageal ganglion (SEG) receives input from mechanosensory neurons in all parts of the head and its sensory appendages. Some mechanosensory neurons project further to the first thoracic ganglion. In addition to receiving input from chemosensory neurons of the maxilla, the SEG may also receive chemosensory input from epipharyngeal sensilla of the labrum.

An accessory olfactory pathway in Lepidoptera: the labial pit organ and its central projections in Manduca sexta and certain other sphinx moths and silk moths.

In the hawkmoth, Manduca sexta, the third segment of each labial palp contains a pit, which houses a densely packed array of sensilla. We have named this structure the labial pit organ (LPO). The sensilla within the pit are typical of olfactory receptors, characterized by a grooved surface, wall pores, and pore tubules. Axons arising from receptor cells that innervate these sensilla project bilaterally to a single glomerulus in each antennal lobe. We have compared this central projection with that in three other species of Manduca (M. quinquemaculata, M. dilucida, and M. lanuginosa) and in the silkmoths Antheraea polyphemus and Bombyx mori. A bilateral projection to a single glomerulus in each antennal lobe is present in all cases. We suggest that the LPO serves as an accessory olfactory organ in adult Lepidoptera.