Central nervous system regeneration: from leech to opossum.

A major problem of neurobiology concerns the failure of injured mammalian spinal cord to repair itself. This review summarizes work done on two preparations in which regeneration can occur: the central nervous system of an invertebrate, the leech, and the spinal cord of an immature mammal, the opossum. The aim is to understand cellular and molecular mechanisms that promote and prevent regeneration. In the leech, an individual axon regrows successfully to re-establish connections with its synaptic target, while avoiding other neurons. Functions that were lost are thereby restored. Moreover, pairs of identified neurons become re-connected with appropriate synapses in culture. It has been shown that microglial cells and nitric oxide play key roles in leech CNS regeneration. In the opossum, the neonatal brain and spinal cord are so tiny that they survive well in culture. Fibres grow across spinal cord lesions in neonatal animals and in vitro, but axon regeneration stops abruptly between postnatal days 9 and 12. A comprehensive search has been made in spinal cords that can and cannot regenerate to identify genes and establish their locations. At 9 days, growth-promoting genes, their receptors and key transcription molecules are up-regulated. By contrast at 12 days, growth-inhibitory molecules associated with myelin are prominent. The complete sequence of the opossum genome and new methods for transfecting genes offer ways to determine which molecules promote and which inhibit spinal cord regeneration. These results lead to questions about how basic research on mechanisms of regeneration could be 'translated' into effective therapies for patients with spinal cord injuries.

Optical recording from respiratory pattern generator of fetal mouse brainstem reveals a distributed network.

Unfailing respiration depends on neural mechanisms already present in mammals before birth. Experiments were made to determine how inspiratory and expiratory neurons are grouped in the brainstem of fetal mice. A further aim was to assess whether rhythmicity arises from a single pacemaker or is generated by multiple sites in the brainstem. To measure neuronal firing, a fluorescent calcium indicator dye was applied to embryonic central nervous systems isolated from mice. While respiratory commands were monitored electrically from third to fifth cervical ventral roots, activity was measured optically over areas containing groups of respiratory neurones, or single neurones, along the medulla from the facial nucleus to the pre-Bötzinger complex. Large optical signals allowed recordings to be made during individual respiratory cycles. Inspiratory and expiratory neurones were intermingled. A novel finding was that bursts of activity arose in a discrete area intermittently, occurring during some breaths, but failing in others. Raised CO2 partial pressure or lowered pH increased the frequency of respiration; neurons then fired reliably with every cycle. Movies of activity revealed patterns of activation of inspiratory and expiratory neurones during successive respiratory cycles; there was no evidence for waves spreading systematically from region to region. Our results suggest that firing of neurons in immature respiratory circuits is a stochastic process, and that the rhythm does not depend on a single pacemaker. Respiratory circuits in fetal mouse brainstem appear to possess a high safety factor for generating rhythmicity, which may or may not persist as development proceeds.

Gene expression monitoring for gene discovery in models of peripheral and central nervous system differentiation, regeneration, and trauma.

Gene expression monitoring using gene expression microarrays represents an extremely powerful technology for gene discovery in a variety of systems. We describe the results of seven experiments using Incyte GEM technology to compile a proprietary portfolio of data concerning differential gene expression in six different models of neuronal differentiation and regeneration, and recovery from injury or disease. Our first two experiments cataloged genes significantly up- or down-regulated during two phases of the retinoic acid-induced differentiation of the embryonal carcinoma line Ntera-2. To identify genes involved in neuronal regeneration we performed three GEM experiments, which included changes in gene expression in rat dorsal root ganglia during the healing of experimentally injured sciatic nerve, in regenerating neonatal opossum spinal cord, and during lipopolysaccharide stimulation of primary cultures of rat Schwann cells. Finally we have monitored genes involved in the recovery phase of the inflammatory disease of the rat spinal cord, experimental allergic encephalomyelitis, as well as those responsible for protection from oxidative stress in a glutamate-resistant rat hippocampal cell line. Analysis of the results of the approximately 70,000 data points collected is presented.

Control of respiration in the isolated central nervous system of the neonatal opossum, Monodelphis domestica.

Respiration represents an unusual motor activity with respect to its development. As newly born mammals enter the world, their limb movements are not coordinated; time and experience are required for effective performance to be achieved. Yet the rhythm of respiration is of necessity functionally perfected and unfailing at birth. Inspiratory and expiratory motor neurons are already able to fire at appropriate rates, under the command of rhythmically active neurons in the medulla. In this review, we discuss refinements of control present in the newborn opossum, particularly with respect to mechanisms that allow adaptation of respiration to changes in the level of activity or in the outside environment. Our own studies have been aimed at analyzing respiration at the earliest stages, and at establishing the way in which important variables influence inspiration and expiration. To this end, we have used the central nervous system (CNS) of a neonatal opossum, isolated in its entirety and maintained in culture. Although the opossum is unable to walk and highly immature at birth, its respiration is regular and unfailing. The isolated CNS survives, undergoes development, and maintains its neural activity and fine structure in vitro. Moreover, fictive respiration persists for over a day or longer at rates similar to those of the intact pup. The effects of altered pH, of increased temperature, and of drugs known to alter respiratory rhythm in intact animals can be measured directly, by electrical recordings made from medullary neurons or ventral roots. As in a slice, fluids of different composition can be applied focally, through micropipettes to the surface of the ventral medulla, or diffusely to the brainstem, With highly localized application of procaine hydrochloride (2%) to selected areas of the ventral medulla, the respiratory rhythm is reduced or abolished. As in adult mammals, both the rate and the amplitude of respiration simultaneously increase in response to lowered pH (6.5-.7.1) or to topical application of 1.0 microM carbachol. Conversely, as expected, the rate and amplitude decrease in response to increased pH (pH 7.5-7.7), or 100 microM scopolamine. Two characteristic features of the control of respiration in the neonatal opossum are evident from such tests. First, changes in rate are achieved by changes in the duration of the expiratory phase of respiration. This result suggests that the timing of the respiratory cycle in the neonatal opossum is controlled by an expiratory instead of an inspiratory "off-switch". Second, the rate and the amplitude of the respiratory excursions can be controlled independently, depending on the stimulus. For example, an increase in temperature increases the rate of fictive respiration without changing its amplitude, whereas noradrenaline decreases the rate while increasing the amplitude. Thus, changes of timing and amplitude need not go hand in hand. The opossum CNS offers a favorable preparation for the analysis of neural mechanisms that generate and modulate a motor rhythm, as the animal develops from embryonic to adult stages.

Why does the central nervous system not regenerate after injury?

Spinal cord injuries in humans and in other mammals are never followed by regrowth. In recent years, considerable progress has been made in analyzing mechanisms that promote and inhibit regeneration. The focus of this review is changes that occur in the transition period in development when the central nervous system (CNS) changes from being able to regenerate to the adult state of failure. In our experiments we have used the neonatal opossum (Monodelphis domestica), which corresponds to a 14-day embryonic rat or mouse. The CNS isolated from an opossum pup and maintained in culture shows dramatic regeneration. Fibers grow through and beyond lesions and reform synaptic connections with their targets. Similarly, anesthetized neonatal pups attached to the mother recover the ability to walk after complete spinal cord transection. Although the CNS isolated from a 9-day-old animal will regenerate in vitro, CNS from a 12-day-old will not. This is the stage at which glial cells in the CNS develop. Present research is devoted toward molecular screening to determine which growth-promoting molecules decrease during development, which inhibitory molecules increase, and which receptors on growing axons become altered. Despite progress in many laboratories, major hurdles must be overcome before patients can hope to be treated. Nevertheless, the picture today is not as discouraging as it was: one can think of strategies for research on spinal cord injury so as to promote regeneration and restore function.

Re-establishment of direct synaptic connections between sensory axons and motoneurons after lesions of neonatal opossum CNS (Monodelphis domestica) in culture.

For functional recovery after spinal cord injury, regenerating fibres need to grow and to reform appropriate connections with their targets. The isolated central nervous system of neonatal opossums aged 1-9 days has been used to analyse the precision with which neurons become reconnected during regeneration. In culture these preparations maintain their electrical activity and show rapid outgrowth through spinal cord crushes or cuts. By recording electrically and by staining with horseradish peroxidase, we first demonstrated that direct reflex connections were already present at birth between sensory fibres in one segment and motoneurons in the same segment and in adjacent segments. As in previous experiments, 5 days after the spinal cord had been crushed, labelled sensory fibres grew across the lesion to reach the next segment (Woodward et al. (1993) J. Exp. Biol., 176, 77-88; Varga et al. (1995a) Eur. J. Neurosci., 7, 2119-2129, Varga et al. (1995b) Proc. Natl. Acad. Sci. USA, 92, 10959-10963). Beyond the lesion the labelled axons abruptly changed direction, traversed the spinal cord and terminated on labelled motoneurons in the ventral horn. In preparations that had regenerated dorsal root stimulation once again initiated ventral root reflexes. Electron micrographs revealed synapses made by labelled sensory axons on motoneurons. Double staining of growing sensory axons and radial glial fibres showed close association, suggesting guidance. These results indicate that the original pathway is re-established during repair and that appropriate connections are reformed after injury.

Fine structure and development of dorsal root ganglion neurons and Schwann cells in the newborn opossum Monodelphis domestica.

The aim of these experiments was to determine the state of maturity of dorsal root ganglia and axons in opossums (Monodelphis domestica) at birth and to assess quantitatively changes that occur in early life. Counts made of dorsal root ganglion cells at cervical levels showed that the numbers were similar in newborn and adult animals, approximately 1,600 per ganglion. In cervical dorsal root ganglia of newborn animals, division of neuronal precursors cells had ceased. The number of axons in cervical dorsal roots was similar in newborn and adult animals (about 4,500). For each ganglion cell body, approximately three axons were counted in the dorsal root. At birth, dorsal roots contained several bundles about 30 microns in diameter consisting of small axons (0.05-2 microns in diameter). A few non-neural cells were identified as Schwann cell perikarya, each enclosing a number of neurites. Later, marked changes occurred in Schwann cells and in their relationship to axons in the roots. Thus, at 12 days, an increase occurred in the number of Schwann cells and fibroblasts, and the bundles had enlarged to about 80 microns with little increase in axon diameter (0.1-2 microns). By 18 days, the bundles were larger, and myelination had already started. At 23 days, the dorsal root contained more than 500 myelinated axons that could reach 5 microns in diameter. The adult dorsal root enclosed about 900 myelinated axons. Throughout this time, the relationship between the Schwann cells and axons changed. Together, these results indicate that the number of axons and cell bodies of sensory dorsal root ganglia in opossum do not show major changes after birth. In addition, these results set the stage for quantitative studies of regeneration of dorsal column fibers in injured neonatal opossum nervous system.

Procedures for whole-mount immunohistochemistry and in situ hybridization of immature mammalian CNS.

Whole-mount labeling techniques for staining in invertebrates or lower vertebrates cannot simply be applied to the mammalian central nervous system (CNS) because of its large size. Such techniques if possible would offer advantages over conventional methods based on sections since an immediate and 3-dimensional view of the stained components in a transparent CNS is provided. It thereby becomes possible to survey and count large number of cells and fibers in their natural relationships. The aim of our experiments is to follow developing and regenerating expression of proteins and mRNAs in the CNS of mouse embryos and newborn opossums (Monodelphis domestica). Accordingly, we have devised three techniques applicable to whole-mounts: (i) An effective immunohistochemical procedure. This comprises a peroxidase-antiperoxidase method (PAP-WM) based on protocols initially developed for Xenopus embryos and oocytes, including a variation to detect exogenously applied nucleotide analogs such as 5-bromo-2'-deoxyuridine (PAP[BrdU]-WM). For greater resolution we have introduced a novel gold-silver method (IGSS-WM). (ii) An in situ hybridization procedure (ISH[PAP]-WM) which combines PAP-WM with protocols described for Xenopus. (iii) A deconvolution (optical sectioning) procedure which improves resolution for bright-field microscopy. We show that reliable whole-mount staining can be obtained using isolated CNS aged up to mouse embryonic day 17 and newborn opossum up to 15 days. Examples are shown of preparations in which one can directly localize nerve cells containing neurotransmitters, cytoskeletal proteins, nucleotide analogs and growth factor messages.

Three-dimensional visualization of the distribution, growth, and regeneration of monoaminergic neurons in whole mounts of immature mammalian CNS.

At birth, the opossum, Monodelphis domestica, corresponds roughly to a 14-day-old mouse embryo. The aim of these experiments was to compare the distribution of monoaminergic neurons in the two preparations during development and to follow their regeneration after injury. Procedures that allowed antibody staining to be visible in transparent whole mounts of the entire central nervous system (CNS) were devised. Neurons throughout the brain and spinal cord were stained for tyrosine hydroxylase (TH) and for serotonin (5-HT). At birth, patterns of monoaminergic cells in opossum CNS resembled those found in 14-day mouse embryos and other eutherian mammals. By postnatal day 5, immunoreactive cell bodies were clustered in appropriate regions of the midbrain and hindbrain, and numerous axons were already present throughout the spinal cord. Differences found in the opossum were the earlier presence of TH neurons in the olfactory bulb and of 5-HT neuronal perikarya in the spinal cord. Most, if not all, monoaminergic neurons in opossum were already postmitotic at birth. To study regeneration, crushes were made in cervical cords in culture. By 5 days, 8% of all TH-labeled axons and 14% of serotonergic axons had grown beyond lesions. Distal segments of monoaminergic axons degenerated. In CNS preparations from opossums older than 11 days, no regeneration of monoaminergic fibers occurred. Isolated embryonic mouse CNS also showed regeneration across spinal cord lesions, providing the possibility of using knockout and transgenic animals. Our procedures for whole-mount observation of identified cell bodies and their axons obviates the need for serial reconstructions and allows direct comparison of events occurring during development and regeneration.

Development of walking, swimming and neuronal connections after complete spinal cord transection in the neonatal opossum, Monodelphis domestica.

Development of coordinated movements was quantitatively assessed in adult opossums (Monodelphis domestica) with thoracic spinal cords transected by (1) crushing 7-8 d after birth [postnatal days 7-8 (P7-P8)]; at 2-3 years of age, systematic behavioral tests (e.g., climbing, footprint analysis, and swimming) showed only minor differences between control (n = 5) and operated (n = 10) animals; and (2) cutting on P4-P6; at 1 month these opossums exhibited coordinated walking movements but were unable to right themselves from a supine position, unlike controls (n = 6). When tested at 2 or 6 months, they could right themselves and showed remarkable coordination, albeit with more differences from controls than after a crush. No animals with spinal cords that were crushed at P14-18 survived because of cannibalism by the mother. Morphological studies (n = 10) 3 months-3 years after crush at 1 week showed restoration of structural continuity and normal appearance at the lesion site. Animals with cut rather than crushed cords showed continuity but greater morphological deficits. That lesions were complete was demonstrated by examining morphology and nerve impulse conduction immediately after crushing or cutting the spinal cord in controls. After lumbar spinal cord injection of 10 kDa dextran amine, retrogradely labeled cells were found rostral to the lesion in hindbrain and midbrain nuclei. Conduction was restored across the site of the lesion. Thus complete spinal cord transection in neonatal Monodelphis was followed by development of coordinated movements and repair of the spinal cord, a process that included development of functional connections by axons that crossed the lesion.

Chemosensory and cholinergic stimulation of fictive respiration in isolated CNS of neonatal opossum.

1. The aim of the present experiments was to characterize the central chemical drive of fictive respiration in the isolated CNS of the newborn opossum, Monodelphis domestica. This opossum preparation, in contrast to those of neonatal rats and mice, produces respiratory rhythm of high frequency in vitro. 2. Fictive respiration was recorded from C3-C5 ventral roots of the isolated CNS of 4- to 14-day-old opossums using suction electrodes. At room temperature (21-23 degrees C) the frequency of respiration was 43 +/- 5.3 min-1 (mean +/- S.E.M., n = 50) in basal medium Eagle's medium (BMEM) equilibrated with 5% CO2-95% O2, pH 7.37-7.40. Respiratory discharges remained regular throughout 8 h experiments and continued for more than 20 h in culture. 3. Superfusion of the brainstem confirmed that solutions of pH 6.3-7.2 increased both the amplitude and frequency of respiration. High pH solutions (7.5-7.7) had the opposite effect and abolished the rhythm at pH 7.7. Addition of ACh (50-100 microM) or carbachol (0.01-10 microM) to the brainstem superfusion also increased the amplitude and frequency of respiratory activity, as did physostigmine (50-100 microM) or neostigmine (20-50 microM). Conversely, scopolamine (50-100 microM) reduced the amplitude and frequency of the basal respiratory rhythm by about 30%. 4. H(+)- and cholinergic-sensitive areas on the surface of the isolated CNS were explored with a small micropipette (outer tip diameter, 100 microns) filled with BMEM (pH 6.5) or 1 microM carbachol. Carbachol applied to H(+)- and cholinergic-sensitive areas in the ventral medulla mimicked the changes of respiratory pattern produced by low pH application. Responses to altered pH and carbachol were abolished by scopolamine (50 microM). Histochemistry demonstrated several medullary groups of neurons stained for acetylcholinesterase. The superficial location of one of these groups coincided with a functional and anatomically well-defined pH- and carbachol-sensitive area placed medial to the hypoglossal roots. 5. Exploration of chemosensitive areas revealed that application of drugs or solutions of different pH to a single well-defined spot could have selective and distinctive effects upon amplitude and frequency of respiratory activity. 6. These results show that fictive respiration in the isolated CNS of the newborn opossum is tonically driven by chemical- and cholinergic-sensitive areas located on the ventral medulla, the activity of which regulates frequency and amplitude of respiration. They suggest that a cholinergic relay, although not essential for rhythm generation, is involved in the central pH chemosensory mechanism, or that cholinergic and chemical inputs converge upon the same input pathway to the respiratory pattern generator.

Neurite outgrowth through lesions of neonatal opossum spinal cord in culture.

The aim of these experiments was to analyze neurite outgrowth during regeneration of opossum spinal cord isolated from Monodelfis domestica and maintained in culture for 3-5 days. Lesions were made by crushing with forceps. In isolated spinal cords of animals aged 3 days, neurites entered the crush and grew along the basal lamina of the pia mater. Growth cones with pleiomorphic appearance containing vesicles, mitochondria and microtubules were abundant in the marginal zone, as were synaptoid contacts with active zones facing basal lamina. In preparations from animals aged 11-12 days, the lesion site was disrupted and contained only degenerating axons, debris and vesicles. Axons and growth cones entered the edge of the lesion but did not extend into it. Lesions in young animals extended over distances of more than 1 mm and contained no radial glia. The damaged area in older preparations was restricted to the crush site with normal astrocytes, oligodendrocytes and neurons immediately adjacent to the lesion. Thus, similar crushes produced more extensive damage in younger spinal cords that were capable of regeneration than in older cords that were not. Dorsal root ganglion fibers labeled with carbocyanine dye (DiI) were observed by video imaging as they grew through lesions. Individual growth cones examined subsequently by electron microscopy had grown again along pial basal lamina. After 5 days in culture dorsal root stimulation gave rise to discharges in ventral roots beyond the lesion indicating that synaptic connections were formed by growing fibers.

Myelin-associated neurite growth-inhibitory proteins and suppression of regeneration of immature mammalian spinal cord in culture.

Neurite outgrowth across spinal cord lesions in vitro is rapid in preparations isolated from the neonatal opossum Monodelphis domestica up to the age of 12 days. At this age oligodendrocytes, myelin, and astrocytes develop and regeneration ceases to occur. The role of myelin-associated neurite growth-inhibitory proteins, which increase in concentration at 10-13 days, was investigated in culture by applying the antibody IN-1, which blocks their effects. In the presence of IN-1, 22 out of 39 preparations from animals aged 13-17 days showed clear outgrowth of processes into crushes. When 34 preparations from 13-day-old animals were crushed and cultured without antibody, no axons grew into the lesion. The success rate with IN-1 was comparable to that seen in younger animals but the outgrowth was less profuse. IN-1 was shown by immunocytochemistry to penetrate the spinal cord. Other antibodies which penetrated the 13-day cord failed to promote fiber outgrowth. To distinguish between regeneration by cut neurites and outgrowth by developing uncut neurites, fibers in the ventral fasciculus were prelabeled with carbocyanine dyes and subsequently injured. The presence of labeled fibers in the lesion indicated that IN-1 promoted regeneration. These results show that the development of myelin-associated growth-inhibitory proteins contributes to the loss of regeneration as the mammalian central nervous system matures. The definition of a critical period for regeneration, coupled with the ability to apply trophic as well as inhibitory molecules to the culture, can permit quantitative assessment of molecular interactions that promote spinal cord regeneration.

The critical period for repair of CNS of neonatal opossum (Monodelphis domestica) in culture: correlation with development of glial cells, myelin and growth-inhibitory molecules.

A comparison was made of neurite growth across spinal cord lesions in the isolated central nervous system (CNS) of newborn opossums (Monodelphis domestica) at various stages of development. The aim was to define the critical period at which growth after injury ceases to occur, with emphasis on growth-inhibitory proteins, myelin and glial cells. In postnatal opossums 3-6 days old (P3-6), repair was observed 5 days after lesions were made in culture at the cervical level (C7) by crushing with forceps. Through-conduction of action potentials was re-established and axons stained by Dil grew into and beyond the crush. In a series of 66 animals 29 showed repair. In 28 animals at P11-12 with comparable lesions repair was observed in five preparations. At P13-14, the CNS was still viable in culture, but none of the 25 preparations examined showed any axonal growth into the crush or conduction through it. The rostro-caudal gradient of development permitted lesions to be made in mature cervical and immature lumbar regions of P11-12 spinal cord. Growth across crushes occurred in lumbar but not in cervical segments of the same preparation. The development of glial cells and myelin was assessed by electron microscopy and by staining with specific antibodies (Rip-1 and myelin-associated glycoprotein) in cervical segments of neonatal P6-14 opossums. At P8, oligodendrocytes and thin myelin sheaths started to appear followed at P9 by astrocytes stained with antibody against glial fibrillary acidic protein. By P14, astrocytes, oligodendrocytes and well-developed myelin sheaths were abundant. The cervical crush sites of P12 cords contained occasional astrocytes but no oligodendrocytes. Specific antibodies (IN-1) to neurite growth-inhibiting proteins (NI-35/250) associated with oligodendrocytes and myelin in the rat CNS cross-reacted with opossum proteins. Assays using the spreading of 3T3 fibroblasts and IN-1 showed that by P7 inhibitory proteins became apparent, particularly in the hindbrain and cervical spinal cord. The concentrations of NI-35/250 thereafter increased and became abundant in the adult opossum. Our finding of a well-defined critical period, encompassing only 5 days, in CNS preparations that can be maintained in culture offers advantages for analysing mechanisms that promote or prevent CNS repair.

Development and migration of olfactory neurones in the nervous system of the neonatal opossum.

The neonatal opossum (Monodelphis domestica) was used to assess how different populations of cells are generated in the olfactory region, and how they migrate along pathways to the central nervous system. Developing nerve cells were immunocytochemically labelled using antisera directed against two specific markers of olfactory receptor neurones: olfactory marker protein (OMP) and the dipeptide carnosine. In new-born opossums both carnosine and OMP are already co-expressed in primary olfactory neurones and in those axons that extend towards the olfactory bulb. Expression of these markers in olfactory receptor neurones during the first postnatal days reflects the advanced developmental state of this system compared to other regions of the central nervous system (such as the cortex and cerebellum), which are highly immature and less developed in comparison with those of new-born rats or mice. A second, distinct population of carnosine/OMP expressing cells was also identified during the first postnatal week. These neurones were present as clusters along the olfactory nerve bundles, on the ventral-medial aspect of the olfactory bulb and in the basal prosencephalon. The distribution of this cell population was compared to another group of well characterized migratory neurones derived from the olfactory placode, which express the decapeptide GnRH (Gonadotropin-releasing hormone, also known as LHRH). GnRH was never co-localized with carnosine/OMP in the same migratory cells. These observations show that distinct cell populations arise from the olfactory placode in the neonatal opossum and that they migrate to colonize the central nervous system by following common pathways.

Repair and recovery following spinal cord injury in a neonatal marsupial (Monodelphis domestica).

1. Repair and recovery following spinal cord injury (complete spinal cord crush) has been studied in vitro in neonatal opossum (Monodelphis domestica), fetal rat and in vivo in neonatal opossum. 2. Crush injury of the cultured spinal cord of isolated entire central nervous system (CNS) of neonatal opossum (P4-10) or fetal rats (E15-E16) was followed by profuse growth of fibres and recovery of conduction of impulses through the crush. Previous studies of injured immature mammalian spinal cord have described fibre growth occurring only around the lesion, unless implanted with fetal CNS. 3. The period during which successful growth occurred in response to a crush is developmentally regulated. No such growth was obtained after P12 in spinal cords crushed in vitro at the level of C7-8. 4. In vivo, in the neonatal (P4-8) marsupial opossum, growth of fibres through, and restoration of, impulse conduction across the crush was apparent 1-2 weeks after injury. With longer periods of time after crushing a considerable degree of normal locomotor function developed. 5. By the time the operated animals reached adulthood, the morphological structure of the spinal cord, both in the region of the crush and on either side of the site of the lesion, appeared grossly normal. 6. The results are discussed in relation to the eventual longterm possibility of devising effective treatments for patients with spinal cord injuries.

Electrical activity, growth cone motility and the cytoskeleton.

The development of the nervous system takes place in two main steps: first an extensive preliminary network is formed and then it is pruned and trimmed to establish the final form. This refinement is achieved by mechanisms that include cell death, selective growth and loss of neurites and the stabilization and elimination of synapses. The focus of this review is on selective neurite retraction during development, with particular emphasis on the role of electrical activity. In many developing vertebrate and invertebrate neurones, the frequency and duration of ongoing impulse activity determine the final arborizations and the pattern of connections. When impulse traffic is silenced, axons fail to retract branches that had grown to inappropriate destinations in the mammalian visual system, cerebellum and neuromuscular junctions. Similarly, in crustaceans, Drosophila melanogaster and leeches, refinements in axonal morphology during development are influenced by impulse activity. From experiments made in culture, it has been possible to mimic these events and to show a clear link between the density of voltage-activated calcium channels in a neurite and its retraction following stimulation. The distribution of these calcium channels in turn is determined by the substratum with which the neurites are in contact or by the formation of synapses. Several lines of evidence suggest that calcium entry into the growth cone leads to collapse by disruption of actin filaments. One candidate for coupling membrane events to neurite retraction is the microfilament-associated protein gelsolin which, in its calcium-activated state, severs actin filaments. Open questions that remain concern the differential effects of activity on dendrites and axons as well as the mechanisms by which the growth cone integrates information derived from stimuli in the cell and in the extracellular environment.

Schoolwork, homework, life's work: the experience of students with and without learning disabilities.

Elementary school students were interviewed about schoolwork, homework, and personal learning projects (e.g., learning about astronomy). Four groups of students were distinguished. Those in the first group experienced school knowledge as an integral part of life and inseparable from their personal projects; students in the second group saw such knowledge as necessary for preparing for life, but as less engaging than their personal projects. For those in the third group, schoolwork was an imposition, contrasting sharply with satisfying personal learning projects. Those in the fourth group lacked absorbing personal learning projects and found schoolwork to be an imposition. Students with learning disabilities (more than students without) fell into the last category. Fostering more favorable motivation and voice (ability to articulate purposes and critique schooling) in such students might involve changing their views of school knowledge, helping them find personal identity-building learning projects, and reducing the dichotomy between schoolwork and personal projects.