Achieving assessor accuracy on the International Standards for Neurological Classification of Spinal Cord Injury.

STUDY DESIGN: A retrospective audit of assessor accuracy using the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) in three multicentre randomised controlled trials (SCIPA: Spinal Cord Injury and Physical Activity) spanning 2010-2014 with standards revised in 2011. OBJECTIVES: To investigate assessor accuracy of neurological classification after spinal cord injury. SETTING: Australia and New Zealand. METHODS: ISNCSCI examinations were undertaken by trained clinicians prior to randomisation. Data were recorded manually and ISNCSCI worksheets circulated to panels, consensus reached and worksheets corrected. An audit team used a 2014 computerised ISNCSCI algorithm to check manual worksheets. A second audit team assessed whether the 2014 computerised algorithm accurately reflected pre- and post-2011 ISNCSCI standards. RESULTS: Of the 208 ISNCSCI worksheets, 24 were excluded. Of the remaining 184 worksheets, 47 (25.5%) were consistent with the 2014 computerised algorithm and 137 (74.5%) contained one or more errors. Errors were in motor (30.1%) or sensory (12.4%) levels, zone of partial preservation (24.0%), motor/sensory scoring (21.5%), ASIA Impairment Scale (AIS, 8.3%) and complete/incomplete classification (0.8%). Other difficulties included classification when anal contraction/sensation was omitted, incorrect neurological levels and violation of the 'motor follows sensory rule in non-testable myotomes' (7.4%). Panel errors comprised corrections that were incorrect or missed or incorrect changes to correct worksheets. CONCLUSION: Given inaccuracies in the manual ISNCSCI worksheets in this long-term clinical trial setting, continued training and a computerised algorithm are essential to ensure accurate scoring, scaling and classification of the ISNCSCI and confidence in clinical trials.

SCIPA Com: outcomes from the spinal cord injury and physical activity in the community intervention.

STUDY DESIGN: Quasi-experimental translational study with pre- and post-measures. OBJECTIVES: To determine the effects of the Spinal Cord Injury and Physical Activity in the Community (SCIPA Com) intervention on leisure-time physical activity (LTPA) and associated outcomes among participants with spinal cord injury (SCI). SETTING: Young Men's Christian Associations and community fitness centers, Australia and New Zealand. METHODS: SCIPA Com consisted of three stages: (i) training exercise professionals via the Train the Trainers Spinal Cord Injury course; (ii) implementation of supervised physical activity programs twice a week for 30 to 60 min for 8 to 12 weeks; and (iii) follow-up assessments on health outcomes over 9 months. Participants with SCI were classified as active or inactive by baseline LTPA levels and linear mixed methods compared LTPA between groups over time. RESULTS: Sixty-four community-dwelling participants with SCI completed customized physical activity programs. Compared with baseline, there were significant improvements in LTPA (26 min per day, 95% confidence interval (CI): 16.6-35.4; P<0.001), functional goals (2, 95% CI: 1.72-2.37; P<0.001), self-esteem (1.5, 95% CI: 0.72-2.27; P<0.001) and overall quality of life (P<0.05). Over time, LTPA participation was greater among the active compared with the inactive group, although LTPA levels among the inactive improved compared with baseline. CONCLUSIONS: Significant improvements in LTPA participation and health outcomes were observed, especially among inactive individuals with SCI. SCIPA Com is an ecologically valid intervention based on training and support provided to community exercise professionals who, although new to adapted training, delivered effective physical activity programs for those at risk of inactivity. SPONSORSHIP: Transport Accident Commission (Project Number DP172) and the International Postgraduate Research Scholarship (IPRS), Curtin University.

Non-invasive stimulation of the vibrissal pad improves recovery of whisking function after simultaneous lesion of the facial and infraorbital nerves in rats.

We have recently shown that manual stimulation of target muscles promotes functional recovery after transection and surgical repair to pure motor nerves (facial: whisking and blink reflex; hypoglossal: tongue position). However, following facial nerve repair, manual stimulation is detrimental if sensory afferent input is eliminated by, e.g., infraorbital nerve extirpation. To further understand the interplay between sensory input and motor recovery, we performed simultaneous cut-and-suture lesions on both the facial and the infraorbital nerves and examined whether stimulation of the sensory afferents from the vibrissae by a forced use would improve motor recovery. The efficacy of 3 treatment paradigms was assessed: removal of the contralateral vibrissae to ensure a maximal use of the ipsilateral ones (vibrissal stimulation; Group 2), manual stimulation of the ipsilateral vibrissal muscles (Group 3), and vibrissal stimulation followed by manual stimulation (Group 4). Data were compared to controls which underwent surgery but did not receive any treatment (Group 1). Four months after surgery, all three treatments significantly improved the amplitude of vibrissal whisking to 30° versus 11° in the controls of Group 1. The three treatments also reduced the degree of polyneuronal innervation of target muscle fibers to 37% versus 58% in Group 1. These findings indicate that forced vibrissal use and manual stimulation, either alone or sequentially, reduce target muscle polyinnervation and improve recovery of whisking function when both the sensory and the motor components of the trigemino-facial system regenerate.

The AuSpinal: a test of hand function for people with tetraplegia.

STUDY DESIGN: Five-phased reliability and validity study. OBJECTIVES: To develop and test an assessment tool designed to quantify unilateral hand function in people with tetraplegia. SETTING: Seven spinal injury units in Australia. METHODS: The AuSpinal is a new assessment tool comprising seven tasks designed to quantify unilateral hand function in people with tetraplegia. There were five phases in this study: (1) development of the AuSpinal; (2) testing the test-retest and intrarater reliability of repeat ratings of 84 videos as determined by 13 therapists; (3) testing the interrater reliability and internal consistency of simultaneous real-life ratings of eight hands as determined by six therapists; (4) testing the range of scores from cross-sectional data obtained from 50 hands; and (5) quantifying sensitivity to change from longitudinal data collected over the course of rehabilitation from 16 hands. RESULTS: The test-retest, intrarater and interrater reliabilities were high (intraclass correlation coefficients ranged from 0.79 to 0.98, 95% CI ranged from 0.72 to 1.0) with a Cronbach α-value of 0.93. There was a reasonable range in the scores obtained from the cross-sectional data of the 50 hands (interquartile range extended from 6 to 14). There was an obvious and marked change in AuSpinal scores over the course of patients' rehabilitation in 8 of the 16 hands. CONCLUSION: The AuSpinal provides a quick and reliable instrument to test hand function in people with tetraplegia. It is useful for people with poor hand function but requires the addition of more complex tasks for those with good hand function.

Manual stimulation of forearm muscles does not improve recovery of motor function after injury to a mixed peripheral nerve.

Transection and re-anastomosis of the purely motor facial nerve leads to poor functional recovery. However, we have recently shown in rat that manual stimulation (MS) of denervated vibrissal muscles reduces the number of polyinnervated motor endplates and promotes full recovery of whisking. Here, we examined whether MS of denervated rat forearm muscles would also improve recovery following transection and suture of the mixed (sensory and motor) median nerve (median-median anastomosis, MMA). Following MMA of the right median nerve, animals received no postoperative treatment, daily MS of the forearm muscles or handling only. An almost identical level of functional recovery, measured by the force of grip in grams, was reached in all animals by the sixth postoperative week and maintained till 3 months following surgery regardless of the postoperative treatment. Also, we found no differences among the groups in the degree of axonal sprouting, the extent of motor endplate polyinnervation and in the soma size of regenerated motoneurons. Taken together, we show that while MS is beneficial following motor nerve injury, combined strategies will be required for functional recovery following mixed nerve injury.

The effects of maternally administered methadone, buprenorphine and naltrexone on offspring: review of human and animal data.

Most women using heroin are of reproductive age with major risks for their infants. We review clinical and experimental data on fetal, neonatal and postnatal complications associated with methadone, the current "gold standard", and compare these with more recent, but limited, data on developmental effects of buprenorphine, and naltrexone. Methadone is a micro-opioid receptor agonist and is commonly recommended for treatment of opioid dependence during pregnancy. However, it has undesired outcomes including neonatal abstinence syndrome (NAS). Animal studies also indicate detrimental effects on growth, behaviour, neuroanatomy and biochemistry, and increased perinatal mortality. Buprenorphine is a partial micro-opioid receptor agonist and a kappa-opioid receptor antagonist. Clinical observations suggest that buprenorphine during pregnancy is similar to methadone on developmental measures but is potentially superior in reducing the incidence and prognosis of NAS. However, small animal studies demonstrate that low doses of buprenorphine during pregnancy and lactation lead to changes in offspring behaviour, neuroanatomy and biochemistry. Naltrexone is a non-selective opioid receptor antagonist. Although data are limited, humans treated with oral or sustained-release implantable naltrexone suggest outcomes potentially superior to those with methadone or buprenorphine. However, animal studies using oral or injectable naltrexone have shown developmental changes following exposure during pregnancy and lactation, raising concerns about its use in humans. Animal studies using chronic exposure, equivalent to clinical depot formulations, are required to evaluate safety. While each treatment is likely to have maternal advantages and disadvantages, studies are urgently required to determine which is optimal for offspring in the short and long term.

The dance of the growth cones--where to next?

Axon guidance in the developing nervous system is accomplished by a remarkable structure, the axon growth cone. This structure navigates, often over long distances, to find and synapse with target cells. Transformation of the growth cone to a terminal arbor establishes functional circuitry. The navigational properties of growth cones, and their interactions with target tissue, have been studied widely by examining individual cells in vitro, and have also been inferred from histological sections. Recent advances in labelling techniques and imaging of living cells have enabled direct observation of the growing axon tip in intact embryos as well as in slice preparations. To understand how pathways and terminal arbors are formed, the challenge now is to relate the dynamic morphology and behaviour of living growth cones to surrounding cues in the complex environment of the developing embryo.

Changing Pax6 expression correlates with axon outgrowth and restoration of topography during optic nerve regeneration.

Pax6, a member of the highly conserved developmental Pax gene family, plays a crucial role in early eye development and continues to be expressed in adult retinal ganglion cells (RGCs). Here we have used Western blots and immunohistochemistry to investigate the expression of Pax6 in the formation and refinement of topographic projections during optic nerve regeneration in zebrafish and lizard. In zebrafish with natural (12-h light/dark cycle) illumination, Pax6 expression in RGCs was decreased during axon outgrowth and increased during the restoration of the retinotectal map. Rearing fish in stroboscopic illumination to prevent retinotopic refinement resulted in a prolonged decrease in Pax6 levels; return to natural light conditions resulted in map refinement and restoration of normal Pax6 levels. In lizard, RGC axons spontaneously regenerate but remain in a persistent state of regrowth and do not restore topography; visual training during regeneration, however, allows a stabilization of connections and return of topography. Pax6 was persistently decreased in untrained animals but remained increased in trained ones. In both species, changes in expression were not due to cell division or cell death. The results suggest that decreased Pax6 expression is permissive for axon regeneration and extensive searching, while higher levels of Pax6 are associated with restoration of topography.

Enabling dual cellular destinations of polymeric nanoparticles for treatment following partial injury to the central nervous system.

Following neurotrauma, oxidative stress is spread via the astrocytic syncytium and is associated with increased aquaporin 4 (AQP4), inflammatory cell infiltration, loss of neurons and glia and functional deficits. Herein we evaluate multimodal polymeric nanoparticles functionalized with an antibody to an extracellular epitope of AQP4, for targeted delivery of an anti-oxidant as a therapeutic strategy following partial optic nerve transection. Using fluorescence microscopy, spectrophotometry, correlative nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy, in vitro and in vivo, we demonstrate that functionalized nanoparticles are coated with serum proteins such as albumin and enter both macrophages and astrocytes when administered to the site of a partial optic nerve transection in rat. Antibody functionalized nanoparticles synthesized to deliver the antioxidant resveratrol are effective in reducing oxidative damage to DNA, AQP4 immunoreactivity and preserving visual function. Non-functionalized nanoparticles evade macrophages more effectively and are found more diffusely, including in astrocytes, however they do not preserve the optic nerve from oxidative damage or functional loss following injury. Our study highlights the need to comprehensively investigate nanoparticle location, interactions and effects, both in vitro and in vivo, in order to fully understand functional outcomes.

Generation of transgenic mice with mild and severe retinal neovascularisation.

AIM: To generate a mouse model for slow progressive retinal neovascularisation through vascular endothelial growth factor (VEGF) upregulation. METHODS: Transgenic mice were generated via microinjection of a DNA construct containing the human VEGF165 (hVEGF) gene driven by a truncated mouse rhodopsin promoter. Mouse eyes were characterised clinically and histologically and ocular hVEGF levels assayed by ELISA. RESULTS: One transgenic line expressing low hVEGF levels showed mild clinical changes such as focal fluorescein leakage, microaneurysms, venous tortuosity, capillary non-perfusion and minor neovascularisation, which remained stable up to 3 months postnatal. Histologically, there were some disturbance and thinning of inner and outer nuclear layers, with occasional focal areas of neovascularisation. By contrast, three other lines expressing high hVEGF levels presented with concomitantly severe phenotypes. In addition to the above, clinical features included extensive neovascularisation, haemorrhage, and retinal detachment; histologically, focal to extensive areas of neovascularisation associated with retinal folds, cell loss in the inner and outer nuclear layers, and partial retinal detachment were common. CONCLUSIONS: The authors generated four hVEGF overexpressing transgenic mouse lines with phenotypes ranging from mild to severe neovascularisation. These models are a valuable research tool to study excess VEGF related molecular and cellular changes and provide additional opportunities to test anti-angiogenic therapies.

Early development of retinal ganglion cell dendrites in the marsupial Setonix brachyurus, quokka.

The dendritic morphology of retinal ganglion cells was studied in flat-mounted retinae of the marsupial Setonix brachyurus, quokka. In the adults, horseradish peroxidase (HRP) was applied to the vitread surface of flattened retinae. Wide-, large-, medium-, and small-field classes appeared to correspond to gamma, alpha, delta, and beta cells, respectively, in the cat (Boycott and Wässle, J. Physiol. 249:397-419, 1974). To reveal the early stages of dendritic development, HRP was placed on the optic nerve of isolated eye cups from the day of birth to postnatal day (P) 63 when the area centralis is beginning to form (Dunlop and Beazley, Dev. Brain Res. 23:81-90, 1985). Youngest cells lacked dendrites and had an elongate soma in the cytoblastic layer with an endfoot contacting the ventricular surface. Once in the ganglion cell layer, the soma was rounded and dendrites appeared as short, unbranched processes. Most cells were asymmetric or "polarised" with the axon arising from the side nearest the optic disk and dendrites from the opposite side. Polarity was maintained in cells with longer, branched dendrites. A small proportion of cells exhibited a reversed polarity in which the axon arose from the side nearest the retinal edge and dendrites towards the disk. Cells appeared to acquire an approximately symmetric, adult-like tree by the addition of new primary dendrites between the existing ones and the axon hillock. Wide-, large-, medium-, and small-field cells were evident from P6, P25, P31, and P40, respectively. Spines were observed on dendrites and axons during development but were rare in the adult. Some dendro-axons were seen at all ages examined. The existence of an initial axodendritic polarity in retinal ganglion cells supports the hypothesis that the axon hillock is the determinant of dendritic geometry (Maffei and Perry, Dev. Brain Res. 41:185-194, 1988). Polarity may also contribute to the establishment of "radial orientation" in which the long axis of the elliptical dendritic tree of cells outside the area centralis points towards central retina and the weighted centre is displaced towards the retinal periphery (Leventhal and Schall, J. Comp. Neurol. 220:465-475, 1983).

Cell death in the developing retinal ganglion cell layer of the wallaby Setonix brachyurus.

The distribution and number of dying cells in the developing retinal ganglion cell layer of the wallaby Setonix brachyurus were assessed by using cresyl violet stained tissue. The density of dying cells has been expressed per 100 live cells for the entire retinal surface, data being presented as a grid of 500 micron squares. For statistical analysis, retinae were divided into 8 regions; dorsal, ventral, nasal, and temporal quadrants, each further divided into center and periphery. This method allowed comparison of the extent of cell death at different retinal locations as the high density area centralis of live cells developed temporal to the optic disk from 60 days onward. Between 30 and 70 days, dying cells were seen across the entire retina; beyond 100 days very few were seen. Initially, there was a significantly higher incidence of dying cells in the central retina compared to the periphery, whereas from 50 days this situation was reversed. Analysis of the central retina before and during area centralis formation consistently indicated a significantly lower number of dying cells per 100 live cells in temporal compared to other retinal quadrants. This differential pattern suggests that cell death lowers live cell densities less in the emerging area centralis than elsewhere, and therefore must play a part in establishing live cell density gradients. However, we cannot exclude the possibility that other factors are also instrumental. Indeed, factors such as areal growth (Beazley et al., in press) presumably operate at later stages since live cell density gradients continue to be accentuated even after cell death is complete. Numbers of dying cells peaked by 50 days, reaching approximately 1% of the live cell population. At this stage, counts were also maximal for live cells with values up to 30% above the adult range.

A biphasic sequence of myelination in the developing optic nerve of the frog.

We have examined the sequence of myelination along the optic nerve of the frog Litoria (Hyla) moorei from early tadpole life to adulthood. Myelinated axons were counted in electron micrographs of transverse sections taken from behind the eye, at the optic foramen and the chiasm. In tadpoles, myelinated axon numbers were significantly higher at the foramen than at the other levels. By metamorphic climax, numbers had risen at all three levels but more so behind the eye and at the chiasm to become approximately equal along the nerve. After metamorphosis, there was a dramatic increase in myelinated axon numbers, but another pattern was seen; in frogs of 5 cm and 7 cm body length, counts were significantly higher at the chiasm than at the foramen and lowest behind the eye. Thereafter, myelinated axon numbers stabilized at the chiasm but increased behind the eye and at the foramen so that in the most mature stage for this species, 9 cm adults, counts were again similar at the three levels. In addition, total axon numbers, that is, myelinated plus unmyelinated, were assessed from electron micrographs and increased from approximately 8,500 in early tadpoles to 0.65 million in fully mature adults. The proportion of axons that were myelinated showed two peaks, one before and the other after metamorphosis. Measurements of axon diameters from electron micrographs suggested that there was a critical diameter for myelination of 0.3 microns before, and of 0.5 microns after metamorphosis. The data indicate that there is a biphasic sequence of myelination of optic axons, the first phase being pre-metamorphic and the second post-metamorphic. The first phase is initiated at the foramen, and then extends both towards the eye and chiasm and continues until metamorphic climax. During the second phase, myelination originates at the chiasm, spreads towards the eye, and is complete only in the most mature adults. The critical diameter for myelination is smaller in the first phase than in the second.

The evolution of an area centralis and visual streak in the marsupial Setonix brachyurus.

The distribution, morphology, size, and number of cells in the retinal ganglion layer of the marsupial Setonix brachyurus, "quokka," was studied from 25 days postnatal to adulthood using Nissl-stained wholemounts. The total cell population was evenly distributed up to 50 days, but by 75 days highest densities were generally observed in a broad band extending across the nasotemporal axis. At 87 days, a temporally situated area centralis was seen for the first time. This was embedded in a horizontally aligned visual streak, the nasal arm of which contained areas of high density. By 106 days, densities in the area centralis had stabilized while peripheral values were higher than adult levels even at 180 days. In the adult, the area centralis was surrounded by a weak visual streak. Retinal area increased steadily during development to reach 168 mm2 at 180 days, the adult range being 225-250 mm2. All cells in the ganglion layer appeared undifferentiated and rounded at 33 days with soma diameters of 3-6 micrometers; by 70 days diameters had increased to 4-12 micrometers and some cells had axon hillocks containing Nissl substance. From 87 days we distinguished ganglion cells, which constituted 54-63% of the total. These were identified by deeply stained Nissl substance and had diameters of 7-18 micrometers, compared to 7-23 micrometers at 143 days and 7-24 micrometers in the adult; the remaining cells, termed glia/interneurons, were 5-8 micrometers throughout. Only ganglion cells were organized into an area centralis and visual streak. Glia/interneurons were evenly distributed except at the extreme periphery, where their density increased. In sectioned material, the ganglion layer was distinct from 25 days while the neuroblastic layer separated only between 48 and 85 days. From 25 to 250 days the total number of cells in the ganglion layer remained similar to the adult range of 336,000-393,000. At both 87 days and in adults optic axon counts fell between 180,000 and 224,000, close to ganglionic cell estimates. At 25 and 34 days, respectively, optic axon numbers were 75,000 and 172,000. Myelination was absent at 25 and 34 days, 3% at 87 days, and almost 100% in adults. Mechanisms are discussed whereby the area centralis and visual streak may evolve from an even distribution of cells while their number remains constant; migration is considered likely to be important.

Topographic order of retinofugal axons in a marsupial: implications for map formation in visual nuclei.

We studied axon order in the primary visual pathway and in nine retinorecipient nuclei of a small marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata) using animals at postnatal day (P) 40 and P80. Dorsal, ventral, nasal, and temporal axons enter the optic nerve true to their retinal origin being respectively dorsal, ventral, medial, and lateral; the arrangement is retained to the chiasm. Dorsal and ventral axons maintain their respective locations within the chiasm but at the base of the contralateral optic tract undergo a 180 degrees axial rotation, thus reversing the dorsoventral axis with respect to the retina. The alignment is conserved along the optic tract with dorsal and ventral axons mapping directly into appropriate quadrants of each retinorecipient nucleus. Nasal and temporal axons remain segregated as they decussate and lie respectively superficially and deep along the optic tract but with some intermingling. Within each retinorecipient nucleus, the nasotemporal axis is clearly demarcated, being represented in either a rostrocaudal (ventral and dorsal lateral geniculate nuclei; lateral posterior, dorsal terminal, and pretectal nuclei) or caudorostral (medial terminal and caudal pretectal nuclei, intergeniculate nucleus and superior colliculus) direction. The results imply that the dorsoventral axis in the retinorecipient nuclei could be due to preordering within the pathway, whereas the nasotemporal axis is determined by target-based cues. Moreover, cues for the orientation of the nasotemporal axis within retinorecipient nuclei must be localised within individual nuclei rather than as a single organiser, as previously envisaged (Chung and Cooke [1978] Proc. R. Soc. Lond. B. 210:335-373).

Neural development in metatherian and eutherian mammals: variation and constraint.

A model for predicting the timing of neurogenesis in mammals (Finlay and Darlington [1995] Science 268:1578-1584) is here extended to an additional five metatherian species and to a variety of other events in neural development. The timing of both the outgrowth of axonal processes and the establishment and segregation of connections proves to be as highly predictable as neurogenesis. Expressed on a logarithmic scale, late developmental events are as predictable as early ones. The fundamental order of events is the same in eutherian and metatherian animals, but there is a curvilinear relation between the event scales of the two; for metatherians, later events are slowed relative to earlier events. Furthermore, in metatherians, the timing of developmental events is more variable than in eutherians. The slowing of late developmental events in metatherians is associated with their considerably longer time to weaning compared with eutherians.

Axon order in the visual pathway of the quokka wallaby.

Axon order throughout the visual pathway of the quokka wallaby (Setonix brachyurus) was determined after localised retinal applications of the tracers DiI and/or DiASP. Postnatal days (P) 22-90 were studied to encompass the development and refinement of retinal projections. Order was essentially similar at all stages. Axons entered the optic nerve head true to their sector of retinal origin. In the optic nerve, nasal and temporal axons continued to reflect their retinal origin, dominating, respectively, the medial and lateral halves. By contrast, dorsal and ventral axons exchanged locations between the retrobulbar level and one-third the distance along the nerve; thus, the inversion of the dorsoventral retinal axis, imposed by the lens, was corrected. Decussating axons maintained their relative locations through the chiasm. At the base of the optic tract, nasal and temporal axons underwent an axial rotation to lie on the medial and lateral sides, respectively; thus nasal overlapped with ventral axons and temporal with dorsal axons. Axons maintained their alignments throughout the tract, and as a result, nasal and ventral axons invaded the superior colliculus medially, whereas temporal and dorsal axons invaded laterally. Each retinal quadrant terminated preferentially in its retinotopically appropriate sector of the colliculus. The arrangement of axons in the quokka visual pathway displays several novel features. Axon order is distinct throughout, involving a well-demarcated exchange of dorsal and ventral axons in the nerve and an axial rotation of nasal and temporal axons at the base of the tract; these relocations suggest decision regions for growing axons. The organisation presumably underlies the less extensive searching within the developing superior colliculus to generate retinotopic maps in the quokka and also in tammar wallaby [Marotte, J. Comp Neurol. 293:524-539, 1990] than in the rat [Simon and O'Leary, J. Neurosci. 12:1212-1232, 1992].

Development of primary visual projections occurs entirely postnatally in the fat-tailed dunnart, a marsupial mouse, Sminthopsis crassicaudata.

We have examined the development of retinal projections in a diminutive polyprotodont marsupial, the fat-tailed dunnart, Sminthopsis crassicaudata. Here, we document the most immature mammalian visual system at birth described to date. At postnatal day (P) 0, the retinal ganglion cell layer has yet to form, and axons have not entered the optic stalk. By P4, the retinal ganglion cell layer could be distinguished at the posterior pole, and the front of growing axons extended one-third the length of the optic stalk, a distance of approximately 150 microm; a few pioneer growth cones had grown beyond the main axon group but had still to reach the midline. Axons had decussated at the optic chiasm by P10 to penetrate the base of the contralateral optic tract and, by P15, had reached the dorsal lateral geniculate nucleus (dLGN), superior colliculus (SC), and accessory optic system (AOS); ipsilaterally projecting axons matured slightly later. From P20, axons had reached the caudal SC both contralaterally and ipsilaterally and terminated throughout the depth of the retinorecipient layers. After P30, the projections gradually refined. Within the rostral dLGN, segregation into four contralateral and four ipsilateral bands occurred by P50, approximately 5 days after eye opening. The projection to the ipsilateral SC underwent refinement by P50, becoming restricted to its rostral pole, and presented as discrete patches within the stratum opticum. At birth, the dunnart visual system is comparable to early to midembryonic stages [embryonic day (E) 12, E14, E19, E24, and E30, respectively] in the mouse, rat, ferret, cat, and monkey. The extreme immaturity of the neonatal dunnart together with the observation that the entire development of the primary optic pathway occurs postnatally over a protracted period make this marsupial especially valuable for investigating factors that control pathway formation in the early developing mammalian primary visual system.

Optic nerve regenerates but does not restore topographic projections in the lizard Ctenophorus ornatus.

In adult fish and amphibians, the severed optic nerve regenerates and visual behaviour is restored. By contrast, optic axons do not regenerate in the more recently evolved birds and mammals. Here we have investigated optic nerve regeneration in a member of the class Reptilia, phylogenetically intermediate between the fish and amphibians and the birds and mammals. We assessed visual recovery anatomically and behaviourally one year after unilateral optic nerve crush in the adult ornate dragon lizard. Ctenophorus ornatus. Ganglion cell densities and numbers of axons in the optic nerve on either side of the crush site indicated that two-thirds of ganglion cells survived axotomy and regrew their axons. However, myelination fell from a mean of 21% in normals to 5.5% and 3%, proximal and distal to the crush, respectively. Anterograde labelling of the entire optic nerve showed that axons regenerated along essentially normal pathways and that the major projection, as in normals, was to the superficial one-third of the contralateral optic tectum. However, localised retinal injections indicated that regenerated projections lacked retinotopic order. Any one retinal region projected to the entire tectum. This feature presumably explains why the experimental lizards consistently appeared blind to stimuli via the regenerated nerve. Our findings indicate that although axons regenerate along essentially normal pathways in adult lizards, conditions within the visual centres do not allow regenerating optic axons to select appropriate central connections. In a wider context, the result suggests that the ability for regenerating central axons to form topographic maps may also have been lost in the more recently evolved vertebrate classes.

Retinal projections throughout optic nerve regeneration in the ornate dragon lizard, Ctenophorus ornatus.

In goldfish and frog, optic nerve regeneration is successful, with restoration of retinotopic projections in visual brain centres and the return of functional vision within 1-2 months. By contrast, at 1 year after unilateral optic nerve crush in the ornate dragon lizard (Ctenophorus ornatus), the regenerated retinotectal projections lack topographic order, presumably explaining why the lizards are blind via the experimental eye (Beazley et al. [1997] J. Comp. Neurol. 377:105-120). To determine whether other abnormalities are associated with the inability to restore topographic projections in the lizard, we charted anatomically the time course, accuracy, and stability of optic nerve regeneration by examining visual projections with the lipophillic dye 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI) applied to the optic disk at intervals up to 1 year after optic nerve crush; in addition, DiI tracing of small groups of axons was used to examine the topicity of axons projecting to the tectum. Axons re-innervated visual centres from between 1 and 2 months, a time frame comparable with that in goldfish and frog. However, the projections in lizard were found to differ from those in goldfish and frog in three major ways. First, there was considerable variability within the projection patterns both between individual lizards at any one stage and with time. Second, the projections were inaccurate. As in normal lizards, the major projection was to the contralateral optic tectum, although it lacked detectable retinotopic axon order throughout. Furthermore, misrouting occurred such that regenerating axons formed a persistent projection to the ipsilateral side of the brain that was considerably stronger and more widespread than normal. Minor visual centres also became re-innervated but, in addition, regenerating axons formed persistent projections into the opposite optic nerve and to non-retino-recipient regions such as the nucleus rotundus, hypothalamus, and olfactory nerve, as well as the posterior and tectal commissures. Third, the projections appeared unstable. Projections to both tecta were strongest between 3 and 5 months, but they diminished thereafter. The results suggest that, compared with goldfish and frog, in lizards both pathway and target cues are degraded and/or cannot be read adequately; as a consequence, regenerating axons are unable to navigate exclusively to visual centres and cannot re-form stable connections.