Tetracycline-encapsulated P(3HB) microsphere-coated 45S5 Bioglass(®)-based scaffolds for bone tissue engineering.

Bioglass(®)-based scaffolds for bone tissue engineering have been developed, which can also serve as carriers for drug delivery. For this, P(3HB) microspheres (PMSs) loaded with tetracycline were fabricated and immobilised on the scaffold surfaces by a modified slurry dipping technique. The sustained drug delivery ability in simulated body fluid was confirmed by using UV-Vis absorption spectroscopy measurements. The MTT assay using mouse fibroblast cells provided evidence that the tetracycline loaded microspheres produced in this study show limited cytotoxicity. The scaffolds developed in this work provide mechanical support, adequate 3D surface roughness, bioactivity and controlled drug delivery function, and are thus interesting candidates for bone tissue engineering applications.

Ag-doped 45S5 Bioglass®-based bone scaffolds by molten salt ion exchange: processing and characterisation.

There is increasing interest in developing scaffolds with therapeutic and antibacterial potential for bone tissue engineering. Silver is a proven antibacterial agent which bacteria such as MRSA have little or no defense against. Using an ion exchange method, silver ions have been introduced into 45S5 Bioglass(®) based scaffolds that were fabricated using the foam replication technique. This technique allows the introduction of Ag(+) ions onto the surface of the scaffold without compromising the scaffold bioactivity and other physical properties such as porosity. Controlling the amount of Ag(+) ions introduced onto the surface of the scaffold was achieved by tailoring the ion exchange parameters to fabricate samples with repeatable and predictable Ag(+) ion release behavior. In vitro studies in simulated body fluid were carried out to ensure that the scaffolds maintained their bioactivity after the introduction of Ag(+) ions. It was also shown that the addition of low concentrations (2000:1 w/w) of silver ions supported the attachment and viability of human periodontal ligament stromal cells on the 3D scaffolds. This work has thus confirmed ion exchange as an effective technique to introduce Ag(+) ions into 45S5 Bioglass(®) scaffolds without compromising the basic properties of 45S5 Bioglass(®) which are required for applications in bone tissue engineering.

Minimally invasive caries removal using bio-active glass air-abrasion.

OBJECTIVES: Alumina air-abrasion has been used to clean teeth and shown to over-prepare access cavity preparation to caries. This study investigated the working hypothesis that bio-active glass air-abrasion is more self-limiting than alumina for minimally invasive caries removal. METHODS: Human extracted molars were scored visually using ICDAS II criteria, divided into sound and carious groups and air-abraded with alumina (n=10) and bio-active glass (n=10) in each group, using identical operating parameters. The amount of enamel removed was semi-quantitatively assessed using scanning electron microscopy. Operating time was recorded. RESULTS: Bio-active glass abrasion removed extrinsic stain and substantial quantities of enamel from all carious fissures but not from sound, where only minimal microscopic surface modifications were observed. Alumina air-abrasion resulted in faster extrinsic stain and clinically substantial enamel removal in both sound and carious groups equally. CONCLUSIONS: Bio-active glass air-abrasion appeared to show a significant self-limiting tendency towards demineralised enamel and extrinsic stain removal but was slower in comparison to alumina air-abrasion. Self-limiting bio-active glass air-abrasion could be used clinically to clean teeth, detect caries and minimally prepare carious enamel as part of MI caries access or placing a sealant restoration.

Emergence of topography in the developing hamster retinocollicular projection: axial differences and the role of cell death.

The precise ordering of the hamster retinocollicular projection is established over the first two postnatal weeks, coincident with developmental cell death. We have used quantitative retrograde labelling to define topographic precision in the early postnatal projection, to describe its refinement and to assess the contribution played by selective retinal ganglion cell death. The hamster's short gestation period allows the investigation of events occurring prenatally in other rodents. Discrete injections of fluorescent beads in the superior colliculus followed by isodensity contour analysis of labelled retinal cells reveals a dramatic decrease in the extent of retina labelled between postnatal days 2, 6 and 12 (P2, P6, P12): the 20% contour encloses 38.3%, 8.3% and 1.8% of the retina at these ages. Paired injections of two different tracers at variable rostrocaudal (R-C) separations at P2 produced complete overlap of label even when injections were separated by over 1 mm. This was not true for paired mediolateral injections at P2 that were separated by more than 500 microm. Analysis of the segregation of the two tracers ('nearest-neighbour analysis') shows topography improving with age so that by P12 injections separated rostrocaudally by more than 500 microm produced no overlap in the retina. To examine the contribution of selective ganglion cell death to topographic refinement, animals given paired R-C injections at P2 were allowed to survive until P12. Nearest-neighbour analysis reveals significantly more order in the P2-P12 retinae than after overnight survival. Thus, selective cell death plays a small but appreciable role in correction of topographical errors.

A novel real-time confocal imaging technique for examining host-implant interfacial shear failure patterns.

In clinical practice, implant failure usually occurs at the biomaterial-host tissue interface, typically involving both biomechanical and biochemical mechanisms. By definition, any new 'bioactive' material will bond to living bone but, prior to clinical use, interface formation, performance, longevity and failure pattern characterizations are necessary. The common missing link in many biomaterial interface investigations is imaging at the point of presumed loaded failure. The novel real-time confocal technique described here allows bond strength, formation rate, longevity and bone-material interface failure pattern characterization for a wide range of biomaterials capable of forming tissue interfaces, in one real-time imaged microshear stress process, conducted using imaging frame matched load/displacement data acquisition under relatively normal near in vivo environmental conditions. The technique, validated by post-failure scanning electron microscopy imaging, revealed that more slowly reacting melt-derived 45S5 glass materials produced stronger and more stable long-term interfaces than faster reacting microporous bioactive sol-gel glasses.

Real-time confocal imaging, during active air abrasion -- substrate cutting.

Air abrasion cutting, using particulates accelerated in a controlled compressed gas stream, is currently being re-evaluated as a precision tissue removal technique for dental cavity preparation. The minimal vibrations and heat generated during cutting commend the technique for use in the shaping of fragile or brittle materials that are vulnerable to vibrations and thermal stresses. Traditional air abrasion studies have relied solely upon post-procedure imaging, and cutting process details have been inferred from the nature of the residual surface. In this paper, however, a real-time confocal microscopic imaging method is described, which for the first time has allowed prior target structure characterization with subsequent imaging of cutting interactions and substrate failure patterns. Using internally focusing long working distance Hill objective lenses, focusing deep to a protective microscope slide and adhesive interfaces, unhindered remote image sampling within the bulk of specimens such as tooth tissue, acrylic and brittle ceramics was possible. Moreover, areas of active cutting and inactive regions were identified within air abraded cavities during their creation. The characteristics of the finished cut surfaces were demonstrated and confirmed the findings of previous SEM studies. The method allowed direct control over all the known variables influencing cutting with particulate streams.

Responses of neurons in neonatal cortex and thalamus to patterned visual stimulation through the naturally closed lids.

In studies of the developing mammalian visual system, it has been axiomatic that visual experience begins with eye-opening. Any role for neuronal activity earlier in development has been attributed to the patterned spontaneous activity found in retina and lateral geniculate nucleus (LGN). Here we show that, as early as 2 wk before eye-opening, visual stimuli presented through the closed eyelids can drive neuronal activity in LGN and striate cortex of the ferret. At this age, spontaneous activity in cortex is much lower than in LGN, and the visual responses of many cortical, but not geniculate, neurons depend on the orientation of a moving grating. Furthermore the selectivity of cortical neurons to the orientation of gratings presented through the closed eyelids improves with age. Thus neuronal activity patterned by visual experience, rather than by spontaneous retinal activity, is present in visual cortex much earlier than previously thought. This could have important implications for the self-organization of visual cortex.

Spatiotemporal patterning of glutamate receptors in developing ferret striate cortex.

We have studied glutamate receptor levels during very early phases of cortical formation by using quantitative in vitro autoradiography to map the expression of NMDA, AMPA and kainate receptors in the developing primary visual cortex of the ferret. NMDA and non-NMDA receptors exhibit very different developmental profiles in primary visual cortex. NMDA receptor density is low at birth and increases throughout the first 2 postnatal months, rising between threefold (layers II/III) and ninefold (layer VI). In contrast, AMPA receptors are abundant at birth and their density remains constant for the first postnatal month, before rising by a maximum of 1.7-fold (layer I) at around the time of eye-opening (postnatal day 32). Kainate receptors are also present in high levels at birth and their expression levels rise in the early postnatal period by between 1. 5-fold (layer I) and threefold (layers V/VI) to a peak just after eye-opening. The proportion of the total ionotropic glutamate receptor binding contributed by NMDA receptors thus rises from 5% at birth to a maximum of 22% at 2 months of age, while the AMPA receptor contribution falls from 87% to 72% over the same period. Below cortex, all three glutamate receptor subtypes are expressed in the subplate region for the first 3 postnatal weeks. These developmental patterns, combined with the fact that AMPA receptors are densely expressed in the proliferative zones underlying presumptive area 17, indicate that non-NMDA receptor expression levels in primary visual cortex are mostly specified much earlier than those of NMDA receptors.

Signals from the superficial layers of the superior colliculus enable the development of the auditory space map in the deeper layers.

We have examined whether the superficial layers of the superior colliculus (SC) provide the source of visual signals that guide the development of the auditory space map in the deeper layers. Anatomical tracing experiments with fluorescent microspheres revealed that a retinotopic map is present in the newborn ferret SC. Aspiration of the caudal region of the superficial layers of the right SC on postnatal day 0 did not cause a reorganization of this projection. Consequently, recordings made when the animals were mature showed that visual units in the remaining superficial layers in rostral SC had receptive fields that spanned a restricted region of anterior space. Auditory units recorded beneath the remaining superficial layers were tuned to corresponding anterior locations. Both the superficial layer visual map and the deeper layer auditory map were normal in the left, unoperated SC. The majority of auditory units recorded throughout the deeper layers ventral to the superficial layer lesion were also tuned to single sound directions. In this region of the SC, however, we observed much greater scatter in the distribution of preferred sound directions and a significant increase in the proportion of units with spatially ambiguous responses. The auditory representation was degraded, although many of these units were also visually responsive. Equivalent lesions of the superficial layers made in adult ferrets did not alter the topographic order in the auditory representation, suggesting that visual activity in these layers may be involved in aligning the different sensory maps in the developing SC.

Spatial-frequency tuning and geniculocortical projections in the visual cortex (areas 17 and 18) of the pigmented ferret.

We have examined the spatial-frequency selectivity of neurons in areas 17 and 18 of the adult pigmented ferret, by measuring how the amplitude of response depends on the spatial-frequency of moving sinusoidal gratings of optimal orientation and fixed contrast. Neurons in area 17 of the ferret respond optimally to low spatial frequencies [average 0.25 cycles per degree (c/deg)], much lower than the optima for cat area 17. The tuning curves are of the same form as those found in cat and monkey: unimodal with bandwidths in the range 0.8-3.5 octaves. Neurons in area 18 of the ferret respond optimally to even lower spatial frequencies (average 0.087 c/deg) than area 17 neurons, and the distributions of optimal spatial frequency for areas 17 and 18 hardly overlap. In both cortical areas, the bandwidth of the tuning curves is inversely correlated with optimal spatial frequency. This marked difference in tuning between the two cortical areas is probably attributable to differential geniculo-cortical projections. Small injections of fluorescent latex microspheres or horseradish peroxidase (HRP) were made into area 17 or area 18 in order to investigate the populations of geniculate neurons projecting to the two cortical areas. After injections into area 17, labelled neurons are found predominantly in the geniculate A layers, with a few neurons labelled in the C layers. Conversely, after an area 18 injection, similar numbers of labelled neurons are found in the C layers as in the A layers. Soma-size analysis of the neurons in the A-layers suggests the existence of two populations of relay neurons, which project differentially to areas 17 and 18. The different geniculate inputs and the different spatial-frequency tuning in areas 17 and 18 may imply that the two cortical areas process visual information more in parallel than in series.

The development of topography in the hamster geniculo-cortical projection.

Precise point-to-point connectivity is the basis of ordered maps of the visual field. The immaturity of the newborn hamster's visual system has allowed us to examine emerging topography in the geniculo-cortical projection well before thalamic axons have reached their cortical target, layer IV. Using anterograde transneuronal labeling with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), we visualized the ingrowth of the whole population of geniculate fibers in the neonatal hamster. Two days after birth (P2), the bulk of the fibers is in the deep cortical layers and the subplate. At the same age, injections of paired retrograde tracers (red and green fluorescent latex microspheres) into area 17 reveal an unordered projection from the dorsal lateral geniculate nucleus (dLGN) to cortex. Individual labeled cells are found throughout the dLGN, and quantitative analysis reveals no segregation of the red and the green populations. At P6, when the pattern of geniculate back label appears ordered and essentially adult-like, geniculate fibers have reached layer IV. The role of selective cell death in this process was investigated by making a tracer injection at P2 and allowing the animals to survive to P6 or P12, when the map is mature. The results show early labeled neurons that made inappropriate connections when the projection was scattered surviving through the period of geniculate cell death. We conclude that the geniculo-cortical map develops from an initially unordered projection to the subplate and the lower cortical layers. Selective cell death appears not to contribute significantly to this process.

Mechanical properties of bioactive glasses, glass-ceramics and composites.

The application of bioactive glass and glass-ceramics has been widely documented over the past twenty years but the high modulus and low fracture toughness has made them less applicable for clinical, load bearing, applications. The development of non-resorbable polyethylene and polysulphone matrices for these materials has improved the mechanical properties. However, the primary concern of whether the bioactivity of the composites is reduced is still unresolved. The more recent development of resorbable carrier systems, dextran and collagen, for bioactive glasses does not introduce such problems, hence making this form of composite suitable for novel soft tissue applications. The development of a simple quality index has enabled some of the materials described within this paper to be ranked by their ability to replace bone, thus enabling possible new research directions to be emphasized.

Purine modulation of dizocilpine effects on spontaneous alternation.

The Y-maze was used to assess spontaneous alternation behaviour in mice to examine possible interactions between the N-methyl-D-aspartate receptor channel blocker dizocilpine and purine receptor agonists and antagonists. Scopolamine reduced spontaneous alternation. Dizocilpine also produced a dose-dependent reduction in alternation scores, which was accompanied by an increase in locomotion. The selective A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) had no effect when administered alone, or in combination with scopolamine. However, when co-administered with dizocilpine, CPX reversed both the deficit in alternation behaviour and also the increase in locomotion induced by dizocilpine. The A1 selective agonist N6-cyclopentyladenosine (CPA) had no effect on either locomotion or alternation scores when administered alone, but in combination with scopolamine, CPA attenuated the scopolamine-induced deficit. CPA had no significant effect on the dizocilpine-induced deficit. The A2 selective agonist N6-[2-(3, 5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA), had no effect on spontaneous alternation when administered alone, but did cause a depression of locomotion. DPMA had no significant effect when co-administered with scopolamine, but reversed the deficit in spontaneous alternation, and the increase in locomotion induced by dizocilpine. The A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) had no effect when given alone or in combination with scopolamine, but when co-administered with dizocilpine, DMPX reversed the reduction in spontaneous alternation caused by dizocilpine. It is concluded that dizocilpine has a detrimental effect on spontaneous alternation which is mediated partly by A1 and A2 adenosine receptors.

The involvement of adenosine receptors in the effect of dizocilpine on mice in the elevated plus-maze.

It has been claimed that blockade of receptors for N-methyl-D-aspartate (NMDA) can enhance adenosine receptor function on single neurones. Previous work has also indicated that the NMDA channel blocker dizocilpine, and the A1 selective agonist N6-cyclopentyladenosine (CPA) both had anxiolytic profiles in the elevated plus-maze. The anxiolytic effect of dizocilpine was accompanied by an increase in locomotor activity. In the present study, the elevated plus-maze has been used to determine whether dizocilpine's effects on behaviour are mediated through activation of adenosine receptors. When co-administered with dizocilpine (0.05 mg/kg), CPA (0.05 mg/kg) reduced the anxiolytic and locomotor effects of dizocilpine. The A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (CPX, 0.05 mg/kg) had no effect when administered alone. When co-administered with dizocilpine, CPX reversed the anxiolytic and increased locomotor effects induced by dizocilpine. The A2 receptor selective agonist N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)ethyladenosine (DPMA) (1 mg/kg) reversed both the anxiolytic effect and the increased locomotion induced by dizocilpine, while the A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (1 mg/kg) did not. It is concluded that at least part of the anxiolytic and locomotor stimulant properties of dizocilpine may be explained by the release of endogenous adenosine acting at A1, but not A2 receptors.

Interactions between ifenprodil and dizocilpine on mouse behaviour in models of anxiety and working memory.

The N-methyl-D-aspartate (NMDA) receptor polyamine site antagonist, ifenprodil, had no effect on spontaneous alteration or locomotor activity in the Y-maze when given alone. The NMDA receptor/ion channel blocker, dizocilpine, induced a deficit in spontaneous alteration, but when ifenprodil was co-administered with dizocilpine, it showed a strong tendency to attenuate the dizocilpine-induced deficit. In the plus-maze, ifenprodil had an anxiolytic profile which was accompanied by an increase in locomotion. Dizocilpine had an anxiolytic profile in this model and increased locomotor activity. When co-administered with dizocilpine, ifenprodil reduced both the anxiolytic and locomotor effects of dizocilpine. When co-administered with ifenprodil, cyclopentyladenosine (CPA) and 1,3-dipropyl-8-cyclopentylxanthine (CPX) reduced the anxiolytic effect of ifenprodil. CPA and CPX in combination did not reverse the anxiolytic effect of ifenpropil. It is concluded that NMDA antagonists with different sites of action can show distinct behavioural profiles, with dizocilpine but not ifenprodil inducing a deficit in working memory, while both are anxiolytic. Blockade of NMDA receptors by ifenprodil, however, can preclude any response to dizocilpine. The anxiolytic activity of ifenprodil may involve the release of purines acting at adenosine receptors.

Soma and axon diameter distributions and central projections of ferret retinal ganglion cells.

Using a combination of retrograde horseradish peroxidase (HRP) labelling, silver staining, and electron microscopy, we have assessed the relationship between retinal ganglion cell soma size and axon diameter in the adult ferret (Mustela putorius furo). Retinal ganglion cells were labelled following injections of HRP into the lateral geniculate nucleus (LGN), superior colliculus (SC), or LGN+SC. The soma size distributions following LGN, SC, or LGN+SC injections were all unimodal showing considerable overlap between different cell classes. This was confirmed for alpha cells identified on the basis of dendritic filling or from neurofibrillar-stained retinae. Analysis of the soma size and axon diameters of a population of heavily labelled retinal ganglion cells showed a significant correlation between the two. However, the overall distribution of intraretinal axon diameter was bimodal with an extended tail. Analysis of the ganglion cell distributions in the adult ferret indicates that beta cells comprise about 50.5-55%, gamma 42.5-47%, and alpha 2.5% of the ganglion cell population. This implies that the proportion of gamma, beta, alpha cells in both cat and ferret retina is highly conserved despite differences in visual specialization in the two species.

The development of topographically-aligned maps of visual and auditory space in the superior colliculus.

The role of the superior colliculus in attending and orienting to sensory stimuli is facilitated by the presence within this midbrain nucleus of superimposed maps of different sensory modalities. We have studied the steps involved in the development of topographically-aligned maps of visual and auditory space in the ferret superior colliculus. Injections of fluorescent beads into the superficial layers showed that the projection from the contralateral retina displays topographic order on the day of birth (PO). Recordings made from these layers at the time of eye opening, approximately 1 month later, revealed the presence of an adult-like map of visual space. In contrast, the auditory space map in the deeper layers emerged gradually over a much longer period of postnatal life. In adult ferrets in which one eye had been deviated laterally just before eye opening, the auditory spatial tuning of single units recorded in the contralateral superior colliculus was shifted by a corresponding amount, so that the registration of the visual and auditory maps was maintained. Chronic application of the NMDA-receptor antagonist MK801 disrupted the normal development of the auditory space map, but had no effect on the visual map in either juvenile or adult animals, or on the auditory map once it had matured. These findings indicate that visual cues may play an instructive role, possibly via a Hebbian mechanism of synaptic plasticity, in the development of appropriately tuned auditory responses, thereby ensuring that the neural representations of both modalities share the same coordinates. Changes observed in the auditory representation following partial lesions of the superficial layers at PO suggest that these layers may provide the source of the visual signals responsible for experience-induced plasticity in auditory spatial tuning.

Neonatal monocular enucleation and the geniculo-cortical system in the golden hamster: shrinkage in dorsal lateral geniculate nucleus and area 17 and the effects on relay cell size and number.

We have examined the effects of neonatal monocular enucleation on the volume of the dorsal lateral geniculate nucleus (dLGN), the area of area 17, and the size and numbers of geniculate relay neurons identified by retrograde transport of HRP from cortex. Compared to values for normal animals, the only significant change contralateral to the remaining eye was an increase in relay cell radius. The effects ipsilateral to the remaining eye were more widespread: we found significant reductions in the volume of the dLGN (27% reduction), the area of striate cortex (22%), and the number (16%) and average soma radius (6%) of geniculate relay neurons. The relay neurons were also more densely packed, suggesting that other geniculate cell types were affected similarly, although this was not explicitly examined. These changes were not uniform throughout the nucleus, and as such, reflected the changes in retinal input. The greatest reduction in cell size occurred in the region of the ipsilateral dLGN receiving the most sparse retinal input subsequent to enucleation. Nor was the shrinkage of the dLGN uniform, being most apparent in the coronal plane especially along the axis orthogonal to the pia; there appeared to be little change in the anteroposterior extent. Shrinkage in area 17 ipsilateral to the remaining eye was the same (about 22%) whether it was defined by myelin staining or transneuronal transport of WGA-HRP. These results show that the transneuronal changes seen in the organization of visual cortex after early monocular enucleation in rodents are associated with only a moderate loss of geniculate relay cells.

Manufacture and release characteristics of Elvax polymers containing glutamate receptor antagonists.

Implantable sustained-release polymers offer an alternative to osmotic minipumps for the local delivery of drugs to specific brain areas. Here we describe the production of Elvax polymers containing a range of glutamate receptor antagonists and the quantitative characterization of their release properties. Sections of Elvax (200 or 400 microns), prepared by a dimethyl sulphoxide-based method, containing the NMDA antagonist MK-801 or the non-NMDA antagonist CNQX exhibited similar release profiles: an initial 2-week burst followed by a slow decline in release rate over the next 6 weeks. Differences in slice preparation method and thickness or drug concentration and solubility all led to alterations in the level of drug release, but not the overall exponential nature of the release curve. Elvax sections prepared by an aqueous method containing the NMDA antagonists CPP or APV displayed more constant but much lower levels of release than those from the dimethyl sulphoxide-based method. The in vitro release characteristics were compared with in vivo release of MK-801 and the close correspondence observed indicates that the in vitro release data is an accurate predictor of the drug release behaviour of implanted Elvax slices.

Postnatal changes in the uncrossed retinal projection of pigmented and albino Syrian hamsters and the effects of monocular enucleation.

Anterograde and retrograde tracing techniques have been used to study the uncrossed retinal projection in neonatal pigmented and albino Syrian hamsters. The total number of retinal ganglion cells projecting ipsilaterally peaks at postnatal days 2-4 (P2-P4) and declines to adult values by P12. The change in cell numbers has a similar time course in albino and pigmented animals. Although the population of uncrossed cells in the temporal retina of albino hamsters is always less than that in pigmented hamsters, no difference between the colour phases was found for the population of uncrossed cells in nasal retina. Differential cell death also contributes to the adult albino decussation pattern in hamsters: The relative loss of cells from temporal retina in albinos (72%) is greater than that in pigmented animals (56%). The additional loss in albinos does not appear to depend on binocular interactions: The same proportion (30%) of uncrossed cells is "rescued" from death by neonatal monocular enucleation in both colour phases. Flat-mount preparations showing the distribution of uncrossed fibres reveal that a distinct focus of terminals emerges in rostral superior colliculus, which is topographically appropriate for a binocular mapping, at the peak of uncrossed ganglion cell numbers (P4). Comparison of uncrossed terminal distributions and ganglion cell death reveals considerable refinement of the terminals prior to the main phase of cell death. Monocular enucleations performed some time after birth have a greater effect on uncrossed terminal distributions than on cell death. These observations suggest that independent mechanisms may be involved in the regulation of terminal distributions and of cell numbers in the developing uncrossed retinal pathways.