Forever young: how to control the elongation, differentiation, and proliferation of cells using nanotechnology.

Within the emerging field of stem cells there is a need for an environment that can regulate cell activity, to slow down differentiation or proliferation, in vitro or in vivo while remaining invisible to the immune system. By creating a nanoenvironment surrounding PC12 cells, Schwann cells, and neural precursor cells (NPCs), we were able to control the proliferation, elongation, differentiation, and maturation in vitro. We extended the method, using self-assembling nanofiber scaffold (SAPNS), to living animals with implants in the brain and spinal cord. Here we show that when cells are placed in a defined system we can delay their proliferation, differentiation, and maturation depending on the density of the cell population, density of the matrix, and the local environment. A combination of SAPNS and young cells can be implanted into the central nervous system (CNS), eliminating the need for immunosuppressants.

Using nanotechnology to design potential therapies for CNS regeneration.

The nanodelivery of therapeutics into the brain will require a step-change in thinking; overcoming the blood brain barrier is one of the major challenges to any neural therapy. The promise of nanotechnology is that the selective delivery of therapeutics can be delivered through to the brain without causing secondary damage. There are several formidable barriers that must be overcome in order to achieve axonal regeneration after injury in the CNS. The development of new biological materials, in particular biologically compatible scaffolds that can serve as permissive substrates for cell growth, differentiation and biological function is a key area for advancing medical technology. This review focuses on four areas: First, the barriers of delivering therapies to the central nervous system and how nanotechnology can potentially solve them; second, current research in neuro nanomedicine featuring brain repair, brain imaging, nanomachines, protein misfolding diseases, nanosurgery, implanted devices and nanotechnologies for crossing the blood brain barrier; third, health and safety issues and fourth, the future of neuro nanomedicine as it relates to the pharmaceutical industry.

Behavioral testing and preliminary analysis of the hamster visual system.

The dependence of visual orienting ability in hamsters on the axonal projections from retina to midbrain tectum provides experimenters with a good model for assessing the functional regeneration of this central nervous system axonal pathway. For reliable testing of this behavior, male animals at least 10-12 weeks old are prepared by regular pretesting, with all procedures carried out during the less active portion of the daily activity cycle. Using a sunflower seed attached to a small black ball held at the end of a stiff wire, and avoiding whisker contact, turning movements toward visual stimuli are video recorded from above. Because at the eye level, the nasal-most 30 degrees of the visual field can be seen by both the eyes, this part of the field is avoided in assessments of a single side. Daily sessions consist of ten presentations per side. Measures are frequency of responding and detailed turning trajectories. Complete assessment of the functional return of behavior in this testing paradigm takes 3-6 months to complete.

A role for tectal midline glia in the unilateral containment of retinocollicular axons.

Retinal fibers approach close to the tectal midline but do not encroach on the other side. Just before the entry of retinal axons into the superior colliculus (SC), a group of radial glia differentiates at the tectal midline; the spatiotemporal deployment of these cells points to their involvement in the unilateral containment of retinotectal axons. To test for such a barrier function of the tectal midline cells, we used two lesion paradigms for disrupting their radial processes in the neonatal hamster: (1) a heat lesion was used to destroy the superficial layers of the right SC, including the midline region, and (2) a horizontally oriented hooked wire was inserted from the lateral edge of the left SC toward the midline and was used to undercut the midline cells, leaving intact the retinorecipient layers in the right SC. In both cases, the left SC was denervated by removing its contralateral retinal input. Animals were killed 12 hr to 2 weeks later, after intraocular injections of anterograde tracers to label the axons from the remaining eye. Both lesions resulted in degeneration of the distal processes of the tectal raphe glia and in an abnormal crossing of the tectal midline by retinal axons, leading to an innervation of the opposite ("wrong") tectum. The crossover occurred only where glial cell attachments were disrupted. These results document that during normal development, the integrity of the midline septum is critical in compartmentalizing retinal axons and in retaining the laterality of the retinotectal projection.

Target-specific morphology of retinal axon arbors in the adult hamster.

The B fragment of cholera toxin (CT-B) provides a highly sensitive anterograde tracer for labeling retinofugal axons, revealing dense projections to known central retinorecipient nuclei, and sparse but distinct inputs to regions that have not been traditionally recognized as targets of direct retinal projections. In hamsters, we can identify CT-B labeled retinal axons in more than 25 cell groups in the mesencephalon, diencephalon, and basal telencephalon. CT-B labeling additionally delineates the complete arbor morphology, especially in regions that receive a sparse input, offering hitherto unknown views of retinal axon ramifications. We present here the terminal morphology of retinal axons in the lateral geniculate body and superior colliculus, verifying earlier studies, and also document novel findings on the configuration of retinal axon endings in the ventral nucleus of the lateral geniculate body, intergeniculate leaflet, suprachiasmatic nucleus, and in the nuclei of the accessory optic tract. Additionally, the trajectory and terminal morphology of retinal afferents to the hypothalamus, preoptic area, and basal telencephalon are detailed. The results are discussed in the context of possible functional roles for some of these projections.

Target- as well as source-derived factors direct the morphogenesis of anomalous retino-thalamic projections.

Neonatal tectal lesions in hamsters result in the elimination of a major central target of retinal axons, massively denervate the lateral posterior nucleus of the thalamus (LP), and lead to a marked increase of the retino-LP projection. In such animals, retino-LP axons show all of the normally-occurring terminal types. In addition, large clusters of varicosities, whose tubular configuration resembles the major type of tecto-LP terminals observed in normal animals, are also noted if the tectal lesion is made on the day after birth (P1). If, however, the neonatal lesion occurs on P5 rather than on P1, terminals resembling normal tecto-LP endings are rarely observed; rather, the distribution and morphology of retino-LP terminals bear a greater resemblance to those seen in normal hamsters, but the size and complexity of the terminals, particularly those that form string-like arrangements, is significantly increased. Our findings suggest that the altered morphology of some abnormally induced retino-LP terminals may be orchestrated by target-associated signals. However, there are age-related limitations on the degree to which afferent systems can vary their terminal morphology; these restrictions may derive from the target, or may be a function of intrinsic changes within the cells of origin of the afferent fibers.

Afferents from the colliculus, cortex, and retina have distinct terminal morphologies in the lateral posterior thalamic nucleus.

We have examined the morphology of afferent endings that originate in three distinct cell groups and terminate in the lateral posterior nucleus of the thalamus (LP). Retino-LP projections were sparse, occurred throughout the nucleus, and could be classified into 1) simple en passant varicosities and terminal swellings found on poorly branched fibers in all LP subdivisions, 2) string-like configurations of varicosities detected largely in the medial subdivision of the LP, and 3) terminals resembling retinogeniculate endings occurring mainly in the rostral part of the superficial subdivision of the LP adjacent to the dorsal nucleus of the lateral geniculate body. Cortico-LP terminals fell into three classes: 1) single varicosities decorating the tips of short appendages on fine preterminal and terminal axons; 2) tiny, round varicosities studding the axon shaft; and 3) boutons of variable shape visible on medium-caliber corticothalamic fibers. Tecto-LP terminals exhibited a large variation in morphology and density. Those found most commonly could be classified into two groups: 1) individual swellings and 2) terminal clusters arranged in a tubular configuration that enclosed a central channel, most likely occupied by the dendrite of a postsynaptic neuron. An unusual tecto-LP terminal consisted of an ovoid swelling (up to 20 microns in the long axis) from which emerged several long, thin extensions and was seen at the tips of large-diameter axons. These results show that, despite having overlapping projection zones, each set of afferents that projects to the LP elaborates terminal specializations that are structurally distinct from others projecting to the same target area.

Bcl-2 promotes regeneration of severed axons in mammalian CNS.

Most neurons of the mammalian central nervous system (CNS) lose the ability to regenerate severed axons in vivo after a certain point in development. At least part of this loss in regenerative potential is intrinsic to neurons. Although embryonic retinal ganglion cells (RGCs) can grow axons into tectum of any age, most RGCs from older animals fail to extend axons into CNS tissue derived from donors of any age, including the embryonic tectum. Here we report that the proto-oncogene bcl-2 plays a key role in this developmental change by promoting the growth and regeneration of retinal axons. This effect does not seem to be an indirect consequence of its well-known anti-apoptotic activity. Another anti-apoptotic drug, ZVAD, supported neuronal survival but did not promote axon regeneration in culture. This finding could lead to new strategies for the treatment of injuries to the CNS.

The optic tract in embryonic hamsters: fasciculation, defasciculation, and other rearrangements of retinal axons.

The early development of the optic tract in hamsters was studied by labeling retinal axons with Dil applied to the eye, and then examining the labeled axons in flatmount preparations of the rostral brain stem. This technique permits a panoramic view of the entire retinal projection, from the chiasm to the caudal end of the superior colliculus. In the E11 embryo, retinal axons have reached the chiasm. They defasciculate as they emerge from the nerve, prior to reaching the ventral midline of the diencephalon, then converge again as they pass over to the opposite side. At the midline, many axonal trajectories crisscross, implying some shuffling of relative positions. Retinal axons are tightly bundled within the optic tract. Upon reaching the ventral border of the lateral geniculate body (LGB), they splay out over the nucleus, revealing a wavefront of pioneer axons individually distributed across the rostro-caudal extent of the LGB. Later-emerging retinal axons course over the surface of the thalamus in waves; subsequent waves of axons interdigitate between the lead fibers without fasciculating along them. Past the LGB, the axons undergo a second change in relative positions as the ribbon of fibers swerves caudally, prior to entering the superior colliculus. Retinal axons are tipped with growth cones of varying morphologies. No strong correlation is evident between the structural complexity of the growth cone and its position within the tract. In the majority of cases, ipsilaterally and contralaterally directed axons follow a similar developmental course along the optic tract, without any indication of a temporal lag in the ipsilateral projection as claimed in earlier reports. Understanding the changes in spatial distribution of embryonic retinal axons as they navigate along the optic tract provides a further step towards elucidating how point-to-point projections form in developing sensory systems.

Intrinsic changes in developing retinal neurons result in regenerative failure of their axons.

The failure of mature mammalian central nervous system axons to regenerate after transection is usually attributed to influences of the extraneuronal milieu. Using explant cocultures of retina and midbrain tectum from hamsters, we have found evidence that these influences account for failure of regrowth of only a small minority of retinal axons. For most of the axons, there is a programmed loss of ability to elongate in the central nervous system. We show that there is a precipitous decline in the ability of retinal axons to reinnervate tectal targets when the retina is derived from pups on or after postnatal day 2, even when the target is embryonic. By contrast, embryonic retinal axons can regrow into tectum of any age, overcoming growth-inhibiting influences of glial factors.

Glial environment in the developing superior colliculus of hamsters in relation to the timing of retinal axon ingrowth.

We have examined the developmental changes of glial cell organization in the superior colliculus of embryonic and neonatal hamsters in reference to the known sequence of retinal axon ingrowth and arborization in the midbrain. Immunolocalization of vimentin, a marker for neuronal and glial cell precursors, reveals a uniform distribution of radially oriented cells, with perikarya located at the ventricular surface and thin, elongated processes fanning out toward the pia. These vimentin-positive cells, referred to as the lateral radial cells, are present in the tectum from embryonic day (E) 10 (earliest day examined) until approximately postnatal day (P) 5. Vimentin expression in the lateral radial cells decreases markedly during the second week of postnatal life: application of DiI to the ventricular surface reveals that the pial attachment of the lateral radial cells is withdrawn and that the radial processes are gradually pulled back toward the ventricular zone. By P14, virtually no vimentin-positive radial cells are detectable in the superior colliculus. At no time during development are the lateral radial cells immunopositive for the glial fibrillary acidic protein (GFAP); however, shorter, vimentin-positive astrocytic profiles can be seen in the tectum around the time the radial fibers have been withdrawn, suggesting that at least some radial cells are transformed into astrocytes that will colonize the mature colliculus. At approximately E12, a second group of cells, referred to as the midline radial glia, is detected at the tectal midline. These cells are tightly bundled, forming a raphe in the tectum. They are intensely vimentin positive from E13 until at least P14. From the time of birth, the midline radial cells also exhibit intense immunoreactivity for GFAP. The lateral radial cells are present in the superior colliculus prior to and during the period of neurogenesis but remain well past the time when collicular neuronal migration is completed. Pial processes of the lateral radial cells are present within the superficial tectal layers during the time retinal axons are entering this target; they may be involved in directing the growth and initial collateralization of retinotectal axons. Their withdrawal from retinorecipient collicular zones begins at about the time arbors are being elaborated on retinal axons. In contrast, the midline glia become distinct just prior to the time retinal axons enter the superior colliculus and persist during the time retinotectal projections are being fully established. These raphe glia may be involved in maintaining the laterality of the retinotectal projection.

The amyloid precursor protein is developmentally regulated and correlated with synaptogenesis.

Metabolism of the amyloid precursor protein (APP) may contribute to the molecular changes observed in Alzheimer's disease, but the function of the protein in the non-pathologic nervous system remains unknown. In vitro studies have suggested that APP can participate in cellular adhesion and may thus contribute to neuronal differentiation in cultured cells. Here we show, in the primary visual pathway of the hamster, that APPs are developmentally regulated proteins rapidly transported to the growing tips of nerve fibers. Transmembrane forms of higher molecular weight (120 and 140 kDa) are preferentially associated with the rapid elongation of axons. Interestingly, another full-length form of 110 kDa and a soluble form of 100 kDa which lacks the C-terminal domain increase at the time of end-arbor formation and synaptogenesis and then decline when mature connections are established, suggesting that target recognition and synaptic contact may result in a signal for APP cleavage in the CNS in vivo.

Changes in rapidly transported proteins associated with development of abnormal projections in the diencephalon.

The development of the hamster visual system is accompanied by striking changes in the pattern of proteins that are synthesized in retinal ganglion cells and rapidly transported to their nerve terminals. To determine whether any of these protein changes are regulated by interactions between the developing nerve endings and the cells with which they form synapses, we induced retinofugal axons to form abnormal projections in the lateral posterior (LP) nucleus of the thalamus and dense patches of hyperinnervation in the lateral geniculate nucleus (LGN) by removing their principal target, the superior colliculus (SC), the day after birth. Under these experimental conditions, two rapidly transported proteins, including the neural cell adhesion molecule, NCAM, showed significant changes in their time course of expression. NCAM, identified here using a monospecific antibody, is normally synthesized and transported at high levels at early stages of development and then declines during the second and third postnatal weeks. However, this decline was delayed when optic fibers were re-routed. A second rapidly transported protein, M(r) = 67 kDa, pI = 4.7, normally shows a rise in its synthesis and transport during terminal arbor formation and a subsequent decline, but it also remained elevated for a prolonged period when the SC was absent. These findings cannot be accounted for by a simple delay in the retinal ganglion cells' program of axonal growth, since other rapidly transported proteins, including the growth-associated protein GAP-43, showed a normal developmental time-course when the SC was removed. Target interactions therefore appear to influence the retinal ganglion cells' expression of different proteins in a specific fashion.

Antibody neutralization of neurite growth inhibitors from oligodendrocytes results in expanded pattern of postnatally sprouting retinocollicular axons.

After early postnatal ablation of one superior colliculus together with the ipsilateral eye in Syrian hamsters, retinofugal fibers abnormally cross the tectal midline and innervate the remaining superior colliculus. The fibers of this aberrant decussation are confined mainly to the superficial gray layer, with little ingrowth or termination in the deeper stratum opticum; laterally, most termination is in the superficial part of the superficial gray. Establishment of this abnormal pattern is temporally correlated with the appearance of oligodendrocytes at progressively more superficial locations in the colliculus. Oligodendrocytes express, on their surface, molecules that are inhibitory to neurite growth. This raises the possibility that their differential distribution in the superior colliculus during growth of retinal fibers is causally involved in the generation of the observed termination pattern. We tested this hypothesis by applying the monoclonal antibody IN-1, which neutralizes this inhibitory activity, during the time of postnatal fiber growth and terminal arbor formation. We found that in the presence of IN-1, but not a control antibody, recrossing retinofugal fibers, observed at postnatal day 12, traverse the stratum opticum as well as the superficial gray, with greater depth of termination in superficial gray and stratum opticum. This pattern resembles that of the normal contralateral retinotectal projection. The results indicate that neurite growth inhibitors expressed by oligodendrocytes are responsible for restricting the innervation of a target area in postnatal plasticity.

Oligodendrocytes and myelin formation along the optic tract of the developing hamster: an immunohistochemical study using the Rip antibody.

The monoclonal antibody Rip recognizes an antigen specific to oligodendrocytes and their processes (Friedman et al: Glia 2:380, 1989). We have used this antibody to document the appearance of oligodendrocytes and the sequence of ensheathment of axons along the optic tract (OT) and within its major target areas in neonatal (P3-P21) and adult hamsters. Myelination of axons in the visual pathway follows an overall proximo-distal gradient. On P3, immunopositive, pre-ensheathing oligodendrocytes are detected in the OT ventral to the lateral geniculate body (LGB) whereas myelin segments are present around OT axons by P5. The first pre-ensheathing oligodendrocytes are detected medially in the LGB on P7 and myelinated axons in the overlying OT by P11. In the superior colliculus, pre-ensheathing oligodendrocytes are present in the optic fiber layer (SO) on P7, but not in the superficial gray layer (SGS) until P11. Myelination of axons within SO proceeds along a marked rostro-caudal gradient. On P14, axons in rostral SO are heavily myelinated; thereafter, ensheathment continues caudally within the SO and the SGS. The progressive invasion of oligodendrocytes along the proximo-distal axis of the optic pathway, and the corresponding myelination of OT axons, are discussed in the context of a possible inhibitory role of oligodendrocytes in regulating the regenerative propensity of retinotectal axons.

Orienting behavior in hamsters with lesions of superior colliculus, pretectum, and visual cortex.

We examined cortical and subcortical mediation of visual locomotor orienting function by comparing the behavior of hamsters with discrete bilateral lesions affecting the pretectum, superior colliculus (SC), or visual cortex (VC). Orienting and approach to stationary targets was evaluated by measuring the accuracy of hamsters' approaches to small black apertures, located at eye level along the wall of a circular white arena. Hamsters with bilateral ablation of the visual cortex were slightly impaired for approaches to central field targets, whereas those with ibotenic acid lesions of the pretectum (which spares fibers of passage and thus leaves tectal afferents intact) were totally unimpaired. Hamsters with transection of the brachium of SC (BSC) at the prectectal-SC (PT-SC) border were severely impaired in their ability to approach stationary targets in central and peripheral fields. Thus, we did not detect any of the central field sparing that has been reported by others for rodents with similar lesions. Several possible reasons for the disparity between our results and those of others are discussed. Overall, our results indicate that in hamsters the SC is essential for normal visually guided approach to dark, stationary targets throughout the visual field. Further, our results and qualitative observations indicate that the approach errors are most likely due to deficits of visuomotor integration rather than to a lack of visual scanning.

Aberrant retinal projections to midbrain targets mediate spared visual orienting function in hamsters with neonatal lesions of superior colliculus.

Rodents, cats, and most nonmammalian vertebrates with bilateral tectal deafferentation or ablation in adulthood are extremely deficient at orienting to visual stimuli; yet animals with neonatal lesions of superficial layers of the superior colliculus (SC) show partial sparing of this response, particularly for targets in the central visual field. In this study, we sought to determine whether these spared orienting abilities are mediated by aberrant retinal projections to the remaining intermediate layers of the SC, or whether visual cortex (VC) mechanisms or alternative behavioral strategies are responsible. Neonatal golden hamsters received either bilateral heat lesions of the SC (rlSC), or a heat lesion of the right SC and enucleation of the right eye (rSCrE). This latter procedure causes axons from the left eye to recross the tectal midline and terminate in the "wrong" (left) SC (Schneider 1973). As adults, both groups of hamsters were extremely deficient in visually guided approach to stationary targets, although rlSC-lesioned hamsters showed some sparing for central field targets and rSCrE-lesioned hamsters often made wrong-direction turns for targets in the left peripheral field. We then subjected both groups of neonatally lesioned hamsters to bilateral aspiration lesions of the VC. Retesting showed no change in visual orienting behavior as a result of the cortical lesions. Labeling of the optic tract with horseradish peroxidase (HRP) revealed abundant aberrant retinal projections to remaining intermediate layers of the SC and thalamic nucleus lateralis posterior (LP), as well as supernormal innervation of pretectal nuclei, the dorsal terminal nucleus of the accessory optic tract, and the ventral nucleus of the lateral geniculate body (LGv). We conclude that the spared visual orienting capabilities of hamsters with rlSC and rSCrE lesions are mediated by the aberrant midbrain projections, and that cortical mechanisms are not involved in spared visual orienting functions following these neonatal lesions.

Initial stages of retinofugal axon development in the hamster: evidence for two distinct modes of growth.

In order to characterize differences in growth patterns of axons as they elongate toward their targets and during the initial stages of terminal arbor formation within the targets, we examined the primary visual system of fetal and newborn hamsters using three morphological methods: the Cajal-deCastro reduced silver method, the rapid Golgi technique, and anterograde transport of HRP. Axons emerge from the retina between the 10th and 11th embryonic days (E10-E11). The front of retinal axons crosses the chiasm, extends over the primitive dorsal nucleus of the lateral geniculate body (LGBd) by E13, and advances to the back of the superior colliculus (SC) by E13.5-E14. The rate of axon growth during this advance is nearly 2 mm/day. Collateral sprouts appear on axons around E15.5. In the LGBd and SC, these sprouts arise from multiple sites along the parent axons. Only one or a few of the sprouts continue to grow and branch, while others are eliminated. The net rate of axon collateral advance in this second phase is an order of magnitude slower than during the stage of axon elongation. Thus, formation of CNS projections may involve two qualitatively distinct modes of axon growth. The arborization mode contrasts with the elongation mode by the presence of branching, a lack of fasciculation and a slower average rate of extension. The stereotypic direct advance of axons during elongation also differs from the remodelling which occurs during arborization. The delay between axon arrival at targets and onset of arborization could be a reflection of axons "waiting" for a maturational change to occur in the retina or in targets. Arborization in the LGBd and SC is initiated around the same time, implicating the former possibility. However, a slower differentiation of retinal arbors in the SC, in addition to morphological differences of arbors in the two structures, suggests that alterations in substrate factors also play a critical role in triggering the early stages of arbor formation.