Activity-dependent alteration of early myelin ensheathment in a developing sensory circuit.

Myelination allows for the regulation of conduction velocity, affecting the precise timing of neuronal inputs important for the development and function of brain circuits. In turn, myelination may be altered by changes in experience, neuronal activity, and vesicular release, but the links between sensory experience, corresponding neuronal activity, and resulting alterations in myelination require further investigation. We thus studied the development of myelination in the Xenopus laevis tadpole, a classic model for studies of visual system development and function because it is translucent and visually responsive throughout the formation of its retinotectal system. We begin with a systematic characterization of the timecourse of early myelin ensheathment in the Xenopus retinotectal system using immunohistochemistry of myelin basic protein (MBP) along with third harmonic generation (THG) microscopy, a label-free structural imaging technique. Based on the mid-larval developmental progression of MBP expression in Xenopus, we identified an appropriate developmental window in which to assess the effects of early temporally patterned visual experience on myelin ensheathment. We used calcium imaging of axon terminals in vivo to characterize the responses of retinal ganglion cells over a range of stroboscopic stimulation frequencies. Strobe frequencies that reliably elicited robust versus dampened calcium responses were then presented to animals for 7 d, and differences in the amount of early myelin ensheathment at the optic chiasm were subsequently quantified. This study provides evidence that it is not just the presence but also to the specific temporal properties of sensory stimuli that are important for myelin plasticity.

EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

Continued growth and circuit building in the anamniote visual system.

Fish and amphibia are capable of lifelong growth and regeneration. The two core components of their visual system, the retina and tectum both maintain small populations of stem cells that contribute new neurons and glia to these tissues as they grow. As the animals age, the initial retinal projections onto the tectum are continuously remodeled to maintain retinotopy. These properties raise several biological challenges related to the control of proliferation and differentiation of retinal and tectal stem cells. For instance, how do stem and progenitor cells integrate intrinsic and extrinsic cues to produce the appropriate type and number of cells needed by the growing tissue. Does retinal growth or neuronal activity influence tectal growth? What are the cellular and molecular mechanisms that enable retinal axons to shift their tectal connections as these two tissues grow in incongruent patterns? While we cannot yet provide answers to these questions, this review attempts to supply background and context, laying the ground work for new investigations.

Sonic hedgehog (Shh)-Gli signaling controls neural progenitor cell division in the developing tectum in zebrafish.

Despite considerable progress, the mechanisms that control neural progenitor differentiation and behavior, as well as their functional integration into adult neural circuitry, are far from being understood. Given the complexity of the mammalian brain, non-mammalian models provide an excellent model to study neurogenesis, including both the cellular composition of the neurogenic microenvironment, and the factors required for precursor growth and maintenance. In particular, we chose to address the question of the control of progenitor proliferation by Sonic hedgehog (Shh) using the zebrafish dorsal mesencephalon, known as the optic tectum (OT), as a model system. Here we show that either inhibiting pharmacologically or eliminating hedgehog (Hh) signaling by using mutants that lack essential components of the Hh pathway reduces neural progenitor cell proliferation affecting neurogenesis in the OT. On the contrary, pharmacological gain-of-function experiments result in significant increase in proliferation. Importantly, Shh-dependent function controls neural progenitor cell behavior as sox2-positive cell populations were lost in the OT in the absence of Hh signaling, as evidenced in slow-muscle-omitted (smu) mutants and with timed cyclopamine inhibition. Expressions of essential components of the Hh pathway reveal for the first time a late dorsal expression in the embryonic OT. Our observations argue strongly for a role of Shh in neural progenitor biology in the OT and provide comparative data to our current understanding of progenitor/stem cell mechanisms that place Shh as a key niche factor in the dorsal brain.

Eph/ephrin gradients in the retinotectal system of Rana pipiens: developmental and adult expression patterns.

Eph/ephrin-receptor/ligand A and B families play a variety of roles during CNS development, including patterning the retinotectal projection. However, the alignment of their expression gradients with developing retinotectal maps and gradients of cellular development is not well understood in species whose midbrain tecta undergo a protracted anterior to posterior development. By using anatomical tracing methods and (3)H-thymidine neuronography, we have mapped the retinotectal projection and the spatiotemporal progression of tectal cellular development onto Eph/ephrin expression patterns in the tectum of larval Rana pipiens, as studied by means of in situ affinity analysis with fusion proteins. EphA expression is maximal in anterior tectum (and temporal retina); ephrin-A expression is maximal at the posterior pole (and nasal retina). EphB expression is graded in the early larva, where it is maximal in the posterior tectum just anterior to the posterior pole (and in the ventral retina). Tectal EphB expression becomes uniform at later stages and remains so in the adult, although its retinal expression remains maximal ventrally. In the early larva, EphA, EphB, and ephrin-A protein gradients are parallel to each other and align with the temporonasal axis of the retinal projection. The early EphB expression maximum overlaps the boundary between the mantle layer of newly postmitotic cells and the posterior, epithelial region of cell proliferation, suggesting that the expression maximum is associated with the initial migrations of the postmitotic cells. Ephrin-B expression was detected in the olfactory bulb and dorsal retina at all ages, but not in the tectum.

The zebrafish zic2a-zic5 gene pair acts downstream of canonical Wnt signaling to control cell proliferation in the developing tectum.

Wnt growth factors acting through the canonical intracellular signaling cascade play fundamental roles during vertebrate brain development. In particular, canonical Wnt signaling is crucial for normal development of the dorsal midbrain, the future optic tectum. Wnts act both as patterning signals and as regulators of cell growth. In the developing tectum, Wnt signaling is mitogenic; however, the mechanism of Wnt function is not known. As a step towards better understanding this mechanism, we have identified two new Wnt targets, the closely linked zic2a and zic5 genes. Using a combination of in vivo assays, we show that zic2a and zic5 transcription is activated by Tcf/Lef transcription factors in the dorsal midbrain. Zic2a and Zic5, in turn, have essential, cooperative roles in promoting cell proliferation in the tectum, but lack obvious patterning functions. Collectively these findings suggest that Wnts control midbrain proliferation, at least in part, through regulation of two novel target genes, the zic2a-zic5 gene pair.

The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis.

The mechanisms that regulate the growth of the brain remain unclear. We show that Sonic hedgehog (Shh) is expressed in a layer-specific manner in the perinatal mouse neocortex and tectum, whereas the Gli genes, which are targets and mediators of SHH signaling, are expressed in proliferative zones. In vitro and in vivo assays show that SHH is a mitogen for neocortical and tectal precursors and that it modulates cell proliferation in the dorsal brain. Together with its role in the cerebellum, our findings indicate that SHH signaling unexpectedly controls the development of the three major dorsal brain structures. We also show that a variety of primary human brain tumors and tumor lines consistently express the GLI genes and that cyclopamine, a SHH signaling inhibitor, inhibits the proliferation of tumor cells. Using the in vivo tadpole assay system, we further show that misexpression of GLI1 induces CNS hyperproliferation that depends on the activation of endogenous Gli1 function. SHH-GLI signaling thus modulates normal dorsal brain growth by controlling precursor proliferation, an evolutionarily important and plastic process that is deregulated in brain tumors.

Developmental pattern of plasminogen activator activity in chick brain hemispheres.

Plasminogen activators play key roles in several developmental events. In previous works we demonstrated the existence of typical developmental patterns of protease activity in the chick optic lobe and cerebellum. The aim of this work is to study the temporal pattern of development of plasminogen activator activity in the brain hemispheres. Plasminogen activator activity was assayed in soluble fractions derived by ultracentrifugation from Triton X-100 treated membrane fractions by using a radial fibrinolytic assay. Employing different inhibitors and anti-plasminogen activators antibodies we showed that developing brain hemispheres express only one type of enzyme which corresponds to the urokinase-type. Other results indicate that the protease activity displays a temporal pattern which completely differs from those of general parameters of development. This suggests that the plasminogen activator activity is developmentally regulated and could display specific functions during particular stages of development.

Ontogeny of two different benzodiazepine binding sites in the chick optic lobe.

The developmental time-course of type I and type II benzodiazepine receptors in the chick optic lobe was determined using a triazolopyridazine, CL 218872. At embryonic day 13 most of the binding sites corresponded to type II (98.23%), while type I represented only a minor proportion (1.77%). During development there was an increase in type I binding sites which reached 62.88% in adulthood, while type II binding sites decreased to 37.12%. These results demonstrate a differential ontogeny of two benzodiazepine receptor subtypes. Changes in the benzodiazepine binding population may account for the variability in the GABA-benzodiazepine receptor interaction during chick optic lobe development.

Current Triton X-100 treatments do not allow a complete plasminogen activator extraction from developing nervous tissue.

Determinations of plasminogen activator (PA) activity are usually performed in Triton X-100-treated tissue homogenates or crude membrane fractions. Such preparations usually involve a single Triton X-100 treatment. In the present paper we describe the pattern of variability of PA activity measured in different fractions obtained from the developing chick CNS by a repetitive procedure of Triton X-100 treatment and ultracentrifugation. To further characterize this PA activity we have also performed zymographic analyses during the embryonic development and the early postnatal life. Our results show that: a) a single Triton X-100 treatment does not completely extract the enzyme and this lead to an underestimation of the total PA activity; b) the PA activity is associated with the particulate component of the total tissue homogenate requiring its complete solubilization more drastic Triton X-100 treatments; c) better estimations of total and specific activities are obtained by using soluble fractions derived by ultracentrifugation from Triton X-100-treated membrane fractions; d) the developing chick optic lobe expresses only one kind of PA molecule along the entire development; e) the level of PA activity vary characteristically during the ontogeny and the early postnatal life indicating the existence of a developmentally regulated mechanism of PA expression.

Effect of a simple visual pattern on the early postnatal development of GABA receptor sites in the chick optic lobe.

It is known that manipulation of the visual environment results in changes in the developmental pattern of several neurotransmitter receptors and that the GABA receptor shows a high degree of plasticity in differential illumination experiments. In the present paper we investigated whether exposure to a visual pattern has a developmental effect on GABA receptor expression during early postnatal life. Two groups of newly hatched chicks were used: one was exposed to a simple and specific visual pattern and the other was deprived of any visual pattern. GABA receptors at each developmental stage were determined by binding experiments performed in a crude membrane fraction. Saturation studies were carried out in a fraction enriched in synaptic membranes. The developmental pattern of both high and low affinity GABA binding sites was affected by the visual pattern. This effect displays its maximal expression by the end of the first postnatal week. The modification in receptor expression was due to a change in the receptor density while the affinity was not affected. The change in receptor density induced by the presence of a visual pattern was highest at the end of the first postnatal week suggesting that at that time there is a brief period of higher plasticity for GABA receptor expression in the visual system than at other times. Our results also suggest that variations in GABA receptor density could be instrumental in adaptative changes in the visual system in response to variations in the environmental stimulation.

Benzodiazepine receptor sites in the chick optic lobe: development and pharmacological characterization.

To investigate the interaction between gamma-aminobutyric acid (GABA) and benzodiazepine (BZD) receptor sites during development, the time-course of appearance of flunitrazepam (FNZ) binding sites and their pharmacological characterization were studied in developing chick optic lobe. At the earliest stage examined, embryonic day (Ed) 12, the receptor density was 30.9% (0.05 +/- 0.01 pmol/mg protein) of that found in the chick optic lobes of adult chicks. The adult value was achieved on Ed 16 (0.16 +/- 0.01 pmol/mg protein). After this stage there was a sharp and transient increase in specific [3H]FNZ binding of about two-fold reaching a maximal value between hatching and the postnatal day (pnd) 2 (0.33 +/- 0.01 pmol/mg protein). Scatchard analysis at different stages of development revealed the presence of a single population of specific FNZ binding sites. The increase in [3H]FNZ binding during development was due to a large number of binding sites while their affinity remained unchanged. Competition experiments in the chick optic lobe revealed that the order of potency for displacement of specific [3H]FNZ binding paralleled the pharmacological potency of the BZDs tested. The IC50s for clonazepam, flunitrazepam, Ro 15-1788 and chlordiazepoxide were 3.02, 4.30, 0.32, and 4778.64 nM respectively. Ro 5-4864, a potent inhibitor of BZD binding to peripheral tissues, had no effect on specific [3H]FNZ binding indicating that only central BZD binding sites are present in the chick optic lobe. The peak of maximal expression of BZD receptor sites precedes in 5-6 days the peak of GABA receptor sites indicating a precocious development of BZD receptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamic acid decarboxylase in different areas of the developing chick central nervous system.

The temporal course of the development of GAD activity in GABAergic neurons was studied in the chick retina, optic lobe and cerebellum. The developmental pattern of GAD activity was similar in the three areas studied, showing typical sigmoideal curves, which reached a maximal value at the 3rd post-hatching day. Kinetic studies during development revealed that Km remained unchanged while Vmax increased 3-fold in the retina (48.99 +/- 0.84 nmol/hr/mg protein), almost 4-fold in the optic lobe (162.77 +/- 4.32 nmol/hr/mg protein) and 3.5 fold in the cerebellum (69.30 +/- 1.26 nmol/hr/mg protein). The developmental pattern of GAD activity in homogenates of the three areas studied from dark-reared and light-reared chicks with respect to normal light-dark cycle animals showed no significant differences. These results indicate that the increase in GAD activity during development are not due to a change in the affinity for its substrate but rather to changes in the concentration of the enzyme. The developmental pattern of GAD activity in the chick visual system was not affected by environmental conditions suggesting that the developmental profile is light-independent.

Development of topographic connections between the isthmic nuclei and optic tecta in the frog Limnodynastes dorsalis.

In the frog Limnodynastes dorsalis, the pattern of topographic connections between the isthmic nuclei and optic tecta was determined by anterograde and retrograde transport of horseradish peroxidase from localised tectal regions. In both larvae and adults, reciprocal mapping of the uncrossed isthmo-tectal and tecto-isthmal projections was evidenced by the juxtaposition of labelled tecto-isthmal terminations with labelled cells in the cortex and medulla of the ipsilateral isthmic nucleus. The crossed isthmo-tectal projection was revealed by labelled cells in the cortex and medulla of the nucleus contralateral to the injection. In adults, rostral tectal areas projected to rostral and ventral regions of the ipsilateral isthmic nucleus. Following more caudal tectal injections, labelled cells were found in progressively more dorsal locations within the nucleus. Labelled cells in the contralateral nucleus were found in the rim cortex abutting a neuropil and in medullary cells adjacent to this region. Connections between ventral isthmic regions and most rostral tectum and between dorsomedial nucleus and caudomedial tectum were similar in both nuclei. However, for isthmic areas projecting to rostromedial and mid-tectum, the location of labelled cells in the contralateral nucleus was inverted with respect to the ipsilateral nucleus. This inversion would allow both nuclei to project to visually corresponding regions of each tectum. During larval stages the basic adult topography was established despite the continued neurogenesis of both isthmic nuclei and optic tecta. In late larval stages a rim neuropil appeared adjacent to the cortical region in the isthmic nuclei where labelled cells of the crossed isthmotectal projection were found. Prior to this stage labelled cells abutted labelled medullary cells. The appearance of this neuropil was approximately temporally correlated with the onset of electrophysiologically detectable responses in the ipsilateral visuotectal projection. Formation of the rim neuropil may relate to maturation of the tecto-isthmo-tectal connections which underlie this visual projection.

Intratectal targeting by optic fibers in goldfish under impulse blockade.

Regenerating optic fibers in goldfish were tested for their capacity to grow from an inappropriate region of the tectum to their appropriate part of the tectum when silenced by periodic intraocular injections of tetrodotoxin. For this, fibers normally innervating the lateral posterior quadrant of one tectum were surgically redirected into the medial anterior end of the opposite host tectum. This starting position corresponds to the path normally taken by fibers to innervate the medial tectum. For comparison, fibers normally innervating the medial posterior quadrant of the opposite tectum were surgically redirected into this same position in the host tectum. To prevent cueing with resident optic fibers, host fibers were eliminated by removing the eye supplying the host tectum. The innervation by these deflected fibers was determined autoradiographically using quantitative microdensitometry. Electrically silent fibers were found to navigate toward their appropriate region just as well as fibers with impulse activity. Thus, the capacity of fibers to read positional tectal cues and to bypass foreign tectal regions was not detectably regulated by impulse activity.

Identification of gene products expressed in the developing chick visual system: characterization of a middle-molecular-weight neurofilament cDNA.

A cDNA library enriched for transcripts that accumulate from embryonic day 7 until day 1 posthatching was constructed from poly(A)+ RNA of chick optic lobe tissue. From this library, three recombinants were isolated that correspond to neural mRNAs appearing during the major period of synaptogenesis in the retinotectal system. One of these recombinants (OZ 11) was identified as a middle-molecular weight neurofilament (NF-M) cDNA which, together with two overlapping clones, encodes part of the rod and the entire tail region of NF-M. In situ hybridization revealed NF-M mRNA to be highly expressed in regions of the chick central nervous system, which contain multipolar and/or long-projection neurons.

Effects of light- and dark-rearing on the postnatal development of GABA receptor sites in the chick optic lobe.

To investigate the ability of GABA receptor sites to undergo environmental-dependent plastic changes, the postnatal developmental pattern of GABA receptors was studied under different levels of light stimulation, i.e. normal-, light- and dark-rearing. At hatching the specific binding of [3H]GABA was 1.74 +/- 0.36 pmol/optic lobe. In normally reared chicks the number of GABA binding sites showed a transient increase with the highest value at the 6th day (7.0 +/- 1.32 pmol/optic lobe). This value is higher than the one reached at the adult stage. Between the 3rd and 6th day, there was a 33.7% increase in specific [3H]GABA binding in light-reared compared with normally reared animals (P less than 0.05). In the dark-reared chicks, the specific binding was 36.4% lower than that found in normally reared (P less than 0.02). However, the changes in receptor density were transient since at the 17th day the number of GABA binding sites returned to adult levels. Scatchard analysis revealed that the differences observed in the high affinity GABA binding sites between the three groups were due to modifications in the total number of binding sites while the affinity remained unchanged. The maximal number of binding sites were: 2.71, 7.01 and 1.79 pmol/mg protein in normally, light- and dark-reared chicks, respectively; while the apparent dissociation constants were unaffected: 3.2, 3.4 and 3.6 nM, respectively. These results show that, during postnatal development, different conditions of visual experience produce synaptic changes at the molecular level. These changes probably occur within a period of high plasticity, prior to the end of a critical period.

Ocular migration and the metamorphic and postmetamorphic maturation of the retinotectal system in Xenopus laevis: an autoradiographic and morphometric study.

The growth of the retina and tectum during larval life in Xenopus has previously been studied extensively. These two structures continue to grow in metamorphosing and postmetamorphic animals. During these later stages there are marked changes in eye position. We have used histogenetic and morphometric techniques to monitor retinal and tectal growth in Xenopus aged from stage 58 of larval life to ten years postmetamorphosis. The relative eye migration was quantified with optical techniques. An attempt has been made to relate modes of retinal growth to problems associated with changing interocular geometry. The predominant mode of growth during this period is hypertrophic, that is, it is due largely to an increase in size and complexity of existing elements, rather than the histogenetic creation of new elements. In the retina, however, tritiated thymidine autoradiography reveals that histogenesis does persist until at least six months after metamorphosis. The rate of histogenesis is much decreased compared with that of larval stages but, like that of larval stages, it takes place at the ciliary margin. It is asymmetrically distributed in the retina, most histogenesis occurring at the ventral retinal pole and very little at the dorsal retinal pole. Some histogenesis occurs at the nasal and temporal retinal poles. Thymidine autoradiography and cell counts indicated that little significant histogenesis occurs in the tectum after metamorphic climax. Two possible biological reasons for the asymmetry of retinal histogenesis have been examined. The first was the suggestion that the asymmetry was related to the emergence, during this period, of a binocular visual field secondary to the relative migration of the two eyes. It is shown that the asymmetric pattern of histogenesis is not closely related to those retinal areas that come to view binocular visual space. It was found, however, that the asymmetry of retinal growth from metamorphic climax was that required to stabilize retinal visual field positions with respect to body position. It is suggested that this minimizes the requirements for visuomotor readjustments that would otherwise be necessary to compensate for a situation in which eye position, relative to the body, is changing.

Visual projections in larval Ichthyophis kohtaoensis (Amphibia: gymnophiona).

The visual projection patterns of retinal efferents were studied in larval Ichthyophis kohtaoensis by means of anterogradely transported HRP. Our results show in all larvae a projection contralateral to a thalamic terminal field, a pretectal terminal field, and a basal optic neuropil, but only a sparse innervation of the contralateral tectum. In addition, all larvae possess an uncrossed projection to a thalamic and a pretectal terminal field. The fibers are bilaterally almost confined to the medial optic tract with only a few fibers running in the marginal and basal optic tract. The ipsilateral and contralateral tracts and terminal fields seem to enlarge during larval life. Comparison with other amphibian orders reveals that larval Ichthyophis are unique in that they develop the medial optic tract and the related thalamic and pretectal terminal fields very early in larval life. In addition they possess only a very sparse tectal projection, though it is the largest projection in larval urodeles and anurans. This suggests a selective phylogenetic loss of those ganglion cells or collaterals which project mainly to the tectum in other amphibian orders and a change in the ontogenetic program leading to an earlier development of the medial optic tract in Ichthyophis as compared to urodeles and anurans.