Pathway-specific genetic attenuation of glutamate release alters select features of competition-based visual circuit refinement

A hallmark of mammalian neural circuit development is the refinement of initially imprecise connections by competitive activity-dependent processes. In the developing visual system retinal ganglion cell (RGC) axons from the two eyes undergo activity-dependent competition for territory in the dorsal lateral geniculate nucleus (dLGN). The direct contributions of synaptic transmission to this process, however, remain unclear. We used a genetic approach to reduce glutamate release selectively from ipsilateral-projecting RGCs and found that their release-deficient axons failed to exclude competing axons from the ipsilateral eye territory in the dLGN. Nevertheless, the release-deficient axons consolidated and maintained their normal amount of dLGN territory, even in the face of fully active competing axons. These results show that during visual circuit refinement glutamatergic transmission plays a direct role in excluding competing axons from inappropriate target regions, but they argue that consolidation and maintenance of axonal territory are largely insensitive to alterations in synaptic activity levels.     

Dark rearing alters the normal development of spatiotemporal response properties but not of contrast detection threshold in mouse retinal ganglion cells

The mouse visual system is immature when the eyes open two weeks after birth. As in other mammals, some of the maturation that occurs in the subsequent weeks is known to depend on visual experience. Development of the retina, which as the first stage of vision provides the visual information to the brain, also depends on light‐driven activity for proper development but has been less well studied than visual cortical development. The critical properties for retinal encoding of images include detection of contrast and responsiveness to the broad range of temporal stimulus frequencies present in natural stimuli. Here we show that contrast detection threshold and temporal frequency response characteristics of ON and OFF retinal ganglion cells (RGCs), which are poor at eye opening, subsequently undergo maturation, improving RGC performance. Further, we find that depriving mice of visual experience from before birth by rearing them in the dark causes ON and OFF RGCs to have smaller receptive field centers but does not affect their contrast detection threshold development. The modest developmental increase in temporal frequency responsiveness of RGCs in mice reared on a normal light cycle was inhibited by dark rearing only in ON but not OFF RGCs. Thus, these RGC response characteristics are in many ways unaffected by the experience‐dependent changes to synaptic and spontaneous activity known to occur in the mouse retina in the two weeks after eye opening, but specific differences are apparent in the ON vs. OFF RGC populations. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 692–706, 2014     

Vision drives correlated activity without patterned spontaneous activity in developing Xenopus retina

Developing amphibians need vision to avoid predators and locate food before visual system circuits fully mature. Xenopus tadpoles can respond to visual stimuli as soon as retinal ganglion cells (RGCs) innervate the brain, however, in mammals, chicks and turtles, RGCs reach their central targets many days, or even weeks, before their retinas are capable of vision. In the absence of vision, activity-dependent refinement in these amniote species is mediated by waves of spontaneous activity that periodically spread across the retina, correlating the firing of action potentials in neighboring RGCs. Theory suggests that retinorecipient neurons in the brain use patterned RGC activity to sharpen the retinotopy first established by genetic cues. We find that in both wild type and albino Xenopus tadpoles, RGCs are spontaneously active at all stages of tadpole development studied, but their population activity never coalesces into waves. Even at the earliest stages recorded, visual stimulation dominates over spontaneous activity and can generate patterns of RGC activity similar to the locally correlated spontaneous activity observed in amniotes. In addition, we show that blocking AMPA and NMDA type glutamate receptors significantly decreases spontaneous activity in young Xenopus retina, but that blocking GABA(A) receptor blockers does not. Our findings indicate that vision drives correlated activity required for topographic map formation. They further suggest that developing retinal circuits in the two major subdivisions of tetrapods, amphibians and amniotes, evolved different strategies to supply appropriately patterned RGC activity to drive visual circuit refinement.     

Glaucoma and optic nerve repair

Glaucoma is a leading cause of irreversible blindness worldwide and causes progressive visual impairment attributable to the dysfunction and death of retinal ganglion cells (RGCs). Progression of visual field damage is slow and typically painless. Thus, glaucoma is often diagnosed after a substantial percentage of RGCs has been damaged. To date, clinical interventions are mainly restricted to the reduction of intraocular pressure (IOP), one of the major risk factors for this disease. However, the lowering of IOP is often insufficient to halt or reverse the progress of visual loss, underlining the need for the development of alternative treatment strategies. Several lines of evidence suggest that axonal damage of RGCs occurs primary at the optic nerve head, where axons appear to be most vulnerable. Axonal injury leads to the functional loss of RGCs and subsequently induces the death of the neurons. However, the detailed molecular mechanism(s) underlying IOP-induced optic nerve injury remain poorly understood. Moreover, whether glaucoma pathophysiology is primarily axonal, glial, or vascular remains unclear. Therefore, protective strategies to prevent further axonal and subsequent soma degeneration are of great importance to limit the progression of sight loss. In addition, strategies that stimulate injured RGCs to regenerate and reconnect axons with their central targets are necessary for functional restoration. The present review provides an overview of the context of glaucoma pathogenesis and surveys recent findings regarding potential strategies for axonal regeneration of RGCs and optic nerve repair, focusing on the role of cytokines and their downstream signaling pathways.     

Zic1 and Zic4 regulate zebrafish roof plate specification and hindbrain ventricle morphogenesis

During development, the lumen of the neural tube develops into a system of brain cavities or ventricles, which play important roles in normal CNS function. We have established that the formation of the hindbrain (4th) ventricle in zebrafish is dependent upon the pleiotropic functions of the genes implicated in human Dandy Walker Malformation, Zic1 and Zic4. Using morpholino knockdown we show that zebrafish Zic1 and Zic4 are required for normal morphogenesis of the 4th ventricle. In Zic1 and/or Zic4 morphants the ventricle does not open properly, but remains completely or partially fused from the level of rhombomere (r) 2 towards the posterior. In the absence of Zic function early hindbrain regionalization and neural crest development remain unaffected, but dorsal hindbrain progenitor cell proliferation is significantly reduced. Importantly, we find that Zic1 and Zic4 are required for development of the dorsal roof plate. In Zic morphants expression of roof plate markers, including lmx1b.1 and lmx1b.2, is disrupted. We further demonstrate that zebrafish Lmx1b function is required for both hindbrain roof plate development and 4th ventricle morphogenesis, confirming that roof plate formation is a critical component of ventricle development. Finally, we show that dorsal rhombomere boundary signaling centers depend on Zic1 and Zic4 function and on roof plate signals, and provide evidence that these boundary signals are also required for ventricle morphogenesis. In summary, we conclude that Zic1 and Zic4 control zebrafish 4th ventricle morphogenesis by regulating multiple mechanisms including cell proliferation and fate specification in the dorsal hindbrain.     

Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.     

Degenerative and regenerative responses of injured neurons in the central nervous system of adult mammals.

In adult mammals, the severing of the optic nerve near the eye is followed by a loss of retinal ganglion cells (RGCs) and a failure of axons to regrow into the brain. Experimental manipulations of the non-neuronal environment of injured RGCs enhance neuronal survival and make possible a lengthy axonal regeneration that restores functional connections with the superior colliculus. These effects suggest that injured nerve cells in the mature central nervous system (CNS) are strongly influenced by interactions with components of their immediate environment as well as their targets. Under these conditions, injured CNS neurons can express capacities for growth and differentiation that resemble those of normally developing neurons. An understanding of this regeneration in the context of the cellular and molecular events that influence the interactions of axonal growth cones with their non-neuronal substrates and neuronal targets should help in the further elucidation of the capacities of neuronal systems to recover from injury.     

Autonomous and non-autonomous Shh signalling mediate the in vivo growth and guidance of mouse retinal ganglion cell axons

In non-mammalian vertebrates, the relatively homogeneous population of retinal ganglion cells (RGCs) differentiates and projects entirely to the contralateral side of the brain under the influence of sonic hedgehog (Shh). In mammals, by contrast, there are two different RGC types: the Zic2-positive ipsilateral projecting and the Isl2-positive contralateral projecting. We asked whether the axons of these two populations respond to Shh and if their response differs. We have also analysed whether midline- and RGC-derived Shh contributes to the growth of the axons in the proximal visual pathway. We show that these two RGC types are characterised by a differential expression of Shh signalling components and that they respond differently to Shh when challenged in vitro. In vivo blockade of Shh activity, however, alters the path and distribution mostly of the contralateral projecting RGC axons at the chiasm, indicating that midline-derived Shh participates in funnelling contralateral visual fibres in this region. Furthermore, interference with Shh signalling in the RGCs themselves causes abnormal growth and navigation of contralateral projecting axons in the proximal portion of the pathway, highlighting a novel cell-autonomous mechanism by which Shh can influence growth cone behaviour.     

An instructive role for patterned spontaneous retinal activity in mouse visual map development

Complex neural circuits in the mammalian brain develop through a combination of genetic instruction and activity-dependent refinement. The relative role of these factors and the form of neuronal activity responsible for circuit development is a matter of significant debate. In the mammalian visual system, retinal ganglion cell projections to the brain are mapped with respect to retinotopic location and eye of origin. We manipulated the pattern of spontaneous retinal waves present during development without changing overall activity levels through the transgenic expression of β2-nicotinic acetylcholine receptors in retinal ganglion cells of mice. We used this manipulation to demonstrate that spontaneous retinal activity is not just permissive, but instructive in the emergence of eye-specific segregation and retinotopic refinement in the mouse visual system. This suggests that specific patterns of spontaneous activity throughout the developing brain are essential in the emergence of specific and distinct patterns of neuronal connectivity.     

Developmental retinal ganglion cell death and retinotopicity of the murine retinocollicular projection

During mammalian visual system development, retinal ganglion cells (RGCs) undergo extensive apoptotic death. In mouse retina, approximately 50% of RGCs present at birth (postnatal day 0; P0) die by P5, at a time when axons innervate central targets such as the superior colliculus (SC). We examined whether RGCs that make short‐range axonal targeting errors within the contralateral SC are more likely to be eliminated during the peak period of RGC death (P1‐P5), compared with RGCs initially making more accurate retinotopic connections. A small volume (2.3 nL) of the retrograde nucleophilic dye Hoechst 33342 was injected into the superficial left SC of anesthetized neonatal C57Bl/6J mice at P1 (n = 5) or P4 (n = 8), and the contralateral retina wholemounted 12 hr later. Retrogradely labelled healthy and dying (pyknotic) RGCs were identified by morphological criteria and counted. The percentage of pyknotic RGCs was analyzed in relation to distance from the area of highest density RGC labelling, presumed to represent the most topographically accurate population. As expected, pyknotic RGC density at P1 was significantly greater than P4 (p < 0.05). At P4, the density of healthy RGCs 500–750 µm away from the central region was significantly less, although this was not reflected in altered pyknotic rates. However, at P1 there was a trend (p = 0.08) for an increased proportion of pyknotic RGCs, specifically in temporal parts of the retina outside the densely labelled center. Overall, the lack of consistent association between short‐range targeting errors and cell death suggests that most postnatal RGC loss is not directly related to topographic accuracy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 51–60, 2018     

Transient ipsilateral retinal ganglion cell projections to the brain: Extent,targeting, and disappearance

During development of the mammalian eye, the first retinal ganglion cells (RGCs) that extend to the brain are located in the dorsocentral (DC) retina. These RGCs extend to either ipsilateral or contralateral targets, but the ipsilateral projections do not survive into postnatal periods. The function and means of disappearance of the transient ipsilateral projection are not known. We have followed the course of this transient early ipsilateral cohort of RGCs, paying attention to how far they extend, whether they enter targets and if so, which ones, and the time course of their disappearance. The DC ipsilateral RGC axons were traced using DiI labeling at E13.5 and E15.5 to compare the proportion of ipsi‐ versus contralateral projections during the first period of growth. In utero electroporation of E12.5 retina with GFP constructs was used to label axons that could be visualized at succeeding time points into postnatal ages. Our results show that the earliest ipsilateral axons grow along the cellular border of the brain, and are segregated from the laterally positioned contralateral axons from the same retinal origin. In agreement with previous reports, although many early RGCs extend ipsilaterally, after E16 their number rapidly declines. Nonetheless, some ipsilateral axons from the DC retina enter the superior colliculus and arborize minimally, but very few enter the dorsal lateral geniculate nucleus and those that do extend only short branches. While the mechanism of selective axonal disappearance remains elusive, these data give further insight into establishment of the visual pathways. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1385–1401, 2015