Retinal Ganglion Cell Axon Terminals Research Articles

Topographic axonal projection at single-cell precision supports local retinotopy in the mouse superior colliculus

Retinotopy, like all long-range projections, can arise from the axons themselves or their targets. The underlying connectivity pattern, however, remains elusive at the fine scale in the mammalian brain. To address this question, we functionally mapped the spatial organization of the input axons and target neurons in the female mouse retinocollicular pathway at single-cell resolution using in vivo two-photon calcium imaging. We found a near-perfect retinotopic tiling of retinal ganglion cell axon terminals, with an average error below 30 μm or 2° of visual angle. The precision of retinotopy was relatively lower for local neurons in the superior colliculus. Subsequent data-driven modeling ascribed it to a low input convergence, on average 5.5 retinal ganglion cell inputs per postsynaptic cell in the superior colliculus. These results indicate that retinotopy arises largely from topographically precise input from presynaptic cells, rather than elaborating local circuitry to reconstruct the topography by postsynaptic cells.

MRNA transport, translation, and decay in adult mammalian central nervous system axons

Localized mRNA translation regulates synapse function and axon maintenance, but how compartment-specific mRNA repertoires are regulated is largely unknown. We developed an axonal transcriptome capture method that allows deep sequencing of metabolically labeled mRNAs from retinal ganglion cell axon terminals in mouse. Comparing axonal-to-somal transcriptomes and axonal translatome-to-transcriptome enables genome-wide visualization of mRNA transport and translation and unveils potential regulators tuned to each process. FMRP and TDP-43 stand out as key regulators of transport, and experiments in Fmr1 knockout mice validate FMRP's role in the axonal transportation of synapse-related mRNAs. Pulse-and-chase experiments enable genome-wide assessment of mRNA stability in axons and reveal a strong coupling between mRNA translation and decay. Measuring the absolute mRNA abundance per axon terminal shows that the adult axonal transcriptome is stably maintained by persistent transport. Our datasets provide a rich resource for unique insights into RNA-based mechanisms in maintaining presynaptic structure and function invivo.

The fine tuning of retinocollicular topography depends on reelin signaling during early postnatal development of the rat visual system

During postnatal development, neural circuits are extremely dynamic and develop precise connection patterns that emerge as a result of the elimination of synaptic terminals, a process instructed by molecular cues and patterns of electrical activity. In the rodent visual system, this process begins during the first postnatal week and proceeds during the second and third postnatal weeks as spontaneous retinal activity and finally use-dependent fine tuning takes place. Reelin is a large extracellular matrix glycoprotein able to affect several steps of brain development, from neuronal migration to the maturation of dendritic spines and use-dependent synaptic development. In the present study, we investigated the role of reelin on the topographical refinement of primary sensory connections studying the development of retinal ganglion cell axon terminals in the rat superior colliculus. We found that reelin levels in the visual layers of the superior colliculus are the highest between the second and third postnatal weeks. Blocking reelin signaling with a neutralizing antibody (CR-50) from PND 7 to PND 14 induced a non-specific sprouting of ipsilateral retinocollicular axons outside their typical distribution of discrete patches of axon terminals. Also we found that reelin blockade resulted in reduced levels of phospho-GAP43, increased GluN1 and GluN2B-NMDA subunits and decreased levels of GAD65 content in the visual layers of the superior colliculus. The results suggest that reelin signaling is associated with the maturation of excitatory and inhibitory synaptic machinery influencing the development and fine tuning of topographically organized neural circuits during postnatal development.

Central visual pathways in glaucoma: evidence for distal mechanisms of neuronal self-repair.

As in other age-related neurodegenerative diseases, progression of neurodegeneration in glaucoma involves early axonopathy. In glaucoma, this is marked by degradation of active transport along retinal ganglion cell (RGC) axons projecting from the retina to the brain. In experimental systems, transport degradation occurs first in the most distal site in the RGC projection, the superior colliculus (SC) of the midbrain. Even as degradation progresses from one retinotopic sector to the next, important structures in the affected sectors persist, including synapses from RGC axon terminals onto SC neurons. This structural persistence is accompanied by focally increased brain-derived neurotrophic factor in hypertrophic SC astrocyte glia and defines a therapeutic window of opportunity. Thus, central brain structures in glaucoma may respond to disease-relevant stress by induction of mechanisms useful for maintaining retinal signals.

Analysis of the lateral geniculate nucleus in dichromatic and trichromatic marmosets.

Marmosets are diurnal New World monkeys that show sex-linked cone photopigment polymorphism, whereby all males and some females are dichromats ("red-green colorblind"), but most females show trichromatic color vision. Here we asked whether trichromats express chromatic-specific circuitry in the lateral geniculate nucleus (LGN). The volume of parvocellular (P), magnocellular (M), and koniocellular (K) layers was calculated in Nissl-stained sections from the LGN of adult marmosets (Callithrix jacchus; 10 trichromatic females; 2 dichromatic females; and 13 dichromatic males). Retinal ganglion cell axon terminals within the P and K layers were reconstructed and measured following anterograde tracer (dextran) injections. We show that there is little difference in LGN layer volume with respect to age, weight, or sex of the animals, or between dichromatic and trichromatic phenotypes. The morphology of retinal ganglion cell terminals was largely indistinguishable on comparing dichromats and trichromats, and likewise on comparing terminals representing peripheral or foveal retina. We conclude that the LGN circuits we studied are largely independent of red-green color vision phenotype and visual field location.

Failure of axonal transport induces a spatially coincident increase in astrocyte BDNF prior to synapse loss in a central target

Failure of anterograde transport to distal targets in the brain is a common feature of neurodegenerative diseases. We have demonstrated in rodent models of glaucoma, the most common optic neuropathy, early loss of anterograde transport along the retinal ganglion cell (RGC) projection to the superior colliculus (SC) is retinotopic and followed by a period of persistence of RGC axon terminals and synapses through unknown molecular pathways. Here we use the DBA/2J mouse model of hereditary glaucoma and an acute rat model to demonstrate that retinotopically focal transport deficits in the SC are accompanied by a spatially coincident increase in brain-derived neurotrophic factor (BDNF), especially in hypertrophic astrocytes. These neurochemical changes occur prior to loss of RGC synapses in the DBA/2J SC. In contrast to BDNF protein, levels of Bdnf mRNA decreased with transport failure, even as mRNA encoding synaptic structures remained unchanged. In situ hybridization signal for Bdnf mRNA was the strongest in SC neurons, and labeling for the immature precursor pro-BDNF was very limited. Subcellular fractionation of SC indicated that membrane-bound BDNF decreased with age in the DBA/2J, while BDNF released from vesicles remained high. These results suggest that in response to diminished axonal function, activated astrocytes in the brain may sequester mature BDNF released from target neurons to counter stressors that otherwise would challenge survival of projection synapses.

Synaptic maturation of the Xenopus retinotectal system: Effects of brain‐derived neurotrophic factor on synapse ultrastructure

Synaptogenesis is a dynamic process that involves structural changes in developing axons and dendrites as synapses form and mature. The visual system of Xenopus laevis has been used as a model to study dynamic changes in axons and dendrites as synapses form in the living brain and the molecular mechanisms that control these processes. Brain-derived neurotrophic factor (BDNF) contributes to the establishment and refinement of visual connectivity by modulating retinal ganglion cell (RGC) axon arborization and presynaptic differentiation. Here, we have analyzed the ultrastructural organization of the Xenopus retinotectal system to understand better the maturation of this synaptic circuit and the relation between synapse ultrastructure and the structural changes in connectivity that take place in response to BDNF. Expression of yellow fluorescent protein (YFP) followed by preembedding immunoelectron microscopy was used to identify RGC axons specifically in living tadpoles. Injection of recombinant BDNF was used to alter endogenous BDNF levels acutely in the optic tectum. Our studies reveal a rapid transition from a relatively immature synaptic circuit in which retinotectal synapses are formed on developing filopodial-like processes to a circuit in which RGC axon terminals establish synapses with dendritic shafts and spines. Moreover, our studies reveal that BDNF treatment increases the number of spine synapses and docked vesicle number at YFP-identified synaptic sites within 24 hours of treatment. These fine structural changes at retinotectal synapses are consistent with the role that BDNF plays in the functional maturation of synaptic circuits and with dynamic, rapid changes in synaptic connectivity during development.

Alpha Cells Take Off First

Using a transgenic mouse line in which GFP is expressed in a single population of retinal ganglion cells (RGCs), Huberman and colleagues report in this issue of Neuron that the axon terminals of RGCs exhibit an orderly pattern of distribution in the higher visual centers. This pattern undergoes a developmental refinement, and synchronous activity in the retina regulates columnar but not laminar formation.

Zebrafish Olfactomedin 1 Regulates Retinal Axon ElongationIn Vivoand Is a Modulator of Wnt Signaling Pathway

Olfactomedin 1 (Olfm1) is a secreted glycoprotein belonging to a family of olfactomedin domain-containing proteins. It is involved in the regulation of neural crest production in chicken and promotes neuronal differentiation in Xenopus. Here, we investigate the functions of Olfm1 in zebrafish eye development. Overexpression of full-length Olfm1, and especially its BMY form lacking the olfactomedin domain, increased the thickness of the optic nerve and produced a more extended projection field in the optic tectum compared with control embryos. In contrast, injection of olfm1-morpholino oligonucleotide (Olfm1-MO) reduced the eye size, inhibited optic nerve extension, and increased the number of apoptotic cells in the retinal ganglion cell and inner nuclear layers. Overexpression of full-length Olfm1 increased the lateral separation of the expression domains of eye-field markers, rx3 and six3. The Olfm1-MO had the opposite effect. These data suggest that zebrafish Olfm1 may play roles in the early eye determination, differentiation, optic nerve extension, and branching of the retinal ganglion cell axon terminals, with the N-terminal region of Olfm1 being critical for these effects. Injection of RNA encoding WIF-1, a secreted inhibitor of Wnt signaling, caused changes in the expression pattern of rx3 similar to those observed after Olfm1-MO injection. Simultaneous overexpression of WIF-1 and Olfm1 abolished the WIF-1 effect. Physical interaction of WIF-1 and Olfm1 was demonstrated by coimmunoprecipitation experiments. We concluded that Olfm1 serves as a modulator of Wnt signaling.

Regulation by Glycogen Synthase Kinase-3β of the Arborization Field and Maturation of Retinotectal Projection in Zebrafish

The retinotectal projection is one of the best systems to study the molecular basis of synapse formation in the CNS because of the well characterized topographic connections and activity-dependent refinement. Here, we developed a presynaptic neuron-specific gene manipulation system in the zebrafish retinotectal projection in vivo using the nicotinic acetylcholine receptor beta3 (nAChRbeta3) gene promoter. Enhanced green fluorescent protein (EGFP) expression signals in living transgenic zebrafish lines carrying the nAChRbeta3 gene promoter-directed EGFP expression vector visualized the development of entire retinal ganglion cell (RGC) axon projection to the tectum. Microinjection of the nAChRbeta3 gene promoter-driven double-cassette vectors directing the expression of both dominant-negative glycogen synthase kinase-3beta (dnGSK-3beta) and EGFP enabled us to follow the development of individual RGCs and to examine the effect of the molecule on the axonal arborization and maturation of the same neurons in living zebrafish. We found that the expression of the dominant-negative form of zebrafish GSK-3beta suppressed the arborization field of RGC axon terminals in the tectum as estimated by the reduction of arbor branch length and arbor areas. Furthermore, the suppression of GSK-3beta activity increased the size of vesicle-associated membrane protein 2-EGFP puncta in RGC axon terminals at the early stage of innervation to the tectum. These results suggest that GSK-3beta regulates the arborization field and maturation of RGC axon terminals in vivo.

Regulation of Class I MHC Gene Expression in the Developing and Mature CNS by Neural Activity

To elucidate molecular mechanisms underlying activity-dependent synaptic remodeling in the developing mammalian visual system, we screened for genes whose expression in the lateral geniculate nucleus (LGN) is regulated by spontaneously generated action potentials present prior to vision. Activity blockade did not alter expression in the LGN of 32 known genes. Differential mRNA display, however, revealed a decrease in mRNAs encoding class I major histocompatibility complex antigens (class I MHC). Postnatally, visually driven activity can regulate class I MHC in the LGN during the final remodeling of retinal ganglion cell axon terminals. Moreover, in the mature hippocampus, class I MHC mRNA levels are increased by kainic acid–induced seizures. Normal expression of class I MHC mRNA is correlated with times and regions of synaptic plasticity, and immunohistochemistry confirms that class I MHC is present in specific subsets of CNS neurons. Finally, β2-microglobulin, a cosubunit of class I MHC, and CD3ζ, a component of a receptor complex for class I MHC, are also expressed by CNS neurons. These observations indicate that class I MHC molecules, classically thought to mediate cell–cell interactions exclusively in immune function, may play a novel role in neuronal signaling and activity-dependent changes in synaptic connectivity.

Membrane properties and monosynaptic retinal excitation of neurons in the turtle accessory optic system.

Using an eye-attached isolated brain stem preparation of a turtle, Pseudemys scripta elegans, in conjunction with whole cell patch techniques, we recorded intracellular activity of accessory optic system neurons in the basal optic nucleus (BON). This technique offered long-lasting stable recordings of individual synaptic events. In the reduced preparation (most of the dorsal structures were removed), large spontaneous excitatory synaptic inputs [excitatory postsynaptic potentials (EPSPs)] were frequently recorded. Spontaneous inhibitory postsynaptic potentials were rarely observed except in few cases. Most EPSPs disappeared after injection of lidocaine into the retina. A few EPSPs of small size remained, suggesting that these EPSPs either were from intracranial sources or may have been miniature spontaneous synaptic potentials from retinal ganglion cell axon terminals. Population EPSPs were synchronously evoked by electrical stimulation of the contralateral optic nerve. Their constant onset latency and their ability to follow short-interval paired stimulation indicated that much of the population EPSP's response was monosynaptic. Visually evoked BON spikes and EPSP inputs to BON showed direction sensitivity when a moving pattern was projected onto the entire contralateral retina. With the use of smaller moving patterns, the receptive field of an individual BON cell was identified. A small spot of light, projected within the receptive field, guided the placement of a bipolar stimulation electrode to activate retinal ganglion cells that provided input to that BON cell. EPSPs evoked by this retinal microstimulation showed features of unitary EPSPs. Those EPSPs had distinct low current thresholds. Recruitment of other inputs was only evident when the stimulation level was increased substantially above threshold. The average size of evoked unitary EPSPs was 7.8 mV, confirming the large size of synaptic inputs of this system relative to nonsynaptic noise. EPSP shape was plotted (rise time vs. amplitude), with the use of either evoked unitary EPSPs or spontaneous EPSPs. Unlike samples of spontaneous EPSPs, data from many unitary EPSPs formed distinct clusters in these scatterplots, indicating that these EPSPs had a unique shape among the whole population of EPSPs. In most BON cells studied, hyperpolarization-activated channels caused a slow depolarization sag that reached a plateau within 0.5-1 s. This property suggests that BON cells may be more complicated than a simple site for convergence of direction-sensitive retinal ganglion cells to form a central retinal slip signal for control of oculomotor reflexes.

Retinal ganglion cell terminals in the hamster superior colliculus: An ultrastructural study

The distribution, cross-sectional area, and presynaptic and postsynaptic characteristics of retinal ganglion cell axon terminals in the superior colliculus of normal adult female Syrian hamsters were investigated by quantitative ultrastructural techniques. After an intravitreal injection of horseradish peroxidase, most labelled axon terminals were found in the stratum griseum superficiale and stratum opticum of the contralateral superior colliculus. However, a small proportion (approximately 2%) of retinal ganglion cell axon terminals were located in deeper layers of the superior colliculus between the stratum opticum and the periaqueductal grey matter. Terminals were smaller in the upper two-thirds of the stratum griseum superficiale than in the lower one-third of this layer, the stratum opticum, and the stratum griseum intermedium. Presynaptic characteristics such as the length and number of contacts and the postsynaptic neuronal domains (somata, dendritic spines, or shafts) contacted by retinal ganglion cell axons in the superior colliculus were similar in all layers.

Time of arrival of wheat germ agglutinin-HRP conjugates in superior colliculus after intraocular injections in the rat

Rats received intravitreal injections of wheat germ agglutinin conjugated with horseradish peroxidase (WGHRP) or labeled with fluorescein isothiocyanate (WGFITC). SDS polyacrylamide gel electrophoresis of WGHRP demonstrated that little or no native horseradish peroxidase (HRP) was present in the injected conjugate. Under the conditions employed in these experiments, evidence for the orthograde transport of WGHRP but not of WGFITC was observed. In a separate experiment carried out under similar circumstances, WGFITC was retrogradely transported. Thus WGFITC does not appear to be a sensitive probe for studies of efferent projections. The earliest time of arrival of WGHRP in the contralateral superior colliculus (SC) was determined in order to establish the most rapid rate of anterograde transport of WGHRP. Retinal ganglion cell axon terminals in SC were first labeled 4 h after an intraocular injection of WGHRP which is equivalent to a transport rate of about 108 mm/day. This rate of transport is approximately half that reported for native HRP. It is concluded that the superior sensitivity of WGHRP over native HRP as an anterogradely transported marker cannot be ascribed to a more rapid rate of orthograde transport of WGHRP.