Axonal transport kinetics and posttranslational modification of synapsin I in mouse retinal ganglion cells.

Synapsin I is a neuron-specific phosphoprotein primarily localized at the presynaptic terminals, where it is thought to play an important role in the mechanisms involved in neurotransmitter release. Its interaction with cytoskeletal proteins and with small synaptic vesicles is regulated in vitro by phosphorylation by a calcium/calmodulin-dependent kinase. Here, we present the first evidence that, in the mouse retinal ganglion cells, synapsin I, moving along the axon with the slow component of axonal transport, is phosphorylated in vivo at both the head and tail regions. In addition, our data suggest that, after synapsin I has reached the nerve endings, the relative proportion of differently phosphorylated molecules of synapsin I changes, and that these changes lead to a decrease of the overall content of phosphorus. The more basic forms, here collectively referred to as beta-forms, become predominant at the terminals after 7 d postlabeling, when the bulk of transported synapsin I has entered the superior colliculus. Along the axon, phosphorylation could be functional in preventing synapsin I from forming, with actin, a dense meshwork that would restrict organelle movement. On the other hand, at the terminals, the dephosphorylation-phosphorylation of synapsin I may regulate the clustering of small synaptic vesicles and modulate neurotransmitter release by controlling the availability of small synaptic vesicles for exocytosis.     

Early differentiation of retinal ganglion cells: an axonal protein expressed by premigratory and migrating retinal ganglion cells

A monoclonal antibody, RA4, was developed that recognizes retinal ganglion cell axons in the mature retina. Between embryonic days 3 and 9, the RA4 antigen was associated with cell bodies in certain regions of the retina in addition to the ganglion cell axons. The RA4-positive cells were of 3 types: an apolar cell adjacent to the ventricular surface, a bipolar cell that spanned the thickness of the retina, and a monopolar cell in the ganglion cell layer. Evidence suggests that these cells are premigratory and migrating retinal ganglion cells. The expression of the RA4 antigen is the earliest indicator of ganglion cell differentiation yet reported. The existence of RA4-positive apolar cells along the outer surface of the retina suggests that the ganglion cell phenotype is expressed as soon as the cell becomes postmitotic. Approximately 20% of the migrating ganglion cells were in pairs. The paired cells most likely arose from the terminal division of a germinal cell. One possibility suggested by these data is that a ganglion cell-specific germinal cell arises from a pluripotent germinal cell. Immunoblots and other analyses revealed the RA4 antigen to be a 140 kDa cytoplasmic protein in the retina. RA4 also recognized many long tract axons in the brain. In the brain, the RA4 epitope was observed on proteins with at least 7 different molecular weights. Evidence suggests that different cell types may express the RA4 antigen with slightly different molecular weights.     

Protein synthesis and fast axonal transport in regenerating goldfish retinal ganglion cells

To characterize the fast component of axonal transport in regenerating goldfish optic axons, the incorporation of l-2,3-[³H]proline into newly-synthetized proteins in the cell bodies of the retinal ganglion cells and the amount of transported labeled protein were determined at 2–36 days after cutting the optic tract. Both the incorporation and the amount of transported protein had doubled by 10 days after the lesion and continued to increase to about 5 times normal at 15 days, a time when a large proportion of the regenerating axon population had reached the optic tectum. Near-normal levels were recovered by 36 days. In contralateral control neurons, the incorporation of l-2,3-[³H]proline was unchanged from normal throughout, whereas the amount of labeled transported protein entering control axons was decreased by 55% at 2 and 10 days after the testing lesion, returning to normal by 15 days. An increase in fast transport velocity was seen in the regenerating axons beginning at 10 days after the lesion. However, a similar velocity increase was also seen in the contralateral control axons and in undamaged axons following removal of the cerebral hemispheres. Therefore, the velocity increase was not a specific consequence of axotomy.     

Early maturation of GABAergic synapses in mouse retinal ganglion cells

This study was aimed to characterize the earliest phases of synapse development in mouse retinal ganglion cells (RGCs) by recording spontaneous postsynaptic currents (PSCs). First PSCs were detected at embryonic day 17 and completely suppressed by bicuculline, demonstrating their GABAergic nature. Starting from postnatal day 3 a small fraction of RGCs had rapidly decaying, most likely glutamatergic currents. The present results suggest that functional GABAergic synapses with RGCs appear before birth and that GABAergic synaptic transmission precedes that of glutamate in the retina. In this early period GABA acts in a depolarizing manner and takes over an excitatory function.     

Vinblastine-induced blockage of orthograde and retrograde axonal transport of protein in retinal ganglion cells

The right eyes of adult, albino rats were injected intravitreally with 0.1–30 μg of vinblastine sulfate, and the left eyes were similarly injected with saline. Twenty-four hours later, both eyes were injected with H³-proline and both superior colliculi with horseradish peroxidase. After 24 hr, the animals were killed and brains and retinae processed both for radioautography and the demonstration of peroxidase. Doses of 0.5 μg or more of vinblastine produced blockage of rapid orthograde transport of radioactively labeled protein within optic nerve axons without causing a significant decrease in protein synthesis in ganglion cells of the retina. A higher dose of vinblastine (10 μg or more) was required to block retrograde axonal transport of peroxidase from the superior colliculus to the ganglion cell somata. This suggests vinblastine-sensitive mechanisms for both orthograde and retrograde axonal transport of protein which may involve microtubules or free tubulin within the axoplasm, since vinblastine in low concentration binds specifically to tubulin.     

Protein synthesis and axonal transport in retinal ganglion cells of mice lacking visual receptors

The retinas of mice with hereditary degeneration of visual receptor cells were compared with those of a closely related strain of mice with normal retinas.

(1) The mutants had 20% fewer retinal ganglion cells and the remaining ganglion cells were reduced in size by 10–20%. (2) Tritiated amino acid incorporation into ganglion cell protein was about 35% less in the mutants than in the normals, but the labeled protein disappeared from the cells at the same rate as in the normals. (3) The rate of the fast component of axonal protein transport in the optic nerve was the same in both groups of animals, with a maximum of 190 mm/day. (4) The rate of slow transport, which was between about 1.5 and 3 mm/day in the normals, was reduced by one-third in the mutants. (5) Results obtained with proline and leucine were essentially the same, but indicated a longer lifetime for the proline-containing proteins in both the fast and slow components.     

Anterograde axonal transport of BDNF and NT-3 by retinal ganglion cells: roles of neurotrophin receptors

Retinal ganglion cells (RGCs) transport exogenous neurotrophins anterogradely to the midbrain tectum/superior colliculus with significant downstream effects. We determined contributions of neurotrophin receptors for anterograde transport of intraocularly injected radiolabeled neurotrophins. In adult rodents, anterograde transport of brain-derived neurotrophic factor (BDNF) was receptor-mediated, and transport of exogenous BDNF and neurotrophin-3 (NT-3) was more efficient, per RGC, in rodents than chicks. RT-PCR and Western blot analysis of purified murine RGCs showed that adult RGCs express the p75 receptor. Anterograde transport of BDNF or NT-3 was not diminished in p75 knock-out mice (with unaltered final numbers of RGCs), but BDNF transport was substantially reduced by co-injected trkB antibodies. In chick embryos, however, p75 antisense or co-injected p75 antibodies significantly attenuated anterograde transport of NT-3 by RGCs. Thus, neither BDNF nor NT-3 utilizes p75 for anterograde transport in adult rodent RGCs, while anterograde NT-3 transport requires the p75 receptor in embryonic chicken RGCs.     

Stereotyped axonal arbors of retinal ganglion cell subsets in the mouse superior colliculus

Mouse retinal ganglion cells (RGCs) have been classified into around 20 subtypes based on the shape, size, and laminar position of their dendritic arbors. In most cases tested, RGC subtypes classified in this manner also have distinct functional signatures. Here we asked whether RGC subtypes defined by dendritic morphology have stereotyped axonal arbors in their main central target, the superior colliculus (SC). We used transgenic and viral methods to sparsely label RGCs and characterized both dendritic and axonal arbors of individual RGCs. Axon arbors varied in size, shape, and laminar position. For each of 12 subtypes defined dendritically, however, axonal arbors in the contralateral SC showed considerable stereotypy. We found no systematic relationship between the laminar position of an RGC's dendrites within the inner plexiform layer and that of its axon within the retinorecipient zone of the SC, suggesting that distinct developmental mechanisms specify dendritic and axonal laminar positions. We did, however, note a significant correlation between the dendritic field sizes of RGCs and the laminar position of their axon arbors: RGCs with larger dendritic areas, and hence larger receptive fields, projected to deeper strata within the SC. Finally, combining these new results with previous physiological analyses, we find that RGC subtypes that share similar functional properties, such as directional selectivity, project to similar depths within the SC.     

Basic fibroblast growth factor: receptor-mediated internalization, metabolism, and anterograde axonal transport in retinal ganglion cells.

Basic fibroblast growth factor (bFGF) was radiolabeled and used in axonal transport studies to determine whether certain neuronal populations express functional receptors for bFGF. Unlike 125I-NGF, 125I-bFGF was not retrogradely transported in the adult rat sciatic nerve or from iris to trigeminal ganglion or superior cervical ganglion. However, after intraocular injection of 125I-bFGF into the posterior chamber of the eye of adult rats, radioactivity was detected within the retinal ganglion cell projections. This radioactivity was localized to the ipsilateral optic nerve and in the contralateral lateral geniculate body and the contralateral superior colliculus by using autoradiographic techniques. Direct measurement of the radioactivity in dissected brain regions was used to study the process of 125I-bFGF uptake and transport by retinal ganglion cells. The uptake and transport were specific for biologically active bFGF since neither denatured, biologically inactive 125I-bFGF nor 125I-NGF was taken up and transported. The uptake and transport of 125I-bFGF were saturable phenomena since they were blocked in the presence of excess, unlabeled bFGF. Wheat germ agglutinin, but not heparinase, blocked uptake and transport of 125I-bFGF, a finding that is consistent with the uptake being mediated by high-affinity bFGF receptors. Radioactivity from 125I-bFGF was transported in retinal ganglion cell axons in an anterograde direction at a maximum rate in excess of 1.7 mm/hr. No specific retrograde transport of bFGF to the retina was detected after 125I-bFGF was injected into the superior colliculus. The radioactivity from 125I-bFGF that accumulated in the superior colliculus was lost from this tissue with a half-life of about 22 hr. Autoradiography of proteins separated by SDS-PAGE demonstrated that 125I-bFGF was not substantially degraded in the retina after internalization within retinal ganglion cells. During anterograde transport, however, 125I-bFGF underwent limited proteolytic cleavage resulting in 3 prominent 125I-bFGF derivatives of molecular weights greater than 7000 Da. Although these were the major radioactive species recovered from the superior colliculus after intraocular injection, some intact 125I-bFGF was also detected within the innervated target. These results indicate that retinal ganglion cells express high-affinity receptors for bFGF, that these receptors mediate the internalization of bFGF, that internalized bFGF undergoes limited proteolytic cleavage, and that bFGF and its derivatives are anterogradely transported to the lateral geniculate body and the superior colliculus. These data raise the possibility that bFGF or its derivatives may act as an anterograde trophic factor in the visual system, a system that is known to undergo anterograde transneuronal cell death.     

Protein synthesis and axonal transport in goldfish retinal ganglion cells during regeneration accelerated by a conditioning lesion

Axonal outgrowth in goldfish retinal ganglion cells following a testing lesion of the optic axons is accelerated by a prior conditioning lesion. Changes in protein synthesis and axonal transport were examined during the accelerated regeneration. The conditioning lesion was an optic tract cut made 2 weeks prior to the testing lesion, which consisted of a tract cut at the chiasma, so that nerves subjected to either a conditioning lesion (‘conditioned nerves’) or a sham operation (‘sham-conditioned nerves’) could be examined in the same animal. In the retinal ganglion cells of conditioned nerves, the incorporation of [³H]proline into protein began to increase between 1 and 8 days after the testing lesion. The amount of fast-transported labeled protein was elevated to about 8 × normal by 1 day after the testing lesion but had decreased to about 3–5 × normal at 8 and 22 days. The 8 and 22 day values were not significantly different from those in sham-conditioned nerves or nerves that had received a testing lesion alone. For slow protein transport, the instantaneous amount transported was 15–16 × normal in the conditioned nerves at 1 and 8 days after the testing lesion, and the velocity of slow transport, which was already elevated above normal by 1 day after the testing lesion, was elevated still further by 8 days — to a value in excess of 1.5 mm/day (compared to 0.2–0.4 mm/day in normal animals). We believe that the enhanced outgrowth resulting from the conditioning lesion is due to a transient increase in the amount of fast transport (possibly responsible for a decreased delay in the initiation of sprouting), and a sustained increase in the amount and velocity of slow transport (which may account for an increased rate of elongation).     

Alteration of the neurofilament sidearm and its relation to neurofilament compaction occurring with traumatic axonal injury

Traumatic injury evokes two characteristic forms of focal axonal injury, one of which involves focal perturbation of axolemmal permeability associated with rapid compaction of the underlying axonal neurofilament lattice and microtubular loss. In this process, the neurofilament sidearms have been the subject of intense scrutiny in relation to their role in this NF compaction, with the suggestion that the sidearms, thought to maintain interfilament distance, are proteolytically cleaved and degraded at the time of injury. The current communication addresses the fate of the NF sidearms in such injured axons. Adult cats were subjected to moderate/severe fluid percussion brain injury after intrathecal administration of horseradish peroxidase (HRP). This tracer, excluded by the intact axolemma of uninjured axons, was used to recognize injured axons via HRP intra-axonal uptake/flooding with HRP. Animals were perfused and processed for light microscopic and electron microscopic study of both HRP-containing and non-HRP-containing axons from the same field. HRP-containing axons consistently displayed evidence of traumatically-induced (NF) cytoskeletal collapse. Electron micrographs of HRP-containing axons as well as uninjured, non-HRP-containing axons from the same fields were videographically captured, digitized, enlarged and analysed for NF sidearm length and NF density. HRP-containing axons were found to have increased NF density. Surprisingly, this increased NF density occurred despite the retention of the NF sidearms, which now, however, were reduced in height in comparison to the non-HRP-containing uninjured axons. These observations are not consistent with previously published reports suggesting that overt proteolytic degradation of sidearms was responsible for NF compaction. Based on our findings, we suggest that the NF compaction associated with traumatically-induced axolemmal permeability changes may have its genesis in more subtle sidearm modification, perhaps involving a change in phosphorylation state.     

Persistence of intact retinal ganglion cell terminals after axonal transport loss in the DBA/2J mouse model of glaucoma

Axonal transport defects are an early pathology occurring within the retinofugal projection of the DBA/2J mouse model of glaucoma. Retinal ganglion cell (RGC) axons and terminals are detectable after transport is affected, yet little is known about the condition of these structures. We examined the ultrastructure of the glaucomatous superior colliculus (SC) with three‐dimensional serial block‐face scanning electron microscopy to determine the distribution and morphology of retinal terminals in aged mice exhibiting varying levels of axonal transport integrity. After initial axonal transport failure, retinal terminal densities did not vary compared with either transport‐intact or control tissue. Although retinal terminals lacked overt signs of neurodegeneration, transport‐intact areas of glaucomatous SC exhibited larger retinal terminals and associated mitochondria. This likely indicates increased oxidative capacity and may be a compensatory response to the stressors that this projection is experiencing. Areas devoid of transported tracer label showed reduced mitochondrial volumes as well as decreased active zone number and surface area, suggesting that oxidative capacity and synapse strength are reduced as disease progresses but before degeneration of the synapse. Mitochondrial volume was a strong predictor of bouton size independent of pathology. These findings indicate that RGC axons retain connectivity after losing function early in the disease process, creating an important therapeutic opportunity for protection or restoration of vision in glaucoma. J. Comp. Neurol. 524:3503–3517, 2016. © 2016 Wiley Periodicals, Inc.     

Central projections of melanopsin-expressing retinal ganglion cells in the mouse

A rare type of ganglion cell in mammalian retina is directly photosensitive. These novel retinal photoreceptors express the photopigment melanopsin. They send axons directly to the suprachiasmatic nucleus (SCN), intergeniculate leaflet (IGL), and olivary pretectal nucleus (OPN), thereby contributing to photic synchronization of circadian rhythms and the pupillary light reflex. Here, we sought to characterize more fully the projections of these cells to the brain. By targeting tau-lacZ to the melanopsin gene locus in mice, ganglion cells that would normally express melanopsin were induced to express, instead, the marker enzyme beta-galactosidase. Their axons were visualized by X-gal histochemistry or anti-beta-galactosidase immunofluorescence. Established targets were confirmed, including the SCN, IGL, OPN, ventral division of the lateral geniculate nucleus (LGv), and preoptic area, but the overall projections were more widespread than previously recognized. Targets included the lateral nucleus, peri-supraoptic nucleus, and subparaventricular zone of the hypothalamus, medial amygdala, margin of the lateral habenula, posterior limitans nucleus, superior colliculus, and periaqueductal gray. There were also weak projections to the margins of the dorsal lateral geniculate nucleus. Co-staining with the cholera toxin B subunit to label all retinal afferents showed that melanopsin ganglion cells provide most of the retinal input to the SCN, IGL, and lateral habenula and much of that to the OPN, but that other ganglion cells do contribute at least some retinal input to these targets. Staining patterns after monocular enucleation revealed that the projections of these cells are overwhelmingly crossed except for the projection to the SCN, which is bilaterally symmetrical.     

Axonal transport of proteins in retinal ganglion cells. Characterization of the transport to the superior colliculus

The axonal transport of protein in the retinal ganglion cells of the adult rabbit was followed after intra-ocular injections of [³H]leucine. The labelled protein reached the nerve terminals in the superior colliculus in at least 4 phases. Compared with the lateral geniculate body, the superior colliculus received a considerable portion of the transported protein, and relatively more of this protein was carried to the superior colliculus with rapid phases of axonal transport. Radioautography showed that the label was localized to the 3 most dorsal layers of the superior colliculus. Cell fractionation of the superior colliculus indicated that the axonal terminals of optic origin did not easily form stable synaptosomes during a conventional homogenization and centrigation procedure.     

Tracer coupling of intrinsically photosensitive retinal ganglion cells to amacrine cells in the mouse retina

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are a subtype of ganglion cell in the mammalian retina that expresses the photopigment melanopsin and drives non-image-forming visual functions. Three morphological subtypes of ipRGCs (M1, M2, and M3) have been described based on their dendritic stratifications in the inner plexiform layer (IPL), but the question of their potential interactions via electrical coupling remains unsettled. In this study, we have addressed this question in the mouse retina by, injecting the tracer Neurobiotin into ipRGCs that had been genetically labelled with the fluorescent protein, tdTomato. We confirmed the presence of the M1-M3 subtypes of ipRGCs based on their distinct dendritic stratifications. All three subtypes were tracer coupled to putative amacrine cells situated within the ganglion cell layer (GCL) but not the inner nuclear layer (INL). The cells tracer coupled to the M1 and M2 cells were shown to be widefield GABA-immunoreactive amacrine cells. We found no evidence of homologous tracer coupling of ipRGCs or heterologous coupling to other types of ganglion cells.     

Morphological properties of chick retinal ganglion cells in relation to their central projections

Morphological properties of chick retinal ganglion cells (RGCs) were studied in relation to their central projections in 23 chicks. A total of 217 RGCs were retrogradely labeled by applying a carbocyanine dye (DiI) to the thalamus and optic tectum. The labeled RGCs were classified into six groups on the basis of their somal areas, dendritic fields, and branching patterns. The dendrites of these RGCs extended horizontally in the inner plexiform layer (IPL) forming eight dendritic strata. The RGCs in each group showed certain specificities in their central projections. Group Ic predominantly projected to the tectum. Groups IIs and IIIs showed a high thalamic dominance. Groups Is and IIc were nonspecific with regard to their tectal and thalamic projections. Group IVc showed tectal‐specific projections. Occurrence rates of the dendritic strata increased progressively toward the inner part of the IPL, i.e., DSs (dendritic strata) 1–4 were scantily distributed, DSs 5 and 6 were moderately distributed, and DSs 7 and 8 were the most frequently distributed. A total of 42 dendritic stratification patterns were identified, and of these, 18 patterns were common to the tectal RGCs (tec‐RGCs) and thalamic RGCs (tha‐RGCs). The common patterns were detected very frequently in the tec‐ and tha‐RGCs (≈85%), and the dendritic strata were largely distributed in the inner part of the IPL (DSs 5–8). In contrast, the remaining 24 noncommon stratification patterns showed low occurrence rates (≈15%); however, these dendritic strata were widely distributed in both the outer (DSs 1–4) and inner (DSs 5–8) IPL. J. Comp. Neurol. 514:117–130, 2009. © 2009 Wiley‐Liss, Inc.     

Assessment of possible transneuronal changes in the retina of rats with inherited retinal dystrophy: cell size, number, synapses, and axonal transport by retinal ganglion cells

In pigmented RCS rats with inherited retinal dystrophy, most photoreceptor cells disappear between postnatal days 20 and 100. We have examined the time course of the degeneration of photoreceptor nuclei and synapses and determined whether transneuronal changes occur in the inner nuclear layer (INL), inner plexiform layer (IPL), and retinal ganglion cells following loss of photoreceptor cells in these animals. Electron microscopic photomontages of the entire thickness of the IPL of dystrophic (RCS-p+) and control (RCS-rdy+ p+) rats 334 to 515 days old were prepared, and synapses were counted and identified as either conventional (amacrine) or ribbon (bipolar) types. Neither the incidence of synapses in the IPL nor the ratio of conventional to ribbon synapses differed in the dystrophic and control retinas. Ganglion cell diameter, perimeter, area, and density were measured from drawings of wholemount preparations of dystrophic and control rats 105 days and older. Diameter, perimeter, area and number of ganglion cells were not significantly different in the two genotypes. Anterograde axonal transport was measured by studying the displacement of labeled material as it traveled along ganglion cell axons and accumulated in the superior colliculus. The normal and dystrophic rats showed no significant difference in (1) the rates of rapidly moving components (approximately 110-180 mm/day) and slowly moving components (1.7-2.5 mm/day) or (2) the amount of radioactive material transported to the superior colliculus. The absence of transneuronal changes in retinal ganglion cells of RCS rats contrasts with results obtained earlier in rd mice (Graftstein et al., '72). Unlike the RCS rat, retinal degeneration in rd mice occurs before the maturation of the retina. We hypothesize that the ganglion cells may be more affected by loss of input early in development, and, therefore, ganglion cells of retinal dystrophic rats are less affected despite little or no synaptic input for several months. Furthermore, any reduction in the electrical activity of retinal ganglion cells that might follow loss of photoreceptor cells does not result in a significantly decreased rate of axonal transport.     

Environmental light enhances survival and axonal regeneration of axotomized retinal ganglion cells in adult cats

A segment of peripheral nerve was transplanted to the cut stump of the optic nerve to facilitate axonal regeneration of retinal ganglion cells (RGCs) in adult cats. The cats were reared under different light environment: 12 h light-12 h darkness, additional flash light under conventional light cycle, or 24 h darkness. After 60 days, the density and morphology of RGCs with regenerated axons were examined with retrograde labeling by fluoro-ruby and intracellular injections of Lucifer Yellow. In the retina of cats reared in darkness, densities of RGCs with regenerated axons were 11-42% of those in the retina of cats reared under conventional light and dark cycle. More than half of the labeled RGCs were degenerative in the retina of cats reared in darkness, while most RGCs were normal under conventional environment or flash light. We conclude that environmental light is essential for the survival and axonal regeneration of axotomized RGCs.