Retinal Axon Terminals Research Articles

EphA4 mediates ephrinB1-dependent adhesion in retinal ganglion cells

Eph/ephrin signaling is crucial for organizing retinotopic maps in vertebrates. Unlike other EphAs, which are expressed in the embryonic ventral retina, EphA4 is found in the retinal ganglion cell (RGC) layer at perinatal stages, and its role in mammalian visual system development remains unclear. Using classicin vitrostripe assays, we demonstrate that, while RGC axons are repelled by ephrinB2, they grow on ephrinB1 stripes through EphA4-mediated adhesion. In vivo, retinal axons from EphA4-deficient mice from either sex show impaired arborization in the medial, but not lateral, regions of the superior colliculus that express ephrinB1. Gain-of-function experiments further reveal that ephrinB1-mediated adhesion depends on EphA4 tyrosine kinase activity but it is independent of its Sterile Alpha Motif. Together, our findings suggest that EphA4/ephrinB1 forward signaling likely facilitates adhesion between retinal axon terminals and cells in the medial colliculus, contributing to the establishment of proper connectivity within the visual system.Significance statementThe significance of our findings goes beyond unveiling the mechanisms underlying topographic visual map formation. By discovering a dual role for EphA4 in mediating both repulsion and adhesion, which challenges the conventional understanding of EphA4/ephrin interactions, this work opens up new avenues for exploring EphA4 broader implications in other cellular contexts, including cell differentiation, migration and synaptic plasticity.

Extracellular Engrailed Participates in the Topographic Guidance of Retinal Axons In Vivo

Engrailed transcription factors regulate the expression of guidance cues that pattern retinal axon terminals in the dorsal midbrain. They also act directly to guide axon growth in vitro. We show here that an extracellular En gradient exists in the tectum along the anterior-posterior axis. Neutralizing extracellular Engrailed in vivo with antibodies expressed in the tectum causes temporal axons to map aberrantly to the posterior tectum in chick and Xenopus. Furthermore, posterior membranes from wild-type tecta incubated with anti-Engrailed antibodies or posterior membranes from Engrailed-1 knockout mice exhibit diminished repulsive activity for temporal axons. Since EphrinAs play a major role in anterior-posterior mapping, we tested whether Engrailed cooperates with EphrinA5 in vitro. We find that Engrailed restores full repulsion to axons given subthreshold doses of EphrinA5. Collectively, our results indicate that extracellular Engrailed contributes to retinotectal mapping in vivo by modulating the sensitivity of growth cones to EphrinA.

Observations on the synaptic ribbon morphology in retinas of two human subjects at autopsy

Retinal photoreceptor and bipolar cell axon terminals possess synaptic ribbons (SR) that aid in the trafficking of synaptic vesicles at active zones. In rodents, besides SR, a number of other synaptic elements [e.g., synaptic spheres (SS)] are known to appear when exposed to altered ambient illumination. Here, we report changes of ribbon shape in photoreceptor and bipolar cell axon terminals in retinas of two persons at autopsy who suffered from brain hemorrhage. In both subjects, retinal hemorrhage was present in the outer and inner nuclear layers. SR were bent or swollen and transformed into SS. A count revealed that about 54-60% of the photoreceptor axon terminals over the nasal to temporal retina possessed SS. They were associated with synaptic triads or remained floating in cytoplasm. The bipolar cell axon terminals possessed either SR or sphere-like bodies. As these features were not seen in control retinas of donors who died of other causes, we assume that in hemorrhagic subjects, SR underwent transformation into SS, in which perhaps ischemia (caused due to vascular obstructions by hemorrhage) played a leading role.

Analysis of quantal size of voltage responses to retinal stimulation in the accessory optic system

In the intact vertebrate central nervous system, the quantal nature of synaptic transmission is difficult to measure because the postsynaptic sites may be distributed along a tortuous dendritic tree that cannot be readily clamped spatially to a uniform potential. Titrating the intact brain's extracellular concentration of calcium ions is also challenging because of its strong buffering mechanisms. In this study, using a whole brain with eye attached preparation, quantal neurotransmission was examined in the turtle brainstem in vitro, by recording from accessory optic system neurons that receive direct input from visually responsive retinal ganglion cells. Unitary EPSPs, evoked by microstimulation of a single ganglion cell, were measured during whole cell current-clamp recordings. In this preparation, the neurons exhibit direction-selectivity, despite the hypoxic conditions. Bath application of cadmium to reduce calcium influx also reduced evoked EPSP amplitudes to that of the spontaneous synaptic events. Statistical analyses indicated that these evoked response amplitudes could be well fitted to a Poisson distribution for most brainstem neurons. Therefore, the spontaneous miniature excitatory synaptic events of approximately 1 mV, as also observed during spike blockade of the retina [Kogo, N., Ariel, M., 1997. Membrane properties and monosynaptic retinal excitation of neurons in the turtle accessory optic system. Journal of Neurophysiology 78, 614–627], are likely responses to the neurotransmitter of single vesicles release by retinal axon terminals.

Role of the calcium modulated cyclases in the development of the retinal projections

Transmembrane isoforms of adenylate cyclases (AC) integrate a wide variety of extracellular signals from neurotransmitters to morphogens and can also regulate cAMP production in response to calcium entry. Based on observations in the barrelless mouse strain, the Adcy1 gene (AC1) was involved in the segregation of binocular retinal inputs. To determine the potential role of other AC isoforms we localized the Adcy genes in the visual centres during development, using in situ hybridization. Six different AC subtypes were found in the developing retinal ganglion cell layer (RGC; AC1, AC2, AC3, AC5, AC8, and AC9), and three AC subtypes were expressed in the central brain targets, the dorsal lateral geniculate nucleus (AC1 and AC8), the ventral lateral geniculate nucleus (AC2 and AC8) and the superior colliculus (AC1, AC2, AC8). Using a genetic approach we tested the role of the calcium modulated cyclases AC1, AC5 and AC8 for the segregation retinal fibres. Ipsilateral retinal axons remained exuberant in the AC1(-/-) mice, with overlapping retinal projections from both eyes in the superior colliculus and the visual thalamus. These abnormalities were similar to those of barrelless mouse mutants. No abnormalities were detectable in the AC5(-/-) or the AC8(-/-) mice. Similar abnormalities were noted in the single AC1(-/-) and the AC1/AC8 double-knockout mice (DKO). Thus, only AC1 is required for the maturation of the retinal axon terminals whereas AC5 and AC8 are not needed. The specificity of AC1's action is linked to its cellular localization in the RGCs and to its distinctive functional profile, compared with the other cyclases expressed in the same cells.

Changes in glutamate receptor function in synaptic input to the superficial superior colliculus (SSC) with aging and in retinal degeneration in the Royal College of Surgeons (RCS) rat

Ionotropic and metabotropic glutamate receptors mediate and modulate retinocollicular transmission. The Royal College of Surgeons (RCS) dystrophic strain of rats suffers from a progressive retinal degeneration with age and hence loss of visual function. We investigated whether this loss of function is accompanied by functional changes in a central target of retinal axons, the superficial superior colliculus (SSC). Field potential recordings were made in SSC slices from RCS rats aged either 4–7 weeks or 33–52 weeks. Blockade of GABAergic transmission revealed a field EPSP in response to optic tract stimulation which was sensitive to the NMDA antagonist AP5. In normal non-dystrophic rats the contribution of NMDA receptors to the fEPSP declined with age, whereas in dystrophic animals no such decline was seen. As mGluR8 may be located on terminals of retinal axons, we also assessed the function of this receptor. The mGluR8 agonist DCPG reduced fEPSPs in normal and dystrophic rats in both age groups to a similar extent, although the effect of DCPG declined with age. These findings indicate that the contribution of NMDA receptors to retinocollicular transmission declines with age in normal rats, but that such a decline is not seen in dystrophic rats which have severely reduced visual function. As NMDA receptors are associated with neural plasticity, it may be that this finding represents an increased residual potential for plasticity in dystrophic rats which may be functionally important.

Competitive interactions between retinal ganglion axons for tectal targets explain plasticity of retinotectal projection in the servomechanism model of retinotectal mapping

The mechanism of topographic mapping of retinal ganglion cells to the midbrain was previously elucidated by the servomechanism model, which is based on the fact that cells expressing Eph-receptors respond specifically to surface expressing membrane-bound ephrin-ligands at a critical level. The retina has increased nasal-to-temporal gradient of Eph receptor-density, and the optic tectum/superior colliculus has increased rostral-to-caudal gradient of membrane-bound ephrin-ligand. An axon from the retina has an identification tag of a certain level of Eph-receptor density depending on its retinal position, and adheres to the site on the tectum/superior colliculus expressing ephrin-ligands at a critical ligand-density level. The servomechanism model rigidly defines positions of axon terminals on the midbrain. However, optic nerve regeneration experiments combined with halved retina or tectum show a plastic or flexible mapping (expansion, compression and transposition of tectal projections). To reconcile the discrepancy between the rigid model and the plastic behavior, competition between retinal axon terminals for a target site was introduced to the servomechanism. The servomechanism/competition model succeeded in computer simulations of the plastic mapping of retinal axons on the tectum. Recent experiments of upregulated ligand-density on the tectum during nerve regeneration and the role of axonal competition are discussed.

Competition between Retinal Ganglion Axons for Targets under the Servomechanism Model Explains Abnormal Retinocollicular Projection of Eph Receptor-Overexpressing or Ephrin-Lacking Mice

Topographic mapping of retinal ganglion axons to the midbrain is computed by the servomechanism model, which is based on the experimental result of cell attachment. Cells expressing a certain level of Eph proteins (receptors for ephrin ligands) optimally attach to a surface that expresses a specific level of ephrin ligand density. The retina has an increasing nasal-to-temporal gradient of Eph receptor density, and the optic tectum/superior colliculus has an increasing rostral-to-caudal gradient of membrane-bound ephrin ligand. An axon from the retina has an identification tag of a certain level of Eph receptor density depending on its retinal position and adheres to the site on the tectum/superior colliculus expressing ephrin ligands at a critical ligand density level. Quantitatively, a retinal axon has a receptor density (R) that is determined by its retinal position, and the axon terminal is induced to adhere to the tectal site of ligand density (L = S/R), where S is a constant. Consequently, the servomechanism model defines positions of axon terminals on the midbrain. Abnormal topographic maps are reported in a knock-in experiment with elevated density of Eph receptors and a knock-out experiment lacking ephrin ligands using gene-targeting technology. By adding competition between axon terminals for target sites to the servomechanism model, the abnormal maps became easy to understand. Furthermore, the servomechanism-competition model allowed conjecture of the gradient shapes of receptor and ligand densities and estimation of the capacity of the midbrain surface to accept retinal axon terminals.

Presynaptic Modulation of the Retinogeniculate Synapse

Modulatory projections from brainstem nuclei and intrinsic thalamic interneurons play a significant role in modifying sensory information as it is relayed from the thalamus to the cortex. In the lateral geniculate nucleus (LGN), neurotransmitters released from these modulatory inputs can affect the intrinsic conductances of thalamocortical relay neurons, thus altering their firing properties. Here, we show that in addition to postsynaptic effects, neuromodulators such as serotonin (5-HT) and GABA can act presynaptically to regulate neurotransmitter release at the synapse between retinal ganglion cells (RGCs) and relay neurons in the LGN, the retinogeniculate synapse. Activation of 5HT1 and GABA(B) receptors significantly decreased EPSC amplitude. This inhibition was accompanied by a decrease in the extent of paired-pulse depression, suggesting that it is presynaptic in origin. In addition, fluorometric calcium measurements from retinal axon terminals labeled with Calcium Green-1 dextran revealed that 5HT1 and GABA(B) receptor agonists decreased presynaptic calcium influx. Taken together, our data indicate that serotonin and GABA can act presynaptically to decrease calcium influx at the retinogeniculate synapse and modify transmission of visual information in the LGN.

Disruption of Retinogeniculate Pattern Formation by Inhibition of Soluble Guanylyl Cyclase

During development of the visual system of the ferret, the terminals of retinal ganglion cell axons first segregate to form eye-specific layers and subsequently On-center and Off-center sublayers within the dorsal lateral geniculate nucleus (dLGN). Sublamination requires the activity of the afferent fibers, NMDA receptors, and nitric oxide synthase (NOS). We here report that soluble guanylyl cyclase (sGC), which in turn produces cGMP, is critically involved in the process of sublamination. cGMP expression is upregulated in both retinal terminals and postsynaptic dLGN cells during sublamination, and this expression is controlled by the activity of both NMDA receptors and NOS. Furthermore, the infusion of specific inhibitors of sGC or protein kinase G (PKG), a target of cGMP, prevents sublamination in vivo. We conclude that the sGC-cGMP-PKG pathway acts downstream of NMDA receptors and nitric oxide as an effector of the activity-dependent refinement of connections at this level of the mammalian visual system.

Indirect evidence for an association of 5‐HT1B binding sites with retinal and geniculate axon terminals in the rat suprachiasmatic nucleus

Indirect evidence for an association of 5‐HT1B binding sites with retinal and geniculate axon terminals in the rat suprachiasmatic nucleus

Indirect evidence for an association of 5-HT(1B) binding sites with retinal and geniculate axon terminals in the rat suprachiasmatic nucleus.

The purpose of the present study was to investigate the possible cellular location of 5-HT(1B) receptors on retinal and geniculate afferents in the rat suprachiasmatic nucleus (SCN). Biocular enucleation significantly decreased 5-HT(1B) binding site labeling (35%), specifically in the ventral part of the SCN, while monocular enucleation produced a decrease of smaller magnitude (12%), limited to the ventral part of the contralateral SCN, these results being consistent with the known distribution of retinal afferents in the nucleus. By contrast, bilateral geniculate lesion did not induce any significant variation of 5-HT(1B) binding site labeling in the SCN. Previously, we reported that serotonin (5-HT) synthesis inhibition by parachlorophenylalanine increases 5-HT(1B) binding site labeling in the SCN. Using saturation studies, we have now demonstrated that this upregulation reflected an increase in the total number of 5-HT(1B) binding sites (+41% in the dorsal and +67% in the ventral part of the SCN). Furthermore, we evaluated the effects of bilateral geniculate lesion after 5-HT stores depletion in order to overcome problems of technical resolution limits. The magnitude of upregulation was significantly decreased (27%) after bilateral geniculate lesion, suggesting that part of the 5-HT(1B) receptor population was located on geniculate axon terminals within the SCN. The possible involvement of 5-HT(1B) receptors, according to their cellular locations evidenced in the present study, in photic and nonphotic entrainment of the circadian clock is discussed.

TFMPP, a 5HT 1B receptor agonist, inhibits light-induced phase shifts of the circadian activity rhythm and c-Fos expression in the mouse suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN) receives afferents from the retina and the midbrain raphe. The retinal innervation mediates photic entrainment of the SCN circadian oscillator whereas the serotonergic input arising from the midbrain raphe nuclei appears to modulate retinohypothalamic neurotransmission. We hypothesized that serotonergic innervation of the SCN may modulate retinal input by activation of 5HT 1B presynaptic receptors on retinal axon terminals in the SCN. We tested this hypothesis using the 5HT 1B receptor agonist, 1-[3-(trifluoromethyl)phenyl]-piperazine (TFMPP). Systemic administration of TFMPP prior to light stimulation significantly attenuated light-induced phase shifts of the circadian activity rhythm and Fos expression in the SCN. These results in the mouse support our earlier findings in the hamster [Pickard, G.E., Weber, E.T., Scott, P.A., Riberdy, A.F. and Rea, M.A.,. J. Neurosci., 16 (1996) 8208–8220] and are consistent with the interpretation that 5HT 1B presynaptic receptors participate in the regulation of photic input to the SCN.

Differential reinnervation of retinal bipolar cell dendrites and axon terminals by dopamine interplexiform cells following dopamine depletion with 6-OHDA

Depletion of retinal dopamine in goldfish increases light sensitivity at photopic backgrounds. As horizontal cells appear not to be involved with this effect (Yazulla and Studholme [1995] Vis. Neurosci. 12:827-837), we investigated the innervation patterns of the ON rod/cone bipolar cells (ON-BC) by dopaminergic interplexiform cells (DA-IPCs) normally and during the period of neogeneration of new DA-IPCs at the marginal zone following DA-IPC destruction. DA-IPCs were destroyed via intraocular injection of 6-hydroxydopamine over 2 successive days. Controls and 1 year post-injection retinas were double labeled for protein kinase C and tyrosine hydroxylase (TH) immunocytochemistry to identify the ON-BCs and the DA-IPCs, respectively. Double-labeled 25 microns tissue sections were examined on a confocal laser scanning microscope by using dual channel immunofluorescence acquisition. Image stacks were analyzed for DA-IPC/ON-BC contacts in the distal inner nuclear layer (INL) and inner plexiform layer (IPL). Image stacks were rotated 180 degrees with respect to each other and reanalyzed to determine potential randomness of the contacts. For control retinas there were 1.8 contacts/axon terminal in the IPL (n = 165) and 9.4 contacts/ON-BC in the distal INL (n = 28). At 1 year after injection, reinnervation of TH-immunoreactive boutons in the retina recovered to 16% of control in the IPL but only 10% in the distal INL. Establishment of DA-IPC/ON-BC contacts recovered to 36% of control for ON-BC axon terminals (n = 103), whereas there was no recovery of contacts in the distal INL (n = 30). Reinnervation of ON-BC by DA-IPCs preferentially targets the axon terminals. The absence of reinnervation of bipolar cell dendrites by DA-IPCs may account for the persistence of the increased light sensitivity following retinal dopamine depletion. Thus, dopamine input to ON-BCs in the outer retina maybe involved in setting background sensitivity under photopic conditions.

Differential reinnervation of retinal bipolar cell dendrites and axon terminals by dopamine interplexiform cells following dopamine depletion with 6‐OHDA

Differential reinnervation of retinal bipolar cell dendrites and axon terminals by dopamine interplexiform cells following dopamine depletion with 6‐OHDA

5HT1BReceptor Agonists Inhibit Light-Induced Phase Shifts of Behavioral Circadian Rhythms and Expression of the Immediate–Early Genec-fosin the Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) is a circadian oscillator and a critical component of the mammalian circadian system. It receives afferents from the retina and the mesencephalic raphe. Retinal afferents mediate photic entrainment of the SCN, whereas the serotonergic afferents originating from the midbrain modulate photic responses in the SCN; however, the serotonin (5HT) receptor subtypes in the SCN responsible for these modulatory effects are not well characterized. In this study, we tested the hypothesis that 5HT1B receptors are located presynaptically on retinal axon terminals in the SCN and that activation of these receptors inhibits retinal input. The 5HT1B receptor agonists TFMPP and CGS 12066A, administered systemically, inhibited light-induced phase shifts of the circadian activity rhythm in a dose-dependent manner at phase delay and phase advance time points. This inhibition was not affected by previous systemic application of either the selective 5HT1A receptor antagonist (+)WAY 100135 or by the 5HT2 receptor antagonist mesulergine, whereas pretreatment with the nonselective 5HT1 antagonist methiothepin significantly attenuated the effect of TFMPP. TFMPP also produced a dose-dependent reduction in light-stimulated Fos expression in the SCN, although a small subset of cells in the dorsolateral aspect of the caudal SCN were TFMPP-insensitive. TFMPP (1 mM) infused into the SCN produced complete inhibition of light-induced phase advances. Finally, bilateral orbital enucleation reduced the density of SCN 5HT1B receptors as determined using [125I]-iodocyanopindolol to define 5HT1B binding sites. These results are consistent with the interpretation that 5HT1B receptors are localized presynaptically on retinal terminals in the SCN and that activation of these receptors by 5HT1B agonists inhibits retinohypothalamic input.

Presynaptic localization of μ-opioid receptor-like immunoreactivity in retinal axon terminals within the terminal nuclei of the accessory optic tract: a light and electron microscope study in the rat

Neurophil within the terminal nuclei of the accessory optic tract of the rat showed intense to moderate μ-opioid receptor-like immunoreactivity (MOR-LI). After unilateral enucleation, MOR-LI within the terminal nuclei almost disappeared or was markedly reduced on the side contralateral to the operation. Electron microscopy revealed that MOR-LI axon terminals within the terminal nuclei were filled with round synaptic vesicles and in asymmetric synaptic contact mainly with dendritic profiles, and occassionally with somatic profiles.

Establishing a functional brain circuitry: the development of the retino-tectal projection

Topographic matching between pre- and postsynaptic ganglion cell sheets appears to be a typical way in which different regions of the vertebrate brain are interconnected. The visual system provides a well-suited model to study the processes underlying the generation of 'topographic projections' during development. In this review, we will discuss several mechanisms which are possibly involved in the sorting of retinal axon terminals within their target field, the optic tectum. These mechanisms include target recognition by 'cytochemical matching' between axons and target cells, fiber-fiber interactions, and synapse stabilization and elimination based on impulse activity.

Quantitative immunogold evidence for enrichment of glutamate but not aspartate in synaptic terminals of retino-geniculate, geniculo-cortical, and cortico-geniculate axons in the cat.

A postembedding immunogold procedure was used on thin sections of the dorsal lateral geniculate nucleus (LGN) and perigeniculate nucleus (PGN) of the cat to estimate qualitatively and quantitatively, at the electron-microscopic (EM) level, the intensity of glutamate or aspartate immunoreactivities on identifiable synaptic terminals and other profiles of the neuropil. On sections incubated with a glutamate antibody, terminals of retinal and cortical axons in the LGN, and of collaterals of geniculo-cortical axons in the PGN, contain significantly higher density of immunogold particles than GABAergic terminals, glial cells, dendrites, and cytoplasm of geniculate cells. By contrast, in sections incubated with an aspartate antibody, terminals of retino-geniculate, cortico-geniculate, and geniculo-cortical axons did not show a selective enrichment of immunoreactivity, but instead the density of immunogold particles was generally low in the different profiles of the neuropil, with the exception of nucleoli. These results suggest that glutamate, but not aspartate, is a neurotransmitter candidate in the retino-geniculo-cortical pathways.

Glutamate receptor binding in juvenile and adult Rana pipiens CNS.

Autoradiographic methods were used to map NMDA- and quisqualate-sensitive glutamate binding sites in the brain of mature and juvenile Rana pipiens frogs. NMDA- and quisqualate-sensitive sites were consistently co-localized in the CNS. The highest glutamate binding occurred in the telencephalon, hypothalamus, and cerebellum. Glutamate binding sites were also specifically localized in visual pathways, including the superficial neuropil of the optic tectum, consistent with glutamate being the retinal ganglion cell neurotransmitter. The distribution of glutamate binding sites in the brain of juvenile postmetamorphic frogs was similar to that in adults. In general, Quis binding increased about twofold in adults compared to juveniles, whereas NMDA binding did not show a comparable developmental increase. To test whether glutamate binding sites are located on retinal axon terminals or on tectal cell dendrites in the optic tectum, juvenile postmetamorphic frogs were enucleated unilaterally, and receptor binding was performed following 1, 3, 7, and 14 days survival. The denervated tectal neuropil showed a delayed decrease in NMDA- and quisqualate-sensitive binding, consistent with the receptors being located on postsynaptic tectal cell dendrites.