Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit

Retinal ganglion cells are the projection neurons that link the retina to the brain. Peptide immunoreactive cells in the ganglion cell layer (GCL) of the mammalian retina have been noted but their identity has not been determined. We now report that, in the rabbit, 25-35% of all retinal ganglion cells contain substance P-like (SP) immunoreactivity. They were identified by either retrograde transport of fluorescent tracers injected into the superior colliculus, or by retrograde degeneration after optic nerve section. SP immunoreactive cells are present in all parts of the retina and have medium to large cell bodies with dendrites that ramify extensively in the proximal inner plexiform layer. Their axons terminate in the dorsal lateral geniculate nucleus, superior colliculus and accessory optic nuclei, and these terminals disappear completely after contralateral optic nerve section and/or eye enucleation. In the dorsal lateral geniculate nucleus large, beaded, immunoreactive axons and varicosities make up a narrow plexus just below the optic tract, where they define a new geniculate lamina. The varicosities make multiple synaptic contacts with dendrites of dorsal lateral geniculate nucleus projection neurons and presumptive interneurons in complex glomerular neuropil. This is direct evidence that some mammalian retinal ganglion cells contain substance P-like peptides and strongly suggests that, in the rabbit, substance P (or related tachykinins) may be a transmitter or modulator in a specific population or populations of retinal ganglion cells.     

Generation of cat retinal ganglion cells in relation to central pathways

The ganglion cells of the cat retina form classes distinguishable in terms of perikaryal size, dendritic morphology and functional properties. Further, the axons differ in their diameters, patterns of chiasmatic crossing and in their central connections. Here we define, by 3H-thymidine autoradiography, the order of production of cells of each class and relate the order of the 'birthdates' to the known axonal pathways. The ganglion cell classes are produced in broad waves, which overlap as cells are produced first for central then for peripheral retina. Medium-sized cells are produced before the largest cells, and small ganglion cells are produced throughout the period of cell generation. This sequence of cell production relates to the orderly arrangement of axons in the optic tract, and can also be related to the rules of chiasmatic crossing observed for each ganglion cell class.     

Segregation of functionally distinct axons in the monkey's optic tract

The classical neuro-ophthalmologic literature describes the organization of the primate's optic tract as containing a single topographic representation of the complete contralateral visual hemifield. In contrast, cats have separate visual field representations for the optic axons of the functionally distinct retinal ganglion cell classes. As the line of decussation for each ganglion cell class in the cat occupies a different location on the retinal surface, whereas in primates they are all superimposed, such a species difference might be expected. We report that implants of horseradish peroxidase placed in either the deep or superficial extremes of the monkey's optic tract produce retrograde labelling of distinct retinal ganglion cell classes, and produce anterograde labelling confined to distinct laminae of the lateral geniculate nucleus. Hence, the optic tract of the primate cannot contain a single representation of the contralateral visual hemifield; rather, independent visual field representations for the functionally distinct optic axons must exist. Their anatomical segregation may account for the clinical observation of selective impairments of distinct visual abilities following partial interruption of the optic tract in man.     

Retinotopic order in the absence of axon competition

The retinotectal projection has long been studied experimentally and theoretically, as a model for the formation of topographic brain maps. Neighbouring retinal ganglion cells (RGCs) project their axons to neighbouring positions in the optic tectum, thus re-establishing a continuous neural representation of visual space. Mapping along this axis requires chemorepellent signalling from tectal cells, expressing ephrin-A ligands, to retinal growth cones, expressing EphA receptors. High concentrations of ephrin A, increasing from anterior to posterior, prevent temporal axons from invading the posterior tectum. However, the force that drives nasal axons to extend past the anterior tectum and terminate in posterior regions remains to be identified. We tested whether axon-axon interactions, such as competition, are required for posterior tectum innervation. By transplanting blastomeres from a wild-type (WT) zebrafish into a lakritz (lak) mutant, which lacks all RGCs, we created chimaeras with eyes that contained single RGCs. These solitary RGCs often extended axons into the tectum, where they branched to form a terminal arbor. Here we show that the distal tips of these arbors were positioned at retinotopically appropriate positions, ruling out an essential role for competition in innervation of the ephrin-A-rich posterior tectum. However, solitary arbors were larger and more complex than under normal, crowded conditions, owing to a lack of pruning of proximal branches during refinement of the retinotectal projection. We conclude that dense innervation is not required for targeting of retinal axons within the zebrafish tectum but serves to restrict arbor size and shape.     

Homing behaviour of axons in the embryonic vertebrate brain

In embryonic nervous systems, growing axons must often travel long distances through diverse extracellular terrains to reach their postsynaptic partners. In most embryos, axons grow to their appropriate targets along particular tracts or nerves, as though they were following guidance cues confined to specific pathways. For example, in all vertebrates, axons from the retina invariably grow to the tectum along the well-defined optic tract. Yet, transplant experiments demonstrate that retinal axons make tectal projections even though they enter the brain at locations which are distinctly off the optic tract. Only recently has it become possible to label discreet growing projections in the embryonic vertebrate brain. Thus, it is not yet known whether displaced retinal axons grow directly towards the tectum or find it accidently, through random extension. To resolve this question, pioneering axons from normal and transplanted eyes in embryonic Xenopus were labelled using a short-survival horseradish peroxidase (HRP) method, and their orientation during growth was quantitatively assessed. The finding that the ectopic fibres head towards their distant targets implies that guidance cues are not restricted to specific pathways but are distributed throughout the embryonic brain. The significance of this result is discussed with respect to the ontogeny and evolution of the visual pathway.     

Non-retinotopic arrangement of fibres in cat optic nerve

Fibres in the mammalian optic nerve are generally thought to be organised retinotopically. Recording electrophysiologically from the cat optic nerve, we found little evidence to support this notion, which led us to investigate the problem by anatomical methods. We made a localised injection of horseradish peroxidase into the lateral geniculate body of the cat, labelling a small clump of retinal ganglion cells and their axons in the optic nerve. These fibres, emanating from neighbouring cells in the retina, became widely scattered through the optic nerve, indicating that retinotopic order is essentially lacking.     

Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex

The use of retrograde axonal transport of various substances (for example, enzymes, lectins, synthetic fluorescent compounds) has yielded much information on the organization of neuronal pathways. Each type of retrograde tracer has its own set of attributes which define the scope of problems it can address. We describe here a new class of retrograde tracer, rhodamine-labelled fluorescent latex microspheres (0.02-0.2 micron diameter), which have distinct advantages over other available tracers for in vivo and in vitro applications. When injected into brain tissue, these microspheres show little diffusion and consequently produce small, sharply defined injection sites. Once transported back to neuronal somata, the label persists for at least 10 weeks in vivo and 1 yr after fixation. Microspheres have no obvious cytotoxicity or phototoxicity as assessed by intracellular recording and staining of retrogradely labelled cells in a cortical brain slice preparation. This approach was further used to visualize and compare, in cat visual cortex slices, neurones with different projection patterns, and revealed significant differences in patterns of intrinsic axons and dendrites. These properties of microspheres open new avenues for anatomical and physiological studies of identified projection neurones in slices as well as in dissociated cell cultures.     

Synaptic connectivity of a local circuit neurone in lateral geniculate nucleus of the cat

Although receptive fields of relay cells in the lateral geniculate nucleus of the cat nearly match those of their retinal afferents, only 10-20% of the synapses on these cells derive from the retina and are excitatory. Many more (30-40%) are inhibitory and largely control the gating of retinogeniculate transmission. These inhibitory synapses derive chiefly from two cell types: intrinsic local circuit neurones and cells in the adjacent perigeniculate nucleus. It has been difficult to study the functional organization of these inhibitory pathways; most efforts have relied on indirect approaches. Here we describe the use of direct techniques to study a local circuit neurone by iontophoresing horseradish peroxidase (HRP) into it, which completely labels the soma and processes of cells for subsequent light- and electron microscopic analysis. Although the response properties of the labelled cell are virtually indistinguishable from those of many relay cells, its morphology is typical of 'class 3' neurones (see Fig. 1 legend), which are widely believed to be interneurones (but see ref. 12). Here, we refer to the cell as a 'local circuit neurone', which allows for the possibility of a projection axon, rather than as an 'interneurone', a term that commonly excludes a projection axon. We find that the labelled cell has a myelinated axon, but that the axon loses its myelin within 50 microns of the soma and has not yet been traced further. The dendrites of the labelled cell possess presynaptic terminals that act as intrinsic sources of inhibition on geniculate relay cells. We also characterize other morphological aspects of this inhibitory circuitry.     

Ganglion cell dendrites are presynaptic in catfish retina

The retinal ganglion cells are third-order, spike-generating neurones whose axons transmit the output of the retina to the rest of the brain. It has long been believed that the dendrites of the retinal ganglion cells, like the dendrites of most other Golgi type I neurones, are only postsynaptic. Here we have studied the synapses made onto the ganglion cells in the catfish (Ictalurus punctatus), and we report that the distal dendrites of large-field ganglion cells make conventional chemical synapses onto other inner plexiform layer processes. We have also found that, more than 100 microns away from the cell perikaryon, the synapses made onto and by these ganglion cell dendrites are often tightly clustered. These synaptic clusters appear to be quite regularly spaced along the dendrites. Our results have important implications for the identification of ganglion cell dendrites within the inner plexiform layer as well as for the understanding of the ganglion cell response and receptive field generation.     

Regulation of axon growth in vivo by activity-based competition

The formation of functional neural networks requires precise regulation of the growth and branching of the terminal arbors of axons, processes known to be influenced by early network electrical activity. Here we show that a rule of activity-based competition between neighbouring axons appears to govern the growth and branching of retinal ganglion cell (RGC) axon arbors in the developing optic tectum of zebrafish. Mosaic expression of an exogenous potassium channel or a dominant-negative SNARE protein was used to suppress electrical or neurosecretory activity in subsets of RGC axons. Imaging in vivo showed that these forms of activity suppression strongly inhibit both net growth and the formation of new branches by individually transfected RGC axon arbors. The inhibition is relieved when the activity of nearby 'competing' RGC axons is also suppressed. These results therefore identify a new form of activity-based competition rule that might be a key regulator of axon growth and branch initiation.     

Position-dependent properties of retinal axons and their growth cones

The formation of the very orderly neuronal projection from the retina to the optic tectum is not yet understood, but several mechanisms are thought to be involved in a coordinated fashion. These mechanisms may include mechanical or chemical guidance in channels, guidance by spatial gradients of positional markers, gradients of temporal (maturation) markers or specific inter-axon interactions (see ref. 1 for review). The last-mentioned mechanism could explain the fibre order found in optic nerve and tract. It requires that some or all growing retinal axons can distinguish between retinal axons of various origins and grow preferentially along retinal axons originating from the same area as themselves. The in vitro experiments described here show that growth cones from the temporal half of the chick retina grow preferentially along temporal axons, whereas growth cones from nasal retina do not distinguish between nasal and temporal axons.     

Monoclonal antibodies which recognize different cell types in the rat retina

Seven monoclonal antibodies have been produced against a membrane preparation from adult rat retina. Three antibodies reacted with particular regions of rat photoreceptor cell surfaces: RET-P1 labelled the cell bodies, outer and inner segments (rods but not cones), RET-P2 labelled only outer segments and RET-P3 labelled only the cell bodies. Three antibodies reacted with glial cells; RET-G2 and RET-G3 were specific for Müller cells, RET-G1 also labelled glia elsewhere in brain. The seventh antibody (RET-N1) reacted with many types of neuronal cells.     

Order in the initial retinotectal map in Xenopus: a new technique for labelling growing nerve fibres

Retinal nerve fibres form an orderly map of visual space in several centres in the vertebrate brain. Such topographic maps are a common feature of central nervous system organization, yet the way in which they develop is poorly understood. Early nerve projections in the fetal and neonatal mammalian brain have been found in several cases to be less restricted than those in the adult, suggesting that nerve fibres may initially form a diffuse set of connections in their target structure from which the adult map is sculpted by the elimination of terminals. Indeed, previous electrophysiological data indicate that the retinotectal map in Xenopus laevis might be initially disorganized. We report here, however, that the retinotectal projection is ordered from the beginning of tectal innervation (stage 39/40). We demonstrate this first autoradiographically by tracing groups of growing ganglion cell axons which we labelled by incubating sectors of eye rudiments, before axonal outgrowth, in 3H-proline and replacing them orthotopically. Separate labelling of dorsal and ventral parts of the initial projection showed that retinal fibres are organized topographically, as in the adult, in the tectal rudiment and throughout much of the pathway. Second, we show that visual responses are ordered in the tectum from the first stage that they can be mapped (stage 40). We conclude that the topographic ordering of retinotectal connections develops as a result of directed axonal outgrowth.     

Wnt-Ryk signalling mediates medial-lateral retinotectal topographic mapping

Computational modelling has suggested that at least two counteracting forces are required for establishing topographic maps. Ephrin-family proteins are required for both anterior-posterior and medial-lateral topographic mapping, but the opposing forces have not been well characterized. Wnt-family proteins are recently discovered axon guidance cues. We find that Wnt3 is expressed in a medial-lateral decreasing gradient in chick optic tectum and mouse superior colliculus. Retinal ganglion cell (RGC) axons from different dorsal-ventral positions showed graded and biphasic response to Wnt3 in a concentration-dependent manner. Wnt3 repulsion is mediated by Ryk, expressed in a ventral-to-dorsal decreasing gradient, whereas attraction of dorsal axons at lower Wnt3 concentrations is mediated by Frizzled(s). Overexpression of Wnt3 in the lateral tectum repelled the termination zones of dorsal RGC axons in vivo. Expression of a dominant-negative Ryk in dorsal RGC axons caused a medial shift of the termination zones, promoting medially directed interstitial branches and eliminating laterally directed branches. Therefore, a classical morphogen, Wnt3, acting as an axon guidance molecule, plays a role in retinotectal mapping along the medial-lateral axis, counterbalancing the medial-directed EphrinB1-EphB activity.     

Structure of neurofilaments studied with scanning tunneling microscopy

Neurofilaments (NFs) were isolated from bovine spinal cord. The structure of purified NFs was studied by scanning tunneling microscopy (STM). The STM images showed that NF was composed of a long core filament and numerous sidearms flanking the rod regularly. The diameter of the rod was about 10 nm (10.2 ± 0.8 nm). Most of the sidearms were short and the distance between two adjacent sidearms was approximately 10 nm. There were some long sidearms between two proximal core filaments. The distance between two adjacent long sidearms was 21 nm. A three-quarter-staggered fashion of native NF structure was put forward.     

视神经损伤引起斑马鱼视网膜神经细胞凋亡的研究

用石蜡连续切片苏木精染色法,通过定量分析研究夹伤和切断视神经后,斑马鱼视网膜神经节细胞、视杆和视锥细胞密度的变化。结果发现,在损伤视神经7～21d后,上述3种细胞的细胞核密度均呈减少趋势,节细胞减少的比率大于感光细胞,而感光细胞中视锥细胞所受影响比视杆细胞更为明显;在夹伤和切断视神经两种情况中,后者引起视网膜神经节细胞核密度的减少更为显著。上述结果表明,损伤视神经不但影响与其相连的神经节细胞,而且可逆向跨神经元地影响感光细胞的变化。由上述结果推测,由于损伤视神经使视网膜神经节细胞失去靶组织而引起的各种神经细胞密度减少是视网膜中神经细胞凋亡的表现。     

Guidance of optic nerve fibres by N-cadherin adhesion molecules

The dendritic branches (neurites) of developing neurons migrate along specific pathways to reach their targets. It has been suggested that this migration is guided by factors present on the surface of other neurons or glial cells. The molecular nature of such factors, however, remains to be elucidated. N-cadherin is a cell-surface glycoprotein which belongs to the cadherin family of cell-cell adhesion molecules. This adhesion molecule is expressed in various neuronal cells as well as in glial cells of the central and peripheral nervous systems in vertebrate embryos and recent immunological studies suggested that N-cadherin may play a role in guiding the migration of neurites on myotubes or astrocytes. To further examine this possibility, we used a molecular-genetic approach; that is, we examined the outgrowth of chicken embryonic optic axons on monolayer cultures of Neuro 2a or L cells transfected with the complementary DNA encoding chicken N-cadherin. The data indicate that N-cadherin is used as a guide molecule for the migration of optic axons on cell surfaces.     

日本沼虾胚胎复眼发生的研究

应用光镜技术和透射电镜技术,研究了日本沼虾胚胎复眼的发生.在原肠期,胚区前端的外胚层细胞增殖形成视叶原基.发育至后无节幼体期开始形成视神经节.在前蚤状幼体早期胚胎出现复眼色素，同时构成复眼的细胞开始分化。前蚤状幼体晚期，复眼色素区扩大到复眼直径的一半。复眼由个眼构成，每个个眼由角膜、角膜生成细胞、晶锥、小网膜细胞等组成。超微结构显示，胚胎的复眼结构与成体的相似，但感杆束直径比成体小。至胚胎孵化，眼柄尚未发育完善。     

Induction of functional retinal projections to the somatosensory system

Optic axons can be induced to form permanent, retinotopic connections in the auditory (medial geniculate, MG) and somatosensory (ventrobasal, VB) nuclei of the Syrian hamster thalamus; this occurs when the principal targets of retinofugal axons are ablated in newborn hamsters and alternative terminal space is created by partial deafferentation of MG or VB. The experimentally induced retinal projection to the somatosensory nucleus occurs by the stabilization of an early, normally transient projection. The present study was undertaken to determine whether the anomalous, stabilized retino-VB projection is functional. Newborn hamsters were operated on to produce permanent retino-VB projections and when the animals were mature, neurophysiological recordings were made in the cortical targets of VB, the first and second somatosensory cortices (SI and SII, respectively). Visual stimulation within well-defined receptive fields reliably evoked multi-unit responses in SI and SII of operated, but not normal hamsters. The representations of the visual field in SI and SII showed a partially retinotopic organization. These results demonstrate that optic tract axons can form functional synapses in the thalamic somatosensory nucleus, and suggest that neural structures which normally process information specific to one sensory modality have the potential to mediate function for other modalities.     

Platelet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell

The mitogens which modulate cell-cell interactions during development of the central nervous system are unknown. One of the few interactions sufficiently well understood to allow identification of such molecules involves the two glial lineages which make up the rat optic nerve. One population of glial cells in this tissue, the type-1 astrocytes, secrete a soluble factor(s) which promotes division of a second population of bipotential oligodendrocyte/type-2 astrocyte (O-2A) progenitor cells; these progenitors give rise to oligodendrocytes, which myelinate large axons in the CNS, and type-2 astrocytes, which enwrap bare axons at nodes of Ranvier. Type-1 astrocytes also promote progenitor motility, and inhibit the premature differentiation of progenitors into oligodendrocytes which occur when these cells are grown in the absence of type-1 astrocytes. We have now found that platelet-derived growth factor mimics the effects of type-1 astrocytes on O-2A progenitor cells, and antibodies to PDGF block the effects of type-1 astrocytes.