The transneuronal transport of proline within the mouse visual system: Some characteristics of the [3H]-proline containing material

Following the intraocular injection of tritiated proline in the mouse, the progressive transport of radioactivity in the brain and the nature of the cortical material(s) to which the label is bound was examined. About 35–40% of the radioactivity that was present in the cerebral cortex at four weeks post-injection was extractable with either distilled water or various buffers. By using 0.1% SDS this value can be increased up to 94%. Polyacrylamide gel electrophoresis of the extracted proteins showed that a major part of the radioactivity is accumulated in one band of proteins. This heavily labeled band could not be identified in the homolateral parietooccipital cortex or in the frontal cortex. Similarly, SDS electrophoresis of the SDS-extracted proteins also demonstrated the presence of a major band of proteins whose molecular weight was estimated at approximately 68,000 daltons. The protein was purified by ammonium sulfate precipitation and DEAE-Sephadex separation.     

Retrograde transport of [3H]proline: a widespread phenomenon in the central nervous system

[3H]Proline was injected into spinal cord, pons, inferior colliculus, superior colliculus, lateral geniculate nucleus, pulvinar-LP complex and visual cortex of cats or rats. After 1-3 days' survival the animals were perfused with formalin or mixed aldehydes. Autoradiography showed labeling of cell bodies in most regions known to project to the injection sites. The ability to take up and retrogradely transport [3H]proline appears to be a common property of central neurons. We infer that the transport of this compound is unrelated to its possible status as a neurotransmitter.     

Distribution of ganglion cells in the pigeon retina labeled via retrograde transneuronal transport of the fluorescent dye rhodamine beta-isothiocyanate from the telencephalic visual Wulst

The distribution of retinal ganglion cells (RGCs) providing input to the thalamofugal visual system in the pigeon was studied with an anatomical transneuronal transport technique using the fluorescent dye rhodamine beta-isothiocyanate (RITC). Unilateral injections of RITC made into the telencephalic visual Wulst resulted in the retrograde (1) first-order labeling (FOL) of dorsal thalamic (n. dorsolateralis anterior and n. superficialis parvocellularis: SPC) and brainstem somata as well as (2) second-order labeling of other cell populations within the brain and of retinal ganglion cells in both eyes obtained after transneuronal transfer of the tracer from neurons labeled directly via FOL. The mapping and counting of labeled RGCs in retinal flat-mounts showed that they were mainly distributed within the nasal portion of the retinal yellow field (YF) and that their total numbers were consistently higher (averaging 57%) in the eye contralateral to the tracer injection. Labeled RGCs in the retinal red field (RF) represented 13.4% and 12.0% of total labeled cells in the ipsilateral and contralateral eye, respectively. Moreover, the average densities of labeled cells/mm(2) in the RF and YF were respectively 8.4 and 42.8 (ipsilateral) and 17.9 and 54.0 (contralateral). The preferential distribution of labeled RGCs within the nasal YF supports the notion that the thalamofugal visual system in the lateral-eyed pigeon is mainly concerned with viewing in the lateral visual field. Conversely, the relatively low numbers of labeled RGCs observed within the specialized RF indicate that, unlike the case in frontal-eyed bird species and mammals, this system does not appear to be involved in binocular visual processing.     

Differences in the efficiency and pattern of incorporation of [H]leucine and [H]proline into proteins of adult cat brain

Previous EM autoradiographic studies have shown that injection of [3H]proline ([3H]Pro) into the cat dorsal column nuclei (DCN) results in heavy labeling of macroglia but negligible labeling of DCN neurons. [3H]Leucine ([3H]Leu), in contrast, was extensively incorporated into both neurons and glia. We now report preliminary assessment of differences in molecular labeling patterns produced by the two amino acid precursors in DCN injection sites (24 h following injection; equal amounts of [3H]Pro and [3H]Leu of equal specific radioactivity). [3H]Pro, despite its lack of incorporation into neurons, labeled DCN to a significantly greater extent than [3H]Leu (30.0 dpm/micrograms protein/microCi for [3H]Pro vs 11.7 dpm/micrograms protein/microCi for [3H]Leu). Greater than 90% of the radioactivity from both precursors was recovered in protein as opposed to TCA soluble or ethanol soluble molecules. Of the [3H]Pro- or [3H]Leu-derived radioactivity recovered in protein, greater than 94% was found to remain in the original precursor form. Fluorographic analysis of SDS acrylamide gels showed labeling of a wide variety of individual proteins with either amino acid. However, particular molecular weight classes were relatively more heavily labeled with either [3H]Leu (47, 63, 77 kDa) or [3H]Pro (22, 45, 50, 66, 80 kDa). Overall the results indicate that the difference in cellular distribution of incorporated [3H]Pro and [3H]Leu, as observed by EM autoradiography is a reflection of the extent of labeling and specific labeling pattern of proteins isolated from the tissue.     

Axonal transport and metabolism of [H]fucose- and [S]- Sulfate-labeled macromolecules in the rat visual system

The axonal transport of labeled macromolecules in retinal ganglion cells of rats was investigated from 1 to 20 days following intraocular injection of [³H]fucose and [³⁵S]sulfate. Maximal incorporation of [³H]fucose into acid insoluble material in the retina was at 8 h, followed by a biphasic decline. Transported [³H]fucose (98% as glycoprotein) was in the optic nerve at 1 h, the optic tract and lateral geniculate body by 2 h, and the superior colliculus by 3 h after injection, indicating a rate of transport of approximately 200 mm/day. Radioactivity continued to accumulate in the superior colliculus for at least 8 h and began to decline rapidly by 24 h. Between 3 and 6 days levels rose again in both optic tract and superior colliculus before starting a gradual decline, indicating that a wave of rapidly transported material was delayed in leaving the retina. When proteins in the superior colliculus were fractionated by gel electrophoresis, the composition of the two fucosylated protein transport phases could be partially resolved. Radioactivity in individual gel peaks represented primarily in the first phase decayed with an average half-life of one day, although that in one prominent protein of molecular weight 280,000 turned over with a half-life of the order of 12 h. Radioactive peaks primarily in the second phase decayed with an average half-life of more than a week.Incorporation of [³⁵S]sulfate into acid insoluble material in the retina was maximal at 1–2 h, after which there was a rapid loss of label. The appearance of [³⁵S]sulfate in the optic tract, loateral geniculate body and superior colliculus preceded by a short time that of the [³H]fucose; indicating a shorter retinal processing time for this label. The total transported [³⁵S]sulfate in the superior colliculus peaked by 4–8 h and had fallen by 65% at one day; no prominent second wave of transport was observed as was the case for [³H]fucose. Acid insoluble [³⁵S]sulfate in the superior colliculus was equally divided between glycopeptides and glycosaminoglycans at all times examined, indicating that these macromolecules are transported at the same rate. [³⁵S]Sulfate incorporated into various proteins fractionated by gel electrophoresis had heterogeneous turnover rates, the average being around 12 h. Radioactivity in one group of proteins, of molecular weight around 90,000, decayed with a half-life of only a few hours.     

Transient increase in the in vivo binding of the benzodiazepine antagonist [3H]flumazenil in deafferented visual areas of the adult mouse brain

Flumazer it is an imidazobenzodiazepine, an antagonist of central benzodiazepine (BDZ) receptors. BDZ binding sites are a modulatory component located on the gamma-aminobutyric acid (GABA) receptor macromolecule. We studied the effect of monocular enucleation on [³H]flumazenil binding in deprived and intact visual areas and nonvisual areas of the adult mouse brain under in vivo conditions. [³H]flumazenil binding was examined at seven time points up to 56 days postenucleation. In some monocularly deprived mice, changes in local blood flow accompanied with the BDZ receptor response were evaluated by coinjection of [3H]flumazenil and ^9mTc-HMPAO. Monocular enucleation produced a transient increase in [³H]flumazenil binding in the deprived visual cortex and superior colliculus. At 17 days postenucleation, [³H]flumazenil binding in the anterior and posterior portions of the visual cortex and the superior colliculus increased by 28%, 15% and 23%, respectively, and declined to control levels at 45 days postenucleation. The increase in [³H]flumazenil was accompanied with a decrease in blood flow. Alterations in BDZ receptors and blood flow were selective to deprived visual structures. The regional correlation between the metabolic deficit and the BDZ response provides further support that the increase in BDZ receptor binding is confined to regions of reduced neuronal activity. [¹¹C]flumazenil is an excellent radiotracer for in vivo imaging of benzodiazepine receptors in human brain using positron emission tomography (PET). This study suggests the suitability of [¹¹C]flumazenil for in vivo PET study of BDZ receptor response to deafferentation of visual structures in human brain. © 1994 Wiley-Liss, Inc.     

Localization of high affinity [H]glycine transport sites in the cerebellar cortex

A study was made of [³H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [³H]glycine at the low concentration used. Glial cells also took up [³H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [³H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [³H]GABA uptake (K_i over 600 μM vs theK_t of about 20 μM) and that GABA is an even weaker inhibitor of [³H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [³H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [³H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.     

Axonal transport and metabolism of [3H]fucose- and [35S]-sulfate-labeled macromolecules in the rat visual system.

The axonal transport of labeled macromolecules in retinal ganglion cells of rats was investigated from 1 to 20 days following intraocular injection of [3H]fucose and [35S]sulfate. Maximal incorporation of [3H]fucose into acid insoluble material in the retina was at 8 h, followed by a biphasic decline. Transported [3H]fucose (98% as glycoprotein) was in the optic nerve at 1 h, the optic tract and lateral geniculate body by 2 h, and the superior colliculus by 3 h after injection, indicating a rate of transport of approximately 200 mm/day. Radioactivity continued to accumulate in the superior colliculus for at least 8 h and began to decline rapidly by 24 h. Between 3 and 6 days levels rose again in both optic tract and superior colliculus before starting a gradual decline, indicating that a wave of rapidly transported material was delayed in leaving the retina. When proteins in the superior colliculus were fractionated by gel electrophoresis, the composition of the two fucosylated protein transport phases could be partially resolved. Radioactivity in individual gel peaks represented primarily in the first phase decayed with an average half-life of one day, althouth that in one prominent protein of molecular weight 280,000 turned over with a half-life of the order of 12 h. Radioactive peaks primarily in the second phase decayed with an average half-life of more than a week. Incorporation of [35S]sulfate into acid insoluble material in the retina was maximal at 1-2 h, after which there was a rapid loss of label. The appearance of [35S]sulfate in the optic tract, lateral geniculate body and superior colliculus preceded by a short time that of the [3H]fucose; indicating a shorter retinal processing time for this label. The total transported [35S]sulfate in the superior colliculus peaked by 4-8 h and had fallen by 65% at one day; no prominent second wave of transport was observed as was the case for [3H]fucose. Acid insoluble [35S]sulfate in the superior colliculus was equally divided between glycopeptides and glycosaminoglycans at all times examined, indicating that these macromolecules are transported at the same rate. [35S]Sulfate incorporated into various proteins fractionated by gel electrophoresis had heterogeneous turnover rates, the average being around 12 h. Radioactivity in one group of proteins, of molecular weight around 90,000, decayed with a half-life of only a few hours.     

Bidirectional axonal and transcellular transport of [H]adenosine from the lamprey retina

The axonal transport of [³H]adenosine or its related compounds was examined in the visual system of the lamprey following intraocular injection of the nucleoside. Bidirectional and transcellular transport of the marker was demonstrated from the retina. Two different types of labeling of neuronal bodies were observed, resulting from either retrograde axonal or transneuronal transport of tritiated material. The former label could easily be distinguished from the latter in that it appeared very rapidly, was very intense, and exhibited a marked degree of specificity.     

Effects of monocular occlusion and diffusion on visual system development in the cat

The effects of two forms of monocular deprivation (occlusion or diffusion) on visual system development were investigated. One group of cats monocularly deprived of all form stimulation but permitted diffuse light stimulation (diffusion, n = 4) during development showed a pattern of deficits similar to those reported for monocularly sutured cats. Most cells in the visual cortex were driven exclusively by the non-deprived eye and there were eye-specific deficits in X-cell acuity, proportion of Y-cells, and cell body size (binocular and monocular segment) in the lateral geniculate nucleus (LGN). A second group of cats monocularly deprived of all form and light stimulation (occlusion, n = 4) during development showed a less severe pattern of deficits. There was no acuity loss in LGN X-cells driven by the deprived eye, and cell body shrinkage was of smaller magnitude than in diffusion reared cats and was restricted to the binocular segment. Cortical deficits and LGN Y-cell loss were similar in the two groups. The results are consistent with the idea that monocular occlusion produces only deficits due to binocular competition while monocular diffusion reflects the combined effects of binocular competition and abnormal stimulation.     

The centrifugal visual system of vertebrates: a comparative analysis of its functional anatomical organization

The present review is a detailed survey of our present knowledge of the centrifugal visual system (CVS) of vertebrates. Over the last 20 years, the use of experimental hodological and immunocytochemical techniques has led to a considerable augmentation of this knowledge. Contrary to long-held belief, the CVS is not a unique property of birds but a constant component of the central nervous system which appears to exist in all vertebrate groups. However, it does not form a single homogeneous entity but shows a high degree of variation from one group to the next. Thus, depending on the group in question, the somata of retinopetal neurons can be located in the septo-preoptic terminal nerve complex, the ventral or dorsal thalamus, the pretectum, the optic tectum, the mesencephalic tegmentum, the dorsal isthmus, the raphé, or other rhombencephalic areas. The centrifugal visual fibers are unmyelinated or myelinated, and their number varies by a factor of 1000 (10 or fewer in man, 10,000 or more in the chicken). They generally form divergent terminals in the retina and rarely convergent ones. Their retinal targets also vary, being primarily amacrine cells with various morphological and neurochemical properties, occasionally interplexiform cells and displaced retinal ganglion cells, and more rarely orthotopic ganglion cells and bipolar cells. The neurochemical signature of the centrifugal visual neurons also varies both between and within groups: thus, several neuroactive substances used by these neurons have been identified; GABA, glutamate, aspartate, acetylcholine, serotonin, dopamine, histamine, nitric oxide, GnRH, FMRF-amide-like peptides, Substance P, NPY and met-enkephalin. In some cases, the retinopetal neurons form part of a feedback loop, relaying information from a primary visual center back to the retina, while in other, cases they do not. The evolutionary significance of this variation remains to be elucidated, and, while many attempts have been made to explain the functional role of the CVS, opinions vary as to the manner in which retinal activity is modified by this system.     

Spread of vesicular stomatitis virus along the visual pathways after retinal infection in the mouse

Summary Vesicular stomatitis virus (VSV) was injected into the left eyeball of 3-week-old mice and it infected the retinal ganglion cells. The infection spread rapidly along the visual pathways to the postsynaptic neurons in the contralateral superior collicle (SC) and lateral geniculate body (LGB). The distributional pattern of the viral immunoreactivity indicated an anterograde axonal transport of the infectious material. A subsequent spread to the ganglion cells of the right retina further indicated retrograde axonal VSV transport. Within the retina the infection spread from the ganglion cells to the pigment epithelium. Although a transneuronal spread of the VSV infection was observed, no VSV budding from or uptake in synaptic membranes was demonstrated ultrastructurally in the retina or the superior collicle. In the retina virions budded from the perikaryal and dendritic plasma membranes of the ganglion cells as well as from the nerve cell bodies of the inner and outer nuclear layers, but not from the receptor segments. In the superior collicle budding was also observed from the plasma membranes of nerve cell bodies and dendrites. In contrast, the intraocular injection of Sendai virus caused a limited retinal ganglion cell infection, with no further propagation in the retina or to the SC or LGB.     

Retrograde transport of [H]GABA in the striatotegmental system of the pigeon

The method of retrograde transport of γ-aminobutyric acid (GABA) injected into the nigral complex of the pigeon was used to determine the transmitter-specificity of the striatotegmental projection system in this species. The results indicate that descending GABAergic projections are derived from the nucleus accumbens (Ac), ventral lobus parolfactorius (LPO) and the ventral paleostriatum (VP). In birds VP is considered comparable to the mammalian ventral pallidum/substantia innominata area. These results are interpreted in terms of the similarities and differences of striatal organization in birds, reptiles and mammals.     

Orthograde axonal and transcellular transport of different fluorescent tracers in the primary visual system of the rat

The differential labeling properties of various fluorescent tracers injected intraocularly were investigated using as a model the rat primary visual system. All of the tracers tested (Fast Blue, FB; True Blue, TB; Nuclear Yellow, NY; bisbenzimide, BB; Evans Blue, EB; propidium iodide, PI) produced a retrograde neuronal labeling of oculomotor neurons. However, no such labeling was observed in the medial pretectal nucleus (NPM) considered to be the site of origin of the rat centrifugal visual pathway. Orthograde transport within the axons of the optic tract and their terminal arborizations were visualized directly with FB and TB. No evidence of EB or PI orthograde transport was demonstrated. Furthermore, FB, TB, NY and BB displayed varying degress of leakage from the optic axons and terminals into the extracellular space, there to be taken up by glial (FB, TB, NY, BB) and neuronal (NY, BB) somas of the primary optic system or in adjacent structures including NPM (BB). The neuronal labeling with NY or BB does not result from the retrograde axonal transport but appears to involve an orthograde transneuronal process transport. Some limitations in the use of different fluorescent tracers for determining neuronal connections are discussed.     

Radioautographic evidence for both orthograde and retrograde axonal transport of labeled compounds after intraocular injection of [H]proline in the lamprey (Lampetra fluviatilis)

[³H] Proline injected intraocularly in lampreys has been shown to be bidirectionally transported: 24–96 h after the injection, retinofugal fibers and terminals as well as nerve cell bodies at the origin of the retinopetal system were intensely labeled. These results are at variance with the generally held belief that [³H]proline is taken up only by cell bodies and transported by the anterograde flow. The significance of the retrograde axonal transport of [³H]proline in the lamprey retinopetal system is discussed.