Identification of a novel nicotinic acetylcholine receptor structural subunit expressed in goldfish retina.

A new non-alpha (n alpha) member of the nicotinic acetylcholine receptor (nAChR) gene family designated GFn alpha-2 has been identified in goldfish retina by cDNA cloning. This cDNA clone encodes a protein with structural features common to all nAChR subunits sequenced to date; however, unlike all known alpha-subunits of the receptor, it lacks the cysteine residues believed to be involved in acetylcholine binding. Northern blot analysis shows multiple transcripts hybridizing to the GFn alpha-2 cDNA in goldfish retina but undetectable levels of hybridizable RNA in brain, muscle, or liver. S1 nuclease protection experiments indicate that multiple mRNAs are expressed in retina with regions identical or very similar to the GFn alpha-2 sequence. In situ hybridization shows that the gene encoding GFn alpha-2 is expressed predominantly in the ganglion cell layer of the retina.

Brain peptides and glial growth. II. Identification of cells that secrete glia-promoting factors.

Glia-promoting factors (GPFs) are brain peptides which stimulate growth of specific macroglial populations in vitro. To identify the cellular sources of GPFs, we examined enriched brain cell cultures and cell lines derived from the nervous system for the production of growth factors. Ameboid microglia secreted astroglia-stimulating peptides, while growing neurons were the best source of the oligodendroglia-stimulating factors. These secretion products co-purified by gel filtration, anion exchange chromatography, and reverse-phase high performance liquid chromatography with GPFs isolated from goldfish and rat brain. Our findings suggest that glial growth in the central nervous system is regulated in part by a signaled release of peptides from specific secretory cells.

Glial and neuronal forms of the voltage-dependent sodium channel: characteristics and cell-type distribution.

Two functionally different forms of the voltage-dependent sodium channel were observed in glia and in neurons of the mammalian nervous system. Both forms had identical conductance and tetrodotoxin sensitivity and displayed steady-state inactivation, a strongly voltage-dependent rate of activation, and a faster but weakly voltage-sensitive rate of inactivation. However, the glial form had significantly slower kinetics and a more negative voltage dependence, suggesting that it was functionally specialized for glia. This form was found in most glial types studied, while the neuronal form was observed in retinal ganglion cells, cortical motor neurons, and O2A glial progenitor cells. Both forms occurred in type-2 astrocytes. The presence of the glial form correlated with the RAN-2 surface antigen.

Phosphorylation of neurofilament proteins by protein kinase C.

The low molecular mass (70 kDa) subunit of neurofilaments (NF-L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site-specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two-dimensional alpha-chymotryptic phosphopeptide map analyses of the NF-L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse-radiolabeled with [32P]orthophosphate in vivo.

RNA content of normal and axotomized retinal ganglion cells of rat and goldfish.

The responses of rat and goldfish retinal ganglion cells to axotomy were examined by a quantitative cytochemical method for RNA and by morphometric measurement 1-60 (rat) and 3-90 (goldfish) days after interruption of one optic nerve or tract intracranially. Unoperated control animals were studied also. The RNA content of axotomized neurons of rat fell 7-60 days postoperatively. Additionally, atrophy of the axotomized somas occurred. Over time, neuronal atrophy approximately paralleled the loss of RNA, and mean cell area and RNA content were reduced by about 25% 60 days after axotomy. Incorporation of 3H-uridine by axotomized neurons declined also. Axotomized retinal ganglion cells of goldfish behaved differently from those of the rat and showed increases in RNA content, most conspicuously 14-60 days postoperatively. Enlargement of axotomized fish neurons occurred but was less proportionately than concomitant increases in RNA content. The nonaxotomized ganglion cells of goldfish displayed statistically significant increases in size and RNA content 14-49 days after unilateral optic nerve or tract lesions. In contrast, alterations in rat retinal ganglion cells contralateral to interruption of one optic nerve were of limited and questionable significance. The contrasting reactions to axotomy by the retinal ganglion cells of these two vertebrates, one of which regenerates optic axons and one of which does not, may support the proposition that the somal response to axon injury has an important bearing upon the success or failure of CNS regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of ocular pigmentation to the boundaries of dorsal and ventral retina in a nonmammalian vertebrate.

The goldfish eye and retina are partitioned traditionally into dorsal and ventral sectors by a horizontal meridian that passes through the optic disc and is perpendicular to a vertical meridian that extends from the remnant of the choroid fissure through the optic disc. Axons of retinal ganglion cells (RGCs) situated above the horizontal meridian are thought to reach the optic tectum via the ventrolateral optic tract and axons of RGCs situated below the horizontal meridian are thought to reach the optic tectum via the dorsomedial optic tract. When cobaltous-lysine was applied to small temporal retinal slits that were centered on the traditional horizontal meridian, filled fibers were found in the dorsomedial, but not in the ventrolateral, optic tract (Springer and Mednick, '83). Since cobalt-filled axons should have been found in both optic tracts, the traditional horizontal meridian does not indicate the actual boundary between dorsal and ventral retina. We report here that the goldfish iris contains nasal and temporal pigmentation lines (darts) that are each located approximately 21 degrees above the traditional horizontal retinal meridian. Cobalt applied to retinal slits located just above the darts filled RGC axons in the ventrolateral optic tract and cobalt applied to retinal slits just below the darts filled RGC axons in the dorsomedial optic tract. Converging evidence for the reliability of the darts as indicators of the boundary between dorsal and ventral retina was obtained by applying cobalt to severed RGC axons along the dorsomedial edge of the tectum. Cobalt-filled RGCs were found below the nasal dart.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of substance P-like immunoreactive retinal ganglion cells and their pattern of termination in the optic tectum of chick (Gallus gallus).

Substance P-like immunoreactive (SP-LI) neurons were identified within the inner nuclear layer and ganglion cell layer of the chick retina. The SP-LI cells in the inner nuclear layer consisted of several subtypes of neurons, differing in soma size and dendritic arborization. In the ganglion cell layer a population of moderately labelled SP-LI neurons was also present. About 6-9 microns in diameter and spaced 50-80 microns apart, they formed a regular array across the entire retina, with a density of about 400 cells/mm2 in the superior temporal retina, declining to less than 100 cells/mm2 in the peripheral retina. The total number of SP-LI cells in the ganglion cell layer was approximately 75,000. Individual axons could be followed toward the optic nerve head. Lesions near the optic nerve head resulted in axotomy of ganglion cells within a limited portion of the retina. Two days of postaxotomy there were numerous SP-LI swellings in the proximal segments of axotomized axons. SP-LI neurons in the axotomized zone were larger, more numerous, and showed increased staining of their processes. Fourteen days following a retinal lesion, there was depletion of all SP-LI cells in the ganglion cell layer within the axotomized zone, but the SP-LI neurons in the inner nuclear layer were not noticeably affected. Following a localized injection of rhodamine-coupled latex beads into the optic tectum, a population of retinal ganglion cells (RGCs) in the contralateral retina was retrogradely labelled. Many of these cells also exhibited SP-like immunoreactivity. Examination of the optic tectum indicated the presence of SP-LI fibres in laminae 2-13 (nomenclature of Cajal: Histologie du Systeme Nerveux. Vol. 2. Paris: Maloine, '11), with immunoreactive terminal regions present mainly in laminae 2-4, 7, and 9-13. SP-LI cell bodies were found predominantly in laminae 10-12 and 13. Fourteen days following a retinal lesion, SP-LI processes and terminals were depleted from laminae 2 and 3. Immunoreactive cells and processes in the remaining laminae of the optic tectum were not noticeably altered. The present report confirms the existence of SP-LI retinal ganglion cells in the chick retina and demonstrates their contribution to lamina specific SP-LI arborization in the optic tectum.

Distribution of catecholaminergic cells in the retina of the rat, guinea pig, cat, and rabbit: independence from ganglion cell distribution.

By using an antibody against tyrosine hydroxylase (TH), the rate-limiting enzyme in the production of catecholamines, we have examined the morphology and distribution of catecholaminergic cells in the retinas of the rat, guinea pig, cat, and rabbit. In the albino rat, as reported by others, most TH-immunoreactive (TH-IR) cells were amacrine cells, and formed two morphological classes. Cells of one class (class 1) are stellate amacrine cells, the somata of most being in the inner part of the inner nuclear layer (INL). Cells of the second class (class 2) were seen only as small somata, also in the inner part of the INL. Cells of both classes were found in all areas of the retina, with a distinct but broad concentration around the superior-temporal margin of the retina. A small number of TH-IR interplexiform cells was seen. In the pigmented rat, only class 1 cells were recognized, also concentrating at the superior-temporal margin. In the guinea pig, cat, and rabbit, TH-IR cells also seemed to form one morphological class of amacrine cells, which resembled the class 1 cells of the albino rat. In the guinea pig and cat, their distribution resembled that seen in the rat, with the cells concentrating at the superior-temporal margin of the retina. In the rabbit, TH-IR cells concentrated weakly in the visual streak, but at both ends of the streak the concentration of TH-IR cells extended farther peripherally than the concentration of ganglion cells. Overall, the distribution of TH-IR cells seems largely or (in the rabbit) partially independent of the distribution of ganglion cells. This independence raises questions of the development and function of this group of amacrine cells.

Somatostatin-like immunoreactive material in the rabbit retina: immunohistochemical staining using monoclonal antibodies.

Two mouse monoclonal antibodies to somatostatin-14 were used with avidin-biotin-peroxidase immunohistochemical technique to examine the rabbit retina. In agreement with a previous study using a polyclonal anti-serum, a sparse population (about 1,000 per retina) of neurons in the ganglion cell layer are immunoreactive for somatostatin; the vast majority of these cells are inferior to the myelinated fiber bundle. In addition, the monoclonal antibodies disclose a second neuronal population that forms a circumferential band of immunoreactive neurons around the extreme periphery of the retina. The cells in the body of the inferior retina have dendrites that ramify in the inner plexiform layer. Both the circumferential band of cells and the cells in the body of the inferior retina give off axonlike processes that run in the inner plexiform layer and do not enter the optic nerve. These long, straight varicose fibers form a meshwork that covers the entire retina. The superior retina, which contains only rare immunoreactive cell bodies, has a plexus of stained fibers comparable to that of the inferior retina. The circumferential band of cells is relatively resistant to the neurotoxin kainic acid, explaining a previously reported observation that this toxin depletes only about 50% of the content of somatostatin-like immunoreactivity from the rabbit retina. Moreover, the somatostatin immunoreactive neurons are not labeled by the intraocular injection of the fluorescent dye DAPI, which labels the cholinergic displaced amacrine cells of the rabbit retina. These observations imply that somatostatin-like immunoreactivity is localized to two populations of associational ganglion cells, neurons with cell bodies in the ganglion cell layer, the axons of which remain within the retina.

Amacrine cells in the ganglion cell layer of the cat retina.

Following transection of the optic nerve, ganglion cells in the cat retina undergo retrograde degeneration. However, many small profiles (less than or equal to 10 micron) survive in the ganglion cell layer. Previously considered to be neuroglia, there is now substantial evidence that they are displaced amacrine cells. Their density increases from approximately 1,000 cells/mm2 in peripheral retina to 7,000 cells/mm2 in the central area. Their total number was found to be 850,000, which is five times the number of ganglion cells and also five times the number of astrocytes. Uptake of 3H-muscimol followed by autoradiography labelled 75% of the displaced amacrine cells; hence, the majority seem to be GABAergic. Immunocytochemistry with an antibody directed against choline-acetyl-transferase labelled approximately 10% of the displaced amacrines in the peripheral retina and 17% in the central area. Uptake of serotonin (5-HT) followed by immunocytochemistry was found in 25-30% of displaced amacrines. NADPH diaphorase histochemistry labelled approximately 5% of displaced amacrine cells. The sum of the various percentages make colocalization likely. Intracellular injection of Lucifer Yellow under microscopic control revealed that displaced amacrine cells constitute several morphological types.

The brainstem projection to the lateral geniculate nucleus in the cat: identification of cholinergic and monoaminergic elements.

The pontomesencephalic projection to the dorsal lateral geniculate nucleus (dLGN) of the cat was analyzed by combining retrograde transport of rhodamine-labeled latex spheres and immunohistochemistry. After injections of latex beads into the dLGN, sections of the brainstem were treated immunohistochemically for choline acetyltransferase (ChAT), serotonin (Ser), tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH). Essentially, six regions in the brainstem contained retrogradely labeled cells: the superior colliculus, the parabigeminal nucleus, the dorsal raphe nuclei, the parabrachial area of the central tegmental field, the marginal nucleus of the brachium conjunctivum, and the nucleus coeruleus. Furthermore, isolated retrogradely labeled cells were present in the central nucleus of the raphe, in the cuneiform nucleus, and in the periaqueductal gray. Most serotoninergic double-labeled cells were found in the medial and lateral divisions of the dorsal raphe nuclei, but a few were also present in the central nucleus of the raphe. In the sections immunostained for ChAT, double-labeled cells were located in the central tegmental field, in the marginal nucleus of the brachium conjunctivum, and in the nucleus coeruleus. In the sections treated for TH and DBH, double-labeled cells showed a similar distribution, and like the ChAT(+) cells, they were located mainly in the central tegmental field, in the marginal nucleus of the brachium conjunctivum, and in the nucleus coeruleus. In these regions the cholinergic and noradrenergic cells that projected to the lateral geniculate nucleus were intermingled, the former predominating rostrally and the latter caudally. The majority of retrogradely labeled cells were located in the region of the central tegmental field in the vicinity of the brachium conjunctivum, and most of these cells were also ChAT-immunoreactive. We, therefore, conclude that the cholinergic projection is the most important of the central core projections ascending to the dLGN.

Peptide immunoreactive neurons in the human retina.

The distribution of peptide-immunoreactive neurons in the human retina was investigated. Neurons displaying immunoreactivity towards substance P, vasoactive intestinal polypeptide (VIP), somatostatin, neuropeptide Y (NPY) and peptide histidine-isoleucine (PHI) were found in amacrine cells with cell bodies situated in the innermost part of the inner nuclear layer and nerve fibers ramifying in the inner plexiform layer in a manner differing according to the peptide investigated. Two other cell types were found. In the middle of the inner plexiform layer cell bodies showing immunoreactivity towards substance P, VIP and PHI were found. In the ganglion cell layer there were cell bodies showing immunoreactivity towards substance P, somatostatin, VIP and NPY. Substance P immunoreactive, somatostatin and NPY immunoreactive fibers situated at the border between the inner nuclear and outer plexiform layers and traversing the inner nuclear layer were also found.

Melatonin-binding in the frog retina: autoradiographic and biochemical analysis.

Binding of melatonin was examined in the retina of Rana pipiens. When intact frog retinas were incubated with 3H-melatonin and processed for autoradiography, most of the radioactivity was localized to the melanosomes of the retinal pigment epithelium-choroid (RPE-choroid) and to the outer plexiform layer of the retina. Melanosome-enriched fractions of the RPE-choroid and membrane-enriched fractions of the neural retina demonstrated saturable melatonin binding when incubated with increasing melatonin concentration. Thin-layer chromatography showed that greater than 98% of the bound radioactivity was authentic melatonin. Scatchard analysis revealed a single population of binding sites with apparent Kd values of 6 X 10(-7) M for both the RPE-choroid and neural retina. When various indole analogs were tested for their ability to inhibit 3H-melatonin binding to the neural retina, both 5-methoxytryptophol and 6-chloromelatonin demonstrated complete displacement of melatonin binding. Endogenous retinal melatonin levels were measured by radioimmunoassay. A twofold increase in melatonin levels was observed during the dark period with peak levels at 384.5 +/- 28.8 pgms melatonin/pair retinas. Melatonin levels persisted in constant darkness, but were suppressed in constant light. Our data suggest that in the frog, the sites of action of retinal melatonin are the melanosomes of the RPE-choroid and the outer plexiform layer of the neural retina.

GABA-like immunoreactivity in cholinergic amacrine cells of the rabbit retina.

In the ganglion cell layer of the rabbit retina, the inhibitory transmitter gamma-aminobutyric acid (GABA) and its analogues are accumulated by neurons that appear to match in size and number the population of displaced amacrine cells that synthesize the excitatory transmitter acetylcholine. In this double-label study, we have established directly that the cholinergic amacrine cells, selectively stained with diamidino-phenylindole, are strongly immunoreactive with GABA antisera. The coexistence of two classical transmitters, one excitatory and the other inhibitory, in this defined neuronal population, suggests that stimulation of the cholinergic amacrines may give rise to complex responses in their target neurons.

Differential distribution of phosphorylated and non-phosphorylated neurofilaments within the retina and optic nerve of hamsters.

Retinae and optic nerve sections from adult hamsters were reacted with antibodies against phosphorylated (P) or non-phosphorylated (NP) neurofilament proteins. NP epitopes were observed within ganglion cell bodies and extended into the proximal portion of the optic nerve. P epitopes became prominent within axons as they approached the optic disc and remained throughout the optic nerve. This spatial distribution appears similar to the pattern of myelination within this axonal population.

Action of iontophoretically applied dopamine on cat retinal ganglion cells.

Iontophoretically applied dopamine reversibly altered both the spontaneous firing rates and the light evoked responses of retinal ganglion cells in the intact eye of the cat. The effects of dopamine were the same for all cell classes encountered: on brisk-transient, off brisk-transient, on brisk-sustained, off brisk-sustained, sluggish and non-concentrically organized cells. Dopamine reduced the spontaneous firing rates of all cells. In response to light stimulation, the inhibitory response phase (light off in on ganglion cells, light on in off ganglion cells) was also reduced by dopamine. However, the excitatory response phase (light on in on ganglion cells, light off in off ganglion cells) was only consistently reduced for optimal spot stimulation: for wholefield or annular stimulation the excitatory response phase was reduced in 76% of cells, whereas for the remaining cells it was unchanged or even increased. The net effect of these alterations was to cause a shift in the centre surround balance of the cell output in favour of the centre for 82% of concentrically organized cells. These results are discussed in the context of present anatomical knowledge.

N-acetylaspartylglutamate identified in the rat retinal ganglion cells and their projections in the brain.

N-Acetylaspartylglutamate like immunoreactivity (NAAG-L) was identified in retinal ganglion cell bodies and their axons. The presence of the dipeptide in ganglion cell projection areas, the lateral geniculate nucleus (LGN) and superior colliculus (SC), was confirmed following NAAG purification from these tissues by a high-performance liquid chromatographic method. NAAG-L was identified in the optic tract as well as within fibers and puncta in the LGN and SC. The hypothesis that NAAG is present within ganglion cell axons in the brain was tested by unilateral enucleation which resulted in loss of NAAG and NAAG-L within the contralateral LGN and SC.

N-acetylaspartylglutamate immunoreactivity in neurons of the cat's visual system.

The acidic dipeptide, N-acetylaspartylglutamate (NAAG) was identified immunohistochemically within neurons of the cat's visual system. In the retina, NAAG-like immunoreactivity was observed in some horizontal and amacrine cells at the inner and outer margins of the bipolar cell layer. NAAG-like immunoreactivity was also observed in many retinal ganglion cell bodies, their neurites, and the neuropil of their target areas, the lateral geniculate nucleus (LGN) and the superior colliculus. Additionally, peptide immunoreactivity was also seen in the projection neurons of the LGN, in cells of the pulvinar nucleus, and in the pyramidal cells of layers III and V in areas 17, 18 and 19 of the cerebral cortex. These data suggest that NAAG or a structurally related molecule may have a prominent role in the communication of visual signals at retinal, thalamic and cortical levels.

Co-lamination of cholinergic amacrine cell and displaced ganglion cell dendrites in the chicken retina.

Displaced ganglion cells in the chicken retina were back-labelled with Fast blue injected into the nucleus of the basal optic root, then filled under visual control with Lucifer yellow to reveal the dendritic fields of the cells. In transverse sections, the dendrites of the displaced ganglion cells formed a narrow band in the outer part of the inner plexiform layer. Counter-staining for acetylcholinesterase (AChE) activity showed that the dendrites of the displaced ganglion cells and the type I cholinergic amacrine cells co-laminated in the outer part of the inner plexiform layer.

Substance P-like immunoreactive retinal terminals found in two retinorecipient areas of the Japanese monkey.

Using peroxidase-antiperoxidase immunocytochemistry, we demonstrated a dense accumulation of substance P-like (SP) immunoreactive terminals in the internal layer of the pregeniculate nucleus and in the pretectal olivary nucleus of the Japanese monkey. These distributions of immunostaining were similar to those for retinofugal terminals in the same nuclei. Bilateral eye enucleation markedly decreased SP immunoreactivity in the nuclei. The reduced immunoreactivity probably reflected the loss of SP-containing retinal ganglion cells that sent axons to the pregeniculate and pretectal olivary nuclei.