Displaced Ganglion Cells Research Articles

Melanopsin and calbindin immunoreactivity in the inner retina of humans and marmosets

In primate retina, the calcium-binding protein calbindin is expressed by a variety of neurons including cones, bipolar cells, and amacrine cells but it is not known which type(s) of cell express calbindin in the ganglion cell layer. The present study aimed to identify calbindin-positive cell type(s) in the amacrine and ganglion cell layer of human and marmoset retina using immunohistochemical markers for ganglion cells (RBPMS and melanopsin) and cholinergic amacrine (ChAT) cells. Intracellular injections following immunolabeling was used to reveal the morphology of calbindin-positive cells. In human retina, calbindin-labeled cells in the ganglion cell layer were identified as inner and outer stratifying melanopsin-expressing ganglion cells, and ON ChAT (starburst amacrine) cells. In marmoset, calbindin immunoreactivity in the ganglion cell layer was absent from ganglion cells but present in ON ChAT cells. In the inner nuclear layer of human retina, calbindin was found in melanopsin-expressing displaced ganglion cells and in at least two populations of amacrine cells including about a quarter of the OFF ChAT cells. In marmoset, a very low proportion of OFF ChAT cells was calbindin-positive. These results suggest that in both species there may be two types of OFF ChAT cells. Consistent with previous studies, the ratio of ON to OFF ChAT cells was about 70 to 30 in human and 30 to 70 in marmoset. Our results show that there are species-related differences between different primates with respect to the expression of calbindin.

The morphological characterization of orientation-biased displaced large-field ganglion cells in the central part of goldfish retina.

The vertebrate retina has about 30 subtypes of ganglion cells. Each ganglion cell receives synaptic inputs from specific types of bipolar and amacrine cells ramifying at the same depth of the inner plexiform layer (IPL), each of which is thought to process a specific aspect of visual information. Here, we identified one type of displaced ganglion cell in the goldfish retina which had a large and elongated dendritic field. As a population, all of these ganglion cells were oriented in the horizontal axis and perpendicular to the dorsal-ventral axis of the goldfish eye in the central part of retina. This ganglion cell has previously been classified as Type 1.2. However, the circuit elements which synapse with this ganglion cell are not yet characterized. We found that this displaced ganglion cell was directly tracer-coupled only with homologous ganglion cells at sites containing Cx35/36 puncta. We further illustrated that the processes of dopaminergic neurons often terminated next to intersections between processes of ganglion cells, close to where dopamine D1 receptors were localized. Finally, we showed that Mb1 ON bipolar cells had ribbon synapses in the axonal processes passing through the IPL and made ectopic synapses with this displaced ganglion cell that stratified into stratum 1 of the IPL. These results suggest that the displaced ganglion cell may synapse with both Mb1 cells using ectopic ribbon synapses and OFF cone bipolar cells with regular ribbon synapses in the IPL to function in both scotopic and photopic light conditions.

Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons.

Cryptochromes are ubiquitously expressed in various animal tissues including the retina. Some cryptochromes are involved in regulating circadian activity. Cryptochrome proteins have also been suggested to mediate the primary mechanism in light-dependent magnetic compass orientation in birds. Cryptochrome 1b (Cry1b) exhibits a unique carboxy terminus exclusively found in birds so far, which might be indicative for a specialised function. Cryptochrome 1a (Cry1a) is so far the only cryptochrome protein that has been localised to specific cell types within the retina of migratory birds. Here we show that Cry1b, an alternative splice variant of Cry1a, is also expressed in the retina of migratory birds, but it is primarily located in other cell types than Cry1a. This could suggest different functions for the two splice products. Using diagnostic bird-specific antibodies (that allow for a precise discrimination between both proteins), we show that Cry1b protein is found in the retinae of migratory European robins (Erithacus rubecula), migratory Northern Wheatears (Oenanthe oenanthe) and pigeons (Columba livia). In all three species, retinal Cry1b is localised in cell types which have been discussed as potentially well suited locations for magnetoreception: Cry1b is observed in the cytosol of ganglion cells, displaced ganglion cells, and in photoreceptor inner segments. The cytosolic rather than nucleic location of Cry1b in the retina reported here speaks against a circadian clock regulatory function of Cry1b and it allows for the possible involvement of Cry1b in a radical-pair-based magnetoreception mechanism.

Seasonally Changing Cryptochrome 1b Expression in the Retinal Ganglion Cells of a Migrating Passerine Bird.

Cryptochromes, blue-light absorbing proteins involved in the circadian clock, have been proposed to be the receptor molecules of the avian magnetic compass. In birds, several cryptochromes occur: Cryptochrome 2, Cryptochrome 4 and two splice products of Cryptochrome 1, Cry1a and Cry1b. With an antibody not distinguishing between the two splice products, Cryptochrome 1 had been detected in the retinal ganglion cells of garden warblers during migration. A recent study located Cry1a in the outer segments of UV/V-cones in the retina of domestic chickens and European robins, another migratory species. Here we report the presence of cryptochrome 1b (eCry1b) in retinal ganglion cells and displaced ganglion cells of European Robins, Erithacus rubecula. Immuno-histochemistry at the light microscopic and electron microscopic level showed eCry1b in the cell plasma, free in the cytosol as well as bound to membranes. This is supported by immuno-blotting. However, this applies only to robins in the migratory state. After the end of the migratory phase, the amount of eCry1b was markedly reduced and hardly detectable. In robins, the amount of eCry1b in the retinal ganglion cells varies with season: it appears to be strongly expressed only during the migratory period when the birds show nocturnal migratory restlessness. Since the avian magnetic compass does not seem to be restricted to the migratory phase, this seasonal variation makes a role of eCry1b in magnetoreception rather unlikely. Rather, it could be involved in physiological processes controlling migratory restlessness and thus enabling birds to perform their nocturnal flights.

Retinal projection to the pretectal nucleus lentiformis mesencephali in pigeons (Columba livia).

In birds, the nucleus of the basal optic root (nBOR) and the nucleus lentiformis mesencephali (LM) are retinal-recipient nuclei involved in the analysis of optic flow and the generation of the optokinetic response. The nBOR receives retinal input from displaced ganglion cells (DGCs), which are found at the margin of the inner nuclear and inner plexiform layers, rather than the ganglion cell layer. The LM receives afferents from retinal ganglion cells, but whether DGCs also project to LM remains unclear. To resolve this issue, we made small injections of retrograde tracer into LM and examined horizontal sections through the retina. For comparison, we also had cases with injections in nBOR, the optic tectum, and the anterior dorsolateral thalamus (the equivalent to the mammalian lateral geniculate nucleus). From all LM injections both retinal ganglion cells and DGCs were labeled. The percentage of DGCs, as a proportion of all labeled cells, varied from 2-28%, and these were not different in morphology or size compared to those labeled from nBOR, in which the proportion of DGCs was much higher (84-93%). DGCs were also labeled after injections into the anterior dorsolateral thalamus. The proportion was small (2-3%), and these DGCs were smaller in size than those projecting to the nBOR and LM. No DGCs were labeled from an injection in the optic tectum. Based on an analysis of size, we suggest that different populations of retinal ganglion cells are involved in the projections to LM, nBOR, the optic tectum, and the anterior dorsolateral thalamus.

Expression of voltage‐gated calcium channel α2δ4 subunits in the mouse and rat retina

High-voltage activated Ca channels participate in multiple cellular functions, including transmitter release, excitation, and gene transcription. Ca channels are heteromeric proteins consisting of a pore-forming α(1) subunit and auxiliary α(2)δ and β subunits. Although there are reports of α(2)δ(4) subunit mRNA in the mouse retina and localization of the α(2)δ(4) subunit immunoreactivity to salamander photoreceptor terminals, there is a limited overall understanding of its expression and localization in the retina. α(2)δ(4) subunit expression and distribution in the mouse and rat retina were evaluated by using reverse transcriptase polymerase chain reaction, western blot, and immunohistochemistry with specific primers and a well-characterized antibody to the α(2)δ(4) subunit. α(2)δ(4) subunit mRNA and protein are present in mouse and rat retina, brain, and liver homogenates. Immunostaining for the α(2)δ(4) subunit is mainly localized to Müller cell processes and endfeet, photoreceptor terminals, and photoreceptor outer segments. This subunit is also expressed in a few displaced ganglion cells and bipolar cell dendrites. These findings suggest that the α(2)δ(4) subunit participates in the modulation of L-type Ca(2+) current regulating neurotransmitter release from photoreceptor terminals and Ca(2+)-dependent signaling pathways in bipolar and Müller cells.

Cell bodies of the trigeminal proprioceptive neurons that transmit reflex contraction of the levator muscle are located in the mesencephalic trigeminal nucleus in rats

Since the levator and frontalis muscles lack interior muscle spindles despite being antigravity mixed muscles to involuntarily sustain eyelid opening and eyebrow lifting, this study has proposed a hypothetical mechanism to compensate for this anatomical defect. The voluntary contraction of fast-twitch fibres of the levator muscle stretches the mechanoreceptors in Müller's muscle to evoke proprioception, which continuously induces reflex contraction of slow-twitch fibres of the levator and frontalis muscles. This study confirmed the presence of cell bodies of the trigeminal proprioceptive neurons that transmit reflex contraction of the levator and frontalis muscles. After confirming that severing the trigeminal proprioceptive fibres that innervate the mechanoreceptors in Müller's muscle induced ipsilateral eyelid ptosis, Fluorogold was applied as a tracer to the proximal stump of the trigeminal proprioceptive nerve in rats. Fluorogold labelled the cell bodies of the trigeminal proprioceptive neurons, not in any regions of the rat brain including the trigeminal ganglion, but in the ipsilateral mesencephalic trigeminal nucleus neighbouring the locus ceruleus. Some Fluorogold particles accumulated in the area of the locus ceruleus. The trigeminal proprioceptive neurons could be considered centrally displaced ganglion cells to transmit afferent signal from the mechanoreceptors in Müller's muscle to the mesencephalon, where they may be able to make excitatory synaptic connections with both the oculomotor neurons and the frontalis muscle motoneurons for the involuntary coordination of the eyelid and eyebrow activities, and potentially to the locus ceruleus.

Regional distribution of nitrergic neurons in the inner retina of the chicken

Using both NADPH diaphorase and anti-nNOS antibodies, we have identified-from retinal flatmounts-neuronal types in the inner retina of the chicken that are likely to be nitrergic. The two methods gave similar results and yielded a total of 15 types of neurons, comprising 9 amacrine cells, 5 ganglion cells, and 1 centrifugal midbrain neuron. Six of these 15 cell types are ubiquitously distributed, comprising 3 amacrine cells, 2 displaced ganglion cells, and a presumed orthotopic ganglion cell. The remaining nine cell types are regionally restricted within the retina. As previously reported, efferent fibers of midbrain neurons and their postsynaptic partners, the unusual axon-bearing target amacrine cells, are entirely confined to the ventral retina. Also confined to the ventral retina, though with somewhat different distributions, are the "bullwhip" amacrine cells thought to be involved in eye growth, an orthotopic ganglion cell, and two types of large axon-bearing amacrine cells whose dendrites and axons lie in stratum 1 of the inner plexiform layer (IPL). Intracellular fills of these two cell types showed that only a minority of otherwise morphologically indistinguishable neurons are nitrergic. Two amacrine cells that branch throughout the IPL are confined to an equatorial band, and one small-field orthotopic ganglion cell that branches in the proximal IPL is entirely dorsal. These findings suggest that the retina uses different processing on different regions of the visual image, though the benefit of this is presently obscure.

Calretinin immunoreactivity in the developing retina of sharks: Comparison with cell proliferation and GABAergic system markers

The calcium-binding protein calretinin (CR) has been widely used as a marker of neuronal differentiation. In the present study we analyzed the distribution of CR-immunoreactive (CR-ir) elements in the embryonic and postembryonic retina of two elasmobranchs, the lesser spotted dogfish ( Scyliorhinus canicula) and the brown shyshark ( Haploblepharus fuscus). We compared the distribution of CR with that of a proliferation marker (the proliferating cell nuclear antigen, PCNA) in order to investigate the time course of CR expression during retinogenesis and explored the relationship between CR and glutamic acid decarboxylase (GAD), the synthesizing enzyme of the gamma-aminobutyric acid (GABA), which has been reported to play a role in shark retinogenesis. The earliest CR immunoreactivity was concurrently observed in subsets of: a) ganglion cells in the ganglion cell layer; b) displaced ganglion cells in the inner plexiform layer and inner part of the inner nuclear layer (INLi); c) amacrine cells in the INLi, and d) horizontal cells. This pattern of CR distribution is established in the developing retina from early stage 32, long after the appearance of a layered retinal organization in the inner retina, and coinciding with photoreceptor maturation in the outer retina. We also demonstrated that CR is expressed in postmitotic cells long after they have exited the cell cycle and in a subset of GABAergic horizontal cells. Overall our results provide insights into the differentiation patterns in the elasmobranch retina and supply further comparative data on the development of CR distribution in the retina of vertebrates. This study may help in understanding the possible involvement of CR in aspects of retinal morphogenesis.

Immunohistochemistry of Displaced Sensory Neurons in the Trigeminal Nerve Root

The aim of this study was to examine the morphology and the immunohistochemical features of displaced ganglion cells in the trigeminal nerve root (TNR). Forty human TNRs of 20 persons, obtained during routine autopsy in accordance with the Declaration of Helsinki, were examined following Klüver-Barrera and azan trichrome histological staining, and immunohistochemical reactions against certain neuronal markers, neuropeptides and neurotransmitters. A total number of 61 displaced neurons were investigated, which were present in 80% of individuals studied. Displaced neurons were found in 55.0% of the TNRs, either in the sensory portion (22.5%), motor portion (22.5%) or both (10.0%). Neuronal diameter varied from 12.5 × 25.0 to 45.0 × 63.7 (mean 27.6 × 41.6) µm, and in area between 245 and 2,065 (mean 927) µm². Each neuron was surrounded by 2–17 elongated satellite cells per slice. The immune reaction was positive in all the neurons studied for neuron-specific enolase, protein gene product 9.5, neurofilament protein and synaptophysin, and in some neurons for calcitonin gene-related peptide (CGRP; 24.4%), cholecystokinin (CCK; 13.3%), somatostatin (SST; 17.8%), substance P (SP; 15.6%), vasoactive intestinal polypeptide (4.4%), neuropeptide Y (8.9%), and serotonin (11.1%). The immune reactions were most frequent against the CGRP, SP, CCK and SST. We concluded that displaced neurons in the TNR morphologically and immunohistochemically resembled the sensory neurons in the trigeminal ganglion.

Immunocytochemical study of calretinin and calbindin D-28K expression in the retina of three cartilaginous fishes and a cladistian ( Polypterus)

The distribution of two calcium-binding proteins, calbindin D-28K (CB) and calretinin (CR) was studied in the retina of a cladistian, Polypterus senegalus, and three cartilaginous fishes ( Scyliorhinus canicula, Raja undulata and Torpedo marmorata). Western blot analysis of brain extracts revealed the lack of cross-reactivity of the used antibodies. In Polypterus, CB and CR immunoreactivities were observed in some amacrine and ganglion cells, but scarce cells showed CR/CB colocalization. Furthermore, CR immunoreactivity was present in a number of displaced bipolar cells and in some putative displaced ganglion cells, whereas CB immunoreactivity was found in some cones. No positive retinal structure was observed with the CB antibody used in cartilaginous fishes. Instead, CR was expressed in some amacrine, horizontal and ganglion cells of the dogfish and skate and, in some ganglion cells of the electric ray. The comparative analysis suggests, (1) the presence of CB-positive photoreceptor cells in the retina of cladistians seems to be apomorphic (in jawed fishes) in contrast with the plesiomorphic condition of this character in land vertebrates; (2) the presence of CR in amacrine and ganglion cells is a conserved feature along vertebrate phylogeny, whereas its variable expression in bipolar and horizontal cells represents a derived character; (3) the absence of CB in horizontal cells in cladistians could represent a derived character; and (4) the presence of CR displaced bipolar and putative displaced ganglion cells in Polypterus is shared with basal groups of actinopterygians.

Evoked Expression of the Glutamate Transporter GLT-1c in Retinal Ganglion Cells in Human Glaucoma and in a Rat Model

Glaucoma is a common disease of the eye, a key characteristic consequence of which is the death of retinal ganglion cells. The cause of this loss is unknown, though glutamate-mediated toxicity has been implicated. Glutamate transporters are key regulators of glutamate; therefore, the purpose of the study was to determine whether unusual excitation is associated with unusual expression of one or more transporters. The expression of a splice variant of the glutamate transporter GLT-1 (EAAT2) was examined in normal and glaucomatous retinas from humans and rats. In normal eyes of humans and rats, GLT-1c was expressed only in photoreceptors. In glaucoma, there was additional robust expression of GLT-1c in retinal ganglion cells, including occasional displaced ganglion cells. Conversely, cells such as displaced amacrine cells and amacrine cells were unlabeled. The induction of GLT-1c expression by retinal ganglion cells supports the notion that an anomaly or anomalies in glutamate homeostasis may be evident in glaucoma and that such anomalies selectively influence retinal ganglion cells. By analogy to in vitro experiments in which elevated glutamate levels induce expression of glutamate transporters, the authors hypothesize that expression of GLT-1c may represent an attempt by retinal ganglion cells to protect themselves against elevated levels of glutamate. Such anomalies in glutamate levels cannot be restricted to the ganglion cell layer, as this would not have affected displaced ganglion cells. GLT-1c may be a useful indicator of the extent of stress of the retinal ganglion cells and thus a tool for examining outcomes of potential therapeutic and experimental interventions.

Tracer coupling of neurons in the rat retina inner nuclear layer labeled by Fluorogold

A subpopulation of neurons in the inner nuclear layer (INL) of the rat retina were labeled 9–13 weeks after application of Fluorogold (FG) to the superior colliculus. Neurobiotin injection of FG-labeled cells in the INL of flatmounted living retina revealed that these cells consisted of both displaced ganglion cells and a subset of amacrine cells. Fluorogold-labeled amacrine cells in the INL showed tracer coupling to other presumptive amacrine cells in the INL, but there was no evidence of coupling to neurons in the ganglion cell layer (GCL). As the labeling of amacrine cells by FG may be due to gap junction coupling between ganglion and amacrine cells, these data add to the evidence that tracer coupling between these cells can be unidirectional. Some of the FG-labeled displaced ganglion cells in the INL injected with Neurobiotin also showed tracer coupling to neurons in the INL or GCL.

The effect of retinal growth on the postnatal development and distribution of displaced retinal ganglion cells in the retina of the chameleon (squamata)

Retinal ganglion cells (RGCs) usually increase their dendritic field area with postnatal retinal growth. The mechanisms that regulate the postnatal shape of dendritic arbors in the growing retina are not well understood. Quantitative studies suffer from the difficulty of labeling specific subpopulations of RGCs selectively including their dendritic processes. In this study, we labeled displaced retinal ganglion cells (DGC) that are known to project to the accessory optic system (AOS) in juvenile and adult chameleons by retrograde transport of dextran amines. The complete population of DGCs was quantitatively screened for the effects of postnatal retinal growth on cell morphology, dendritic field coverage, and dendritic arbor size. The adult eye contained 2000 DGCs/retina. This number was already present at birth. The smaller size of the hatchling eye (approximately 1/3 of the adult size) led to higher densities of DGCs. The greatest accumulation of juvenile DGCs (two-fold higher compared to the adult) was found in the periphery of the retina where the greatest surface expansion was observed. DGC dendritic field areas were adjusted proportionally to this expansion in order to maintain a constant dendritic coverage. The increase of dendritic fields was mediated by two putative passive mechanisms: First, an elongation of individual dendrites similar to previous reports of postnatal RGC development in the retina of goldfish and chicks. Second, and more prominent, we observed that neighboring dendrites were pulled apart from each other. This resulted in a looser spacing of the initially tightly packed dendrites of each dendritic arbor. This dispersal of dendrites over a larger area was, due to its passive nature, proportional to the increase of the retinal surface and preserved a constant dendritic coverage irrespective of the animal's age and eye size.

Retinal ganglion cells projecting to the optic tectum and visual thalamus of lizards.

Retinal ganglion cells projecting to the optic tectum and visual thalamus have been investigated in the lizard, Podarcis hispanica. Injections of biotinylated dextran-amine in the optic tectum reveal seven morphological cell varieties including one displaced ganglion cell type. Injections in the visual thalamus yield similar ganglion cell classes plus four giant ganglion cells, including two displaced ganglion cell types. The present study constitutes the first comparison of tectal versus thalamic ganglion cell types in reptiles. The situation found in lizards is similar to that reported in mammals and birds where some cell types projecting to the thalamus are larger than those projecting to the mesencephalic roof. The presence of giant retino-thalamic ganglion cells with specific dendritic arborizations in sublaminae A and B of the inner plexiform layer suggests that parts of the visual thalamus of lizards could be implicated in movement detection, a role that might be played by the ventral lateral geniculate nucleus, which is involved in our tracer injections.

Calbindin and calretinin immunoreactivities in the retina of a chondrostean, Acipenser baeri.

The distribution of calbindin and calretinin in the retina of the sturgeon Acipenser baeri was studied with immunocytochemistry. Western blot analysis of brain extracts, together with immunocytochemical results in the retina and brain, indicated the presence of the two calcium-binding proteins in sturgeon. Calbindin immunocytochemistry revealed only a large displaced bipolar cell type with narrowly stratified axons, similar to some mixed rod and cones bipolar cells described in teleosts. The plexus formed by the axons of these cells in the inner plexiform sublayer was similar to that formed by calbindin-immunoreactive diffuse bipolar cells of some mammals. Calretinin immunocytochemistry also stained these displaced bipolar cells, most ganglion cells including displaced ganglion cells (Dogiel cells), and some amacrine cells of the inner nuclear layer. The distribution of calbindin and calretinin immunoreactivities in the retina of a primitive bony fish indicates that these proteins are highly specific to the cell type.

Displaced retinal ganglion cells project to the accessory optic system in the chameleon ( Chamaeleo calyptratus).

Retinal ganglion cells were successfully labelled in the chameleon by retrograde axonal transport of dextran amines that were applied to the nucleus of the basal optic root (nBOR) in an in vitro preparation. Labelled ganglion cells were restricted to the contralateral eye. Many cells were completely stained including their dendritic trees. With few exceptions, all cells had displaced somata that were located at the inner margin of the inner nuclear layer. The labelled ganglion cells had two to six primary dendrites that branched frequently and formed large unistratified dendritic trees within sublamina 1 of the inner plexiform layer. There was extensive overlap of the dendritic trees of neighbouring cells leading to an estimated coverage factor of 2-4. The dendritic field areas varied in size according to the retinal position of the cells and were highest in the central retina around the fovea with a maximum of 0.14 mm(2) and reached a second maximum at the retinal margin with values of 0.08-0.1 mm(2). The smallest dendritic areas (0.04-0.06 mm(2)) were measured midway between the fovea and retinal margin. The size of the soma area was not correlated to the dendritic field size and increased from 100 to 150 microm(2) near the fovea to 150-300 microm(2) at the retinal margin. There was no evidence for a retinotopic organisation of ganglion cell fibres within the nBOR. All cells were of uniform morphology that was identical to the type of nBOR-projecting displaced ganglion cell (DGC) described previously for the bird retina. Similar to birds, the labelled DGCs were the only source of retinal projection to the nBOR. A small fraction of cells had orthotopic somata located in the ganglion cell layer but were otherwise identical to the labelled DGCs. The similarity of chameleon nBOR-projecting ganglion cells to those described in avian retinas mirrors the close phylogenetic relationship of birds and lizards.

Evidence for spatial regularity among retinal ganglion cells that project to the accessory optic system in a frog, a reptile, a bird, and a mammal

The vertebrate retina contains only five major neuronal classes but these embrace a great diversity of discrete types, many of them hard to define by classical methods. Consideration of their spatial distributions (mosaics) has allowed new types, including large ganglion cells, to be resolved across a wide range of vertebrates. However, one category of large ganglion cells has seemed refractory to mosaic analysis: those that project to the accessory optic system (AOS) and serve vestibulocerebellar mechanisms of motion detection and image stabilization. Whenever AOS-projecting cells have been analyzed by nearest-neighbor methods, their distribution has appeared almost random. This is puzzling, because most aspects of visual processing require the visual scene to be sampled regularly. Here, spatial correlogram methods are applied to distributions of large ganglion cells, labeled retrogradely from the AOS in frogs, turtles, and rats, and to the AOS-projecting displaced ganglion cells of chickens. These methods reveal hidden spatial order among AOS-projecting populations, of a form that can be simulated either by superimposing a single regular mosaic on a random population or, more interestingly, by overlapping three or more regular, similar but spatially independent mosaics. The rabbit is known to have direction-selective ganglion cells (not, however, AOS projecting) that can be subdivided into functionally distinct, regular mosaics by their tracer-coupling patterns even though they are morphologically homogeneous. The present results imply that the direction-selective AOS-projecting ganglion cells of all vertebrates may, likewise, be subdivided into regular, independent mosaics.

Neuronal and glial cell types revealed by NADPH-diaphorase histochemistry in the retina of a teleost fish, the grass goby (Zosterisessor ophiocephalus, Perciformes, Gobiidae).

The grass goby is a mud-burrowing fish with a rich retinal vasculature appropriate to its hypoxic habitat. NADPH-diaphorase histochemistry was performed on retinal sections and wholemounts to reveal cells that contain nitric oxide synthase and so may be presumed to synthesise nitric oxide, a gaseous intercellular messenger with many roles including vasodilation. Structures that were consistently stained by this method included cone ellipsoids, horizontal cells, Müller cells and their processes, large displaced ganglion cells in the inner nuclear layer (identified by their axons), large interstitial ganglion cells in the inner plexiform layer, and capillary endothelial cells. In wholemounts, horizontal cells were seen to form a regular pattern, contacting each other at their dendritic terminals. Some cells in the ganglion cell layer were weakly stained, but stained bipolar and amacrine cells were not seen. The diaphorase-positive large ganglion cells all formed large, sparsely branched dendritic trees, arborizing near the scleral border of the inner plexiform layer. The displaced and interstitial cells seemed to belong to distinct morphological types, the interstitial cells having smaller somata and trees. Analysis of their spatial distributions in one representative retina confirmed this: the displaced cells formed a highly regular mosaic with a mean spacing (nearest-neighbour distance) of 303 microm, whereas the interstitial cells formed a separate mosaic, almost as regular but with a smaller mean spacing of 193 microm, rising to 217 microm in a sample that excluded the area retinae temporalis. Spatial correlogram analysis showed that these two mosaics were spatially independent. Nitric oxide probably has many roles in the retina. The presence of its synthetic enzyme in Müller cells, which communicate with retinal blood vessels, is consistent with a role in the control of retinal blood flow. Its function in large, mosaic-forming retinal ganglion cells is unknown.

Neurogenesis of cholinoceptive neurons in the chick retina

Immunocytochemistry and [3H]thymidine autoradiography were combined in this study to determine the neurogenesis of cholinoceptive cells in the chick retina. After injections of [3H]thymidine between embryonic days 1 and 11, the time of birth of retinal neurons containing either the α3 or the α8 subunit of the nicotinic acetylcholine receptors was determined. The results indicate that the α3-positive neurons in the ganglion cell layer leave the cell cycle from E2 through E7, and those in the inner nuclear layer (amacrine and displaced ganglion cells) from E2 through E9. The α8-positive cells in the ganglion cell layer were born from E1 through E7, and those in the inner nuclear layer (amacrine and bipolar cells) from E2 through E11. These data suggest that the time of birth of cholinoceptive neurons in the chick retina follows the general pattern of cell generation in the chick retina, and that α8-positive cells in the ganglion cell layer start to leave the cell cycle almost one day earlier than the α3-positive cells in the same layer.