Localization of glycine-containing neurons in the Macaca monkey retina

Autoradiography following 3H-glycine (Gly) uptake and immunocytochemistry with a Gly-specific antiserum were used to identify neurons in Macaca monkey retina that contain a high level of this neurotransmitter. High-affinity uptake of Gly was shown to be sodium dependent whereas release of both endogenous and accumulated Gly was calcium dependent. Neurons labeling for Gly included 40-46% of the amacrine cells and nearly 40% of the bipolars. Synaptic labeling was seen throughout the inner plexiform layer (IPL) but with a preferential distribution in the inner half. Bands of labeled puncta occurred in S2, S4, and S5. Both light and postembedding electron microscopic (EM) immunocytochemistry identified different types of amacrine and bipolar cell bodies and their synaptic terminals. The most heavily labeled Gly+ cell bodies typically were amacrine cells having a single, thick, basal dendrite extending deep into the IPL and, at the EM level, electron-dense cytoplasm and prominent nuclear infoldings. This cell type may be homologous with the Gly2 cell in human retina (Marc and Liu: J. Comp. Neurol. 232:241-260, '85) and the AII/Gly2 of cat retina (Famiglietti and Kolb: Brain Res. 84:293-300, '75; Pourcho and Goebel: J. Comp. Neurol. 233:473-480, '85a). Gly+ amacrines synapse most frequently onto Gly- amacrines and both Gly- and Gly+ bipolars. Gly+ bipolar cells appeared to be cone bipolars because their labeled dendrites could be traced only to cone pedicles. The pattern of these labeled dendritic trees indicated that both diffuse and midget types of biopolars were Gly+. The EM distribution of labeled synapses showed Gly+ amacrine synapses throughout the IPL, but these composed only 11-23% of the amacrine population. Most of the Gly+ bipolar terminals were in the inner IPL, where 70% of all bipolar terminals were labeled. These findings are consistent with previous data from cats and humans and suggest that both amacrine and bipolar cells contribute to glycine-mediated neurotransmission in the monkey retina.     

Glycinergic neurons in the human retina

Neurotransmitter-specific properties of glycinergic neurons in the human retina were studied using 11 pairs of eyes from donors ranging from 2 1/2 to 54 years in age. A mean endogenous level of 10.3 nmoles glycine per mg protein was measured by amino acid analysis in retinas isolated within 1 hour postmortem. When retinas were incubated with 3H-glycine (2 microM) and processed for autoradiography, label was found associated with neurons whose somata reside within the inner nuclear layer. Some heavily labeled neurons located at the vitread border of the inner nuclear layer were identified as amacrine cells based on ultrastructural verification of the conventional synaptic contacts made by their processes in distal regions of the inner plexiform layer. In proximal regions of the inner plexiform layer, dendrites of glycine-accumulating amacrine cells were postsynaptic to both ribbon and conventional synaptic contacts, suggesting input from bipolar and other, nonglycinergic amacrine cells. Their density (30 +/- 11 S.D. cells/mm linear retinal expanse) tended to be greater toward the central fundus. A second population of lightly labeled, probable bipolar cells was present in the middle of the inner nuclear layer; the density of this second set of glycine-accumulating cells approximated that of the heavily labeled population from the fovea, centrally, to the ora serrata, peripherally. Release of either accumulated or endogenous glycine was elicited by K+-depolarization in a Ca2+-dependent manner. Tissue fragments exposed for 6 minutes to normal medium, 40 mM K+-substituted medium, or K+-substituted medium with Co2+, release endogenous glycine into each bathing solution in average amounts of 0.6, 2.6, and 0.7 nmoles per mg protein, respectively. Together these data strongly implicate glycine as a neurotransmitter in the human retina.     

Dopaminergic neurons in the human retina

The utilization of dopamine in the adult human retina was examined by using high-affinity uptake, localization, synthesis, and release as neurotransmitter-specific physiological probes. Autoradiographic and histochemical studies have shown that dopamine-accumulating and dopamine-containing cells of the human retina belong to a population of neurons whose somata are located in the proximal regional of the inner nuclear layer. Some of these are amacrine cells which are pre- and postsynaptic to other amacrine cells exclusively in the inner plexiform layer. However, evidence is presented which indicates the existence of interplexiform dopaminergic neurons which send processes to both plexiform layers of the retina. These neurons contain a high concentration of dopamine, take up 3H-dopamine by a hig-affinity mechanism, and release endogenous or accumulated dopamine by a Ca2+-dependent mechanism upon depolarization with high extracellular K+. An endogeneous level of about 20 pmoles dopamine per mg protein was measured in freshly isolated retina using high-pressure liquid chromatography with electrochemical detection. These results demonstrate that mechanisms for dopaminergic neurotransmission are present in the human retina.     

Substance P-immunoreactive neurons in the human retina

Substance P (SP) is a neuropeptide that acts as a neurotransmitter or a neuromodulator in the retina. The aim of this study was to identify the type(s) and the distribution of the SP-immunoreactive (SP-IR) cells in the human retina. We have used an antiserum to SP to immunostain neurons in postmortem human retinae. Immunostained retinae were processedwith the avidin-biotin complex (ABC) to visualize the cells either whole mounted in glycerol or embedded in plastic. Some retinae were also sectioned at 20 μm in order to obtain radial views of stained cells. SP-IR amacrine cells stain intensely and appear to be of a single type in the human retina. They are large-field cells with large cell bodies (16 μm diameter) lying in normal or displaced positions on either side of the inner plexiform layer (IPL). Their sturdy, spiny, and appendage-bearing dendrites stratify in stratum 3 (S3) of the IPL, where many overlapping, fine dendrites intermingle to form a plexus of stained processes. Either cell bodies or primary dendrites emit an “axon-like” process that, typically, divides into two long, fine processes, which run in opposite directions for hundreds of micrometers in S5 and S3 before disappearing as distinct entities in the stained plexus in S3. Long, fine dendrites also pass from the dendritic plexus to run in S5 and down to the nerve fiber layer to end as large varicosities at blood vessel walls. In addition, fine processes are emitted from the dendritic plexus that runs in S1, and some pass up to the outer plexiform layer (OPL) to run therein for short distances. The SP-IR amacrine cell has many similaritiesto the thorny, type 2 amacrine cells described from Golgi studies. In addition to the SP-IR amacrine cells, a presumed ganglion cell type is faintly immunoreactive. Its 20–22 μpm cell body gives rise to a radiate, sparsely branched, widespreading dendritic tree running in S3. Its dendrites and cell body become enveloped by the more intensely SP-IR processes and boutons from the SP-IR amacrine cell type. The SP-IR ganglion cell type most resembles G21 from a Golgi study. © 1995 Wiley-Liss, Inc.     

Localization of GABA, glycine, glutamate and tyrosine hydroxylase in the human retina.

A light microscope study using postembedding immunocytochemistry techniques to demonstrate the common neurotransmitter candidates gamma-aminobutyric acid (GABA), glycine, glutamate, and tyrosine hydroxylase for dopamine has been done on human retina. By using an antiserum to GABA, we found GABA-immunoreactivity (GABA-IR) to be primarily in amacrine cells lying in the inner nuclear layer (INL) or displaced to the ganglion cell layer (GCL). A few stained cells in the INL, which are probably interplexiform cells, were observed to project thin processes towards the outer plexiform layer (OPL). There were heavily stained bands of immunoreactivity in strata 1, 3 and 5 of the inner plexiform layer (IPL). An occasional ganglion cell was also GABA-IR. By using an antiserum to glycine, stained cells were observed at all levels of the INL. Most of these were amacrines, but a few bipolar cells were also glycine-IR. Displaced amacrine cells and large-bodied cells, which are probably ganglion cells, stained in the GCL. The bipolar cells that stained appeared to include both diffuse and midget varieties. The AII amacrine cell of the rod pathway was clearly stained in our material but at a lower intensity than two other amacrine cell types tentatively identified as A8 and A3 or A4. Again, there was stratified staining in the IPL, with strata 2 and 4 being most immunoreactive. An antiserum to glutamate revealed that most of the neurons of the vertical pathways in the human retina were glutamate-IR. Rod and cone photoreceptor synaptic endings labeled as did the majority of bipolar and ganglion cells. The rod photoreceptor stained more heavily than the cone photoreceptor in our material. While both midget and diffuse cone bipolar cell types were clearly glutamate-IR, rod bipolars were not noticeably stained. The most strongly staining glutamate-IR processes of the IPL lay in the outer half, in sublamina a. The antiserum to tyrosine hydroxylase (TOH) revealed two different amacrine cell types. Strongly immunoreactive cells (TOH1) had their cell bodies in the INL and their dendrites ramified in a dense plexus in stratum 1 of the IPL. Fine processes arising from their cell bodies or from the stratum 1 plexus passed through the INL to reach the OPL but did not produce long-ranging ramifications therein. The less immunoreactive amacrines (TOH2) lay in the INL, the center of the IPL or the GCL and emitted thick dendrites that were monostratified in stratum 3 of the IPL.     

(3H) glycine-accumulating neurons of the human retina

Isolated human retinas were incubated in physiological saline containing micromolar (3H) glycine. The types, distributions, and synaptologies of glycine-accumulating neurons were determined by light and electron microscope autoradiography. Two types of amacrine cells were discriminated on the bases of number of processes descending into the inner plexiform layer, density of label in light-microscope autoradiographs, size, and synaptic features: (1) Gly1 amacrine cells have moderate labeling, several oblique dendrites arising from the soma, and electron lucent synaptic terminals containing large presynaptic specializations, nd (2) Gly2 amacrine cells have dense labeling, a single proximal dendrite, and moderately electron-dense terminals with small presynaptic specializations. Gly1 amacrine cells constitute approximately 15% and Gly2 amacrine cells approximately 38% of all cells in the amacrine cell layer. The laminar distribution of label in the inner plexiform layer was measured by scanning microdensitometry, which provided a format for categorizing types of synaptic contacts. Many features of glycine-accumulating amacrine cell contacts were similar to those of cat AII/Gly2 amacrine cells: a diffuse yet bisublaminar distribution of label, concentration of synaptic output in sublamina a, rod bipolar cell input in sublamina b and gap junctions in mid-inner plexiform layer involving labeled cells. The evidence seems to indicate that human Gly2 amacrine cells and cat AII/Gly2 amacrine cells are homologous cell types. finally, some cone bipolar cells were labeled.     

Localization of putative GABAergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographic methods

Putative GABAergic neurons in the larval tiger salamander retina were localized by a comparative analysis of glutamate decarboxylase immunoreactivity (GAD-IR), GABA-like immunoreactivity (GABA-IR), and high-affinity 3H-GABA uptake at the light microscopical level. Preliminary data showed that all GAD-IR neurons were double labeled for GABA-IR. However, because the weak somatic labeling with GAD-IR, we could not determine if the converse were true. Neurons commonly labeled with GABA-IR and 3H-GABA uptake include horizontal cells, type I (outer) and type II (inner) bipolar cells, type I (inner) and type II (outer) amacrine cells, and cell bodies in the ganglion cell layer (GCL). In addition, interplexiform cells were identified with GABA-IR. The presence of GABA-IR ganglion cells was indicated by GABA-IR fibers in the optic fiber layer and optic nerve as well as by a GABA-IR cell in the GCL that included a labeled axon. The percentage of labeled somas in the inner nuclear layer (INL) compared to all cells in each layer was similar for the two methods: 30% in INL 1 (outer layer of somas), 15% in INL 2 (middle layer), 43-52% in INL 3 (inner layer), and about 21-26% in the GCL. Labeled processes were found in three bands in the inner plexiform layer, with the densest band located in the most proximal part. Postembedding labeling of 1-micron Durcupan resin sections for GABA-IR showed the same general pattern as obtained with 10-microns cryostat sections, with additional staining, however, of type II (inner) bipolar cell Landolt's clubs. Extensive colocalization of labeling was indicated, and we conclude that GABA-IR can serve as a valid and reliable marker for GABA-containing neurons in this retina and suggest that GABA serves as a transmitter for horizontal cells, several types of amacrine cell, a type of interplexiform cell, and perhaps a small percentage of type I and type II bipolar cells and ganglion cells.     

Localization and regulation of vasopressin mRNA in human neurons

Vasopressin (prepropressophysin) mRNA is detected in neurons of the supraoptic, suprachiasmatic, and paraventricular nuclei of human postmortem hypothalamic specimens by quantitated in situ hybridization using 35S-labeled single-stranded cDNA probes directed against exon C of the human vasopressin gene. This hybridization displays the anticipated anatomic distribution, as well as several biochemical features supporting its specificity. Hybridization densities in supraoptic neurons, a measure of vasopressin gene expression, display substantial variability from brain-to-brain. We can attribute much of this brain-to-brain variability to differences in antemortem extracellular volume status. This conclusion is based on a) animal models of the human postmortem process, b) animal models of common agonal events, c) good correlations between antemortem volume status and neuronal vasopressin mRNA hybridization densities in human postmortem specimens matched for age and postmortem interval, and d) our inability to correlate human neuronal vasopressin mRNA hybridization densities with other clinical and postmortem features. These results provide an example of antemortem regulation of a human neuroendocrine gene using postmortem tissue.     

Localization of nyctalopin in the mammalian retina

Complete X-linked congenital stationary night blindness (CSNB1) is a hereditary visual disease characterized by abnormalities in both the dark- and light-adapted electroretinogram, consistent with a defect in synaptic transmission between photoreceptors and ON-bipolar cells. The gene responsible for CSNB1, NYX, encodes a novel, leucine-rich repeat protein, nyctalopin. Consistent with its predicted glycosylphosphatidylinositol linkage, we show that recombinant nyctalopin is targeted to the extracellular cell surface in transfected HEK293 cells. Within the retina, strong nyctalopin immunoreactivity is present in the outer plexiform layer, the site of the photoreceptor to bipolar cell synapses. Double labelling of nyctalopin and known synaptic proteins in the outer plexiform layer indicate that nyctalopin is associated with the ribbon synapses of both rod and cone terminals. In the inner plexiform layer, nyctalopin immunoreactivity is associated with rod bipolar cell terminals. Our findings support a role for nyctalopin in synaptic transmission and/or synapse formation at ribbon synapses in the retina.     

Localization of preproenkephalin A mRNA in the neonatal rat retina.

An opioid growth factor, [Met5]-enkephalin, is known to regulate developmental events in the neonatal rat retina. This growth factor interacts with the zeta (zeta) opioid receptor to modulate retinal ontogeny. Both peptide and receptor are present in developing retina, but not in adult retina. We have used in situ hybridization histochemistry to identify and localize preproenkephalin A mRNA in the neonatal rat retina. Preproenkephalin mRNA was localized to the ganglion cell layer, with some radiolabeling found in the neuroblast layer. This result indicates that 1) the mRNA to preproenkephalin A is present during the critical stage of development in the neonatal retina that coincides with the presence of the growth-regulating peptide, [Met5]-enkephalin, and 2) that the source of the opioid growth factor controlling the production of retinal cells appears to be autocrine (i.e., retinal neuroblasts) and paracrine (i.e., ganglion cells) in nature.     

Synaptic organization of the dopaminergic neurons in the rabbit retina.

A number of substances were tested for their ability to label amine-accumulating neurons in the rabbit retina after fixation with OsO₄ or glutaraldehyde and OsO₄. Useful results were obtained with 5,6-dihydroxytryptamine (5,6-DHT) and 6-hydroxydopamine (6-HDA). Labelled processes were characterized by small (40–50 mm) pleomorphic synaptic vesicles containing electron-dense cores, and at times by swelling of mitochondria and by increased electron density of membranes and cytoplasm. Fluorescence microscopy showed that 5,6-DHT labelled both dopaminergic and indoleamine-accumulating neurons. In most experiments, therefore, the indoleamine-accumulating neurons were removed with 5,7-dihydroxytryptamine. In such retinas the dopaminergic processes labelled by 5,6-DHT were found to make synapses of the conventional type, characterized by an accumulation of synaptic vesicles on the presumed presynaptic side and some aggregation of material on the cytoplasmic side of the synaptic membranes and within the synaptic cleft. The dopaminergic processes were found to contact each other and also non-dopaminergic amacrine cells and their processes. Conventional synapses onto dopaminergic processes were observed from both labelled and unlabelled amacrine processes. The input from labelled neurons was observed on varicose dopaminergic processes whereas input from non-labelled elements was found on the intervaricose parts of the dopaminergic processes. No Contacts of dopaminergic processes with bipolar or ganglion cells were observed. Injections of 6-HDA gave the same results, although this drug gave less distinct labelling which made the observations less decisive than with 5,6-DHT. In retinas treated with 5,6-DHT alone (i.e., in which the indoleamine-accumulating neurons remained) numerous processes were observed which were both pre- and postsynaptic to bipolar terminals. These observations suggest that the indoleamine-accumulating processes synapse with bipolar cells. The results show that the dopaminergic neurons form a network involving only amacrine cells, suggesting a regulatory function for them. By analogy with the dopaminergic interplexiform cells of the goldfish retina, it is suggested that the dopaminergic neurons in the rabbit may regulate lateral inhibitory effects mediated by amacrine cells. Furthermore, the finding that the dopaminergic and indoleamine-accumulating cells apparently have a different synaptic organization suggests that it is appropriate to categorize amacrine cells according to their transmitter content as well as their morphology.     

NADPH-diaphorase neurons in the retina of the hamster

NADPH-diaphorase-positive neurons have been demonstrated in the inner nuclear layer and ganglion cell layer of the retina of different mammalian species, but so far no experiments have been conducted to identify whether these cells are amacrine cells and/or retinal ganglion cells. We attempted to solve this problem by studying the NADPH-diaphorase-positive neurons in the hamster retina. From the NADPH-diaphorase histochemical reaction, two distinct types of neurons in the hamster retina were identified. They were named ND(g) and ND(i) cells. The ND(g) cells were cells with larger somata, ranging from 10 to 21 μm in diameter with a mean of 15.58 μm (S.D.= 2.59). They were found in the ganglion cell layer only. The ND(i) cells were smaller, with the somata ranging from 7 to 11 μm and having the mean diameter of 8.77 μm (S.D. = 1.24). Most of the ND(i) cells were found in the inner nuclear layer, and only very few could be observed in the inner plexiform layer. On average, there were 8,033 ND(g) and 5,051 ND(i) cells in the ganglion cell layer and inner nuclear layer, respectively. Two experiments were performed to clarify whether any of the NADPH-diaphorase neurons were retinal ganglion cells. Following unilateral optic nerve section, which leads to the retrograde degeneration of retinal ganglion cells, the numbers of both ND(g) and ND(i) cells did not change significantly for up to 4 months. In addition, when retinal ganglion cells were prelabeled retrogradely (horseradish peroxidase of flurescent microspheres) and retinas were then stained for NADPH diaphorase, no double-labeled neurons were detected. These results indicated that the NADPH-diaphorase neurons in the hamster retina were the amacrine cells in the inner nuclear layer and displaced amacrine cells in the ganglion cell layer. Dendrites of the ND(g) and ND(i) cells were found to stratify in sublaminae 1, 3, and 5 of the inner plexiform layer, with a prominent staining in the sublamina 5. The possible importance of this arrangement in the rod pathway is also discussed. © 1994 Wiley-Liss, Inc.     

Somatostatin-immunoreactive neurons in the adult rabbit retina

Somatostatin (SRIF) is a neuroactive peptide that is distributed throughout the nervous system, including the retina. This peptide has been localized to populations of amacrine cells in a variety of vertebrate species. In the rabbit retina, SRIF immunoreactivity is present in a sparse population of medium to large neurons (13.72 μm in diameter, or 147.84 μ²) in the ganglion cell layer and in a small number of neurons in the inner nuclear layer. These cells display a preferential distribution to the inferior retina, with the highest density near the ventral and ventrolateral retinal margins (11.33 cells/mm²). SRIF-immunoreactive cells have two to five primary processes that arborize in the proximal inner plexiform layer (IPL). These give rise to a plexus of finer processes in the distal IPL. Occasional immunoreactive processes are also present in the outer plexiform layer. In the IPL, these laminar networks are present in all retinal regions. In addition, SRIF-immunoreactive cells often have a fine-caliber axonlike process that eminates from the soma or perisomal region. These processes travel for great distances across the retina in either the nerve fiber layer or in the distal IPL but are never seen to enter the optic nerve head. In addition, the number of SRIF-immunoreactive somata remains unchanged following transection of the optic nerve. Taken together, these data indicate that SRIF-immunoreactive neurons of the rabbit retina are displaced amacrine cells. Furthermore, the sparse distribution of SRIF-immunoreactive somata, the wide-ranging, asymmetric arborization of their cellular processes, and previous pharmacological studies suggest that these neurons mediate a broad modulatory role in retinal function. © 1996 Wiley-Liss, Inc.     

Localization of nitric oxide synthase in the goldfish retina

The localization of nitric oxide synthase and NADPH-diaphorase was studied in the goldfish retina by means of immunohistochemistry or tetrazolium salt technique. Nitric oxide synthase was found in some small neurons of the inner nuclear layer and in large neurons of the ganglion cell layer. The reaction product was localized in the outer plexiform layer and a diffuse labeling was also observed in the inner plexiform layer. In addition to the outer segments of photoreceptors, NADPH-diaphorase labeled several neurons of the inner nuclear layer and some neurons scattered in the ganglion cell layer. Both outer and inner plexiform layers were labeled. Ultrastructural observations showed that the reaction product was found to be bound to the endoplasmic membranes of positive neurons. In the outer plexiform layer the formazan precipitate labeled prevailingly the presynaptic terminals of rods and cones, in the inner plexiform layer both pre- and postsynaptic profiles showed the reaction product.     

Localization of adenylyl cyclase proteins in the rodent retina

The isoforms of adenylyl cyclase that mediate cyclic AMP signaling pathways in the retina are, for the most part, unknown. Therefore, the protein expression patterns of adenylyl cyclase isoforms in the rodent retina were characterized immunocytochemically using antibodies directed against Ca(2+)-stimulated (AC1, AC3 and AC8), Ca(2+)-inhibited (AC9) and Ca(2+)-insensitive (AC2, AC4, AC7) isoforms of adenylyl cyclase. The ganglion cell layer and the inner nuclear layer (INL) were immunoreactive for both Ca(2+)-sensitive (AC1, AC3) and Ca(2+)-insensitive (AC2, AC4) isoforms of adenylyl cyclase. Antibodies against isoforms from all three classes of adenylyl cyclase labeled the inner plexiform layer. In the outer retina, antibodies against Ca(2+)-insensitive isoforms labeled photoreceptors and the outer plexiform layer (OPL). Radial elements in the ONL and INL were AC4-immunoreactive and the nerve fibre layer and optic nerve were AC2-, AC4- and AC9-immunoreactive. Antibodies against AC7 did not label rodent neural retina. These data indicate that there is a heterogeneous distribution of adenylyl cyclase isoforms throughout the rodent retina. Nonetheless, there is a general indication of a greater expression of Ca(2+)-insensitive adenylyl cyclase isoforms in the outer retina, particularly within photoreceptors.     

Characterization of glucagon-expressing neurons in the chicken retina

We recently identified large glucagon-expressing neurons that densely ramify neurites in the peripheral edge of the retina and regulate the proliferation of progenitors in the circumferential marginal zone (CMZ) of the postnatal chicken eye (Fischer et al. [2005] J Neurosci 25:10157-10166). However, nothing is known about the transmitters and proteins that are expressed by the glucagon-expressing neurons in the avian retina. We used antibodies to cell-distinguishing markers to better characterize the different types of glucagon-expressing neurons. We found that the large glucagon-expressing neurons were immunoreactive for substance P, neurofilament, Pax6, AP2alpha, HuD, calretinin, trkB, and trkC. Colocalization of glucagon and substance P in the large glucagon-expressing neurons indicates that these cells are the "bullwhip cells" that have been briefly described by Ehrlich et al. ([1987] J Comp Neurol 266:220-233). Similar to the bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for calretinin, HuD, Pax6, and AP2alpha. Unlike bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for GABA. While glucagon-immunoreactive amacrine cells were negative for substance P in central regions of the retina, a subset of this type of amacrine cell was immunoreactive for substance P in far peripheral regions of the retina. An additional type of glucagon/substance P-expressing neuron, resembling the bullwhip cells, was found in far peripheral and dorsal regions of the retina. Based on morphology, distribution within the retina, and histological markers, we conclude that there may be four different types of glucagon-expressing neurons in the avian retina.     

Age-related alterations in neurons of the mouse retina

The behavioral consequences of age-related alterations in neural function are well documented, but less is known about their cellular bases. To characterize such changes, we analyzed 14 molecularly identified subsets of mouse retinal projection neurons (retinal ganglion cells or RGCs) and interneurons (amacrine, bipolar, and horizontal cells). The retina thinned but expanded with age, maintaining its volume. There was minimal decline in the number of RGCs, interneurons, or photoreceptors, but the diameter of RGC dendritic arbors decreased with age. Together, the increased retinal area and the decreased dendritic area may lead to gaps in RGC coverage of the visual field. Axonal arbors of RGCs in the superior colliculus also atrophied with age, suggesting that the relay of visual information to central targets may decline over time. On the other hand, the laminar restriction of RGC dendrites and the interneuronal processes that synapse on them were not detectably disturbed, and RGC subtypes exhibited distinct electrophysiological responses to complex visual stimuli. Other neuronal types aged in different ways: amacrine cell arbors did not remodel detectably, whereas horizontal cell processes sprouted into the photoreceptor layer. Bipolar cells showed arbor-specific alterations: their dendrites sprouted but their axons remained stable. In summary, retinal neurons exhibited numerous age-related quantitative alterations (decreased areas of dendritic and axonal arbors and decreased density of cells and synapses), whereas their qualitative features (molecular identity, laminar specificity, and feature detection) were largely preserved. Together, these data reveal selective age-related alterations in neural circuitry, some of which could underlie declines in visual acuity.     

Localization of synapse-associated proteins during postnatal development of the rat retina.

The expression of synapse-associated proteins (SAPs) was monitored throughout postnatal development of the rat retina using specific antibodies and immunocytochemistry. The distribution of chapsin-110/postsynaptic density protein (PSD)-93, SAP90/PSD-95, SAP97 and SAP102 immunoreactivity was characterized. All SAPs were found to be expressed in the inner plexiform layer (IPL) from birth on or soon after birth. With the exception of SAP97, the IPL labelling changed from a diffuse pattern staining the whole developing IPL to the typical adult punctate synaptic staining in the second postnatal week. Staining in the outer retina was first observed at postnatal day 5 (P5) for all proteins at the onset of outer plexiform layer (OPL) development. All SAPs showed a differential cellular and temporal distribution being either exclusively pre- or postsynaptically localized. Except for SAP90/PSD-95, immunoreactivity was also detected in the nerve fibre layer throughout postnatal development. Possible functions of the early expression of SAPs well before differentiation and maturation of glutamatergic ribbon synapses are discussed.     

Localization of kainate receptors at the cone pedicles of the primate retina.

In the macaque monkey retina cone pedicles, the output synapses of cone photoreceptors, contain between 20 and 45 ribbon synapses (triads), which are the release sites for glutamate, the cone transmitter. Several hundred postsynaptic dendrites contact individual cone pedicles, and we studied the glutamate receptors expressed and clustered at these contacts, particularly the kainate receptor subunits GluR5, GluR6/7, and KA2. Pre- and postembedding immunocytochemistry and electron microscopy were used to localize GluR5 and GluR6/7 to specific synaptic contacts at the cone pedicle base. The GluR5 subunit was aggregated at bipolar cell flat contacts. The GluR6/7 subunit was aggregated at bipolar cell flat contacts and at the desmosome-like junctions formed by horizontal cell processes underneath the cone pedicles. KA2 immunoreactivity was observed at the invaginating dendritic tips of ON-cone and rod bipolar cells, which we interpret as a cross-reactivity of the KA2 antiserum with some other, unknown protein of the monkey retina. Kainate receptors are preferentially expressed by OFF-cone bipolar cells and to a lesser extent by horizontal cells. We also performed double-labeling experiments with the ribbon-specific marker bassoon and with antibodies against GluR5 and GluR6/7 in order to define the position of the flat bipolar cell contacts with respect to the triads. There was a tendency of GluR6/7 clusters to represent triad-associated contacts, whereas GluR5 clusters represented non-triad-associated contacts. The GluR5 and GluR6/7 subunits were clustered at different bipolar cell contacts. We studied a possible cone-selective expression of the kainate receptor subunits by double labeling cone pedicles for the S-cone opsin and for the different receptor subunits. We observed a reduced expression of both GluR5 and GluR6/7 at the S-cone pedicles. The reduced expression of GluR6/7 was analyzed in more detail and it appears to be a consequence of a horizontal cell-specific expression: H1 horizontal cells express GluR6/7, whereas H2 horizontal cells, which preferentially innervate S-cones, show no expression of GluR6/7.     

Distribution of neurons projecting to the retina of Caiman crocodilus.

Horseradish peroxidase (HRP) was injected into the vitreous of the eye, the orbital cavity, or the optic tectum of Caiman crocodilus. Following intravitreal injections, retrograde transport of the enzyme was observed bilaterally, but predominantly contralaterally, in a large oblong field of cells at the isthmic level of the midbrain, bounded medially by the trochlear nucleus and laterally by the nucleus isthmi. Control injections of HRP into the orbital cavity and eye muscles labelled motoneurons of the extraocular muscles but not cells of this isthmic field. The field is therefore the source of a projection efferent to the retina in Caiman. Injections of HRP into the dorsal optic tectum produced a fine pattern of anterograde labelling in fibers projecting to the region in which retinopetal cells were identified, but no retrograde labelling of cells in this area. In contrast, the nucleus isthmi showed dense anterograde labelling of fibers and terminals as well as retrograde labelling of cells following tectal injections. These results extend recent evidence for a close evolutionary relationship between the order Crocodilia and modern birds, since birds also possess a well-developed retinopetal system derived from a cell group in the isthmic midbrain.