Morphology and distribution of catecholaminergic amacrine cells in the cone-dominated tree shrew retina.

The tree shrew (Tupaia belangeri) has a cone-dominated retina with a rod proportion of only 5%. This is in contrast to the usual mammalian pattern of rod-dominated retinae. Rod bipolar cells are present at relatively low densities in the tree shrew retina, suggesting that a reduced, but normal, rod pathway might be preserved. The present study investigated another common constituent of the rod pathway, the dopaminergic amacrine cells, and analysed their morphology and distribution by light and electron microscopy. Catecholaminergic (presumed dopaminergic) amacrine cells were labelled with an antibody against tyrosine hydroxylase (TH). Intense TH-immunoreactivity was found in perikarya and dendrites of a uniform amacrine cell population. TH-immunoreactive amacrine cell density varies across the retina from 10 cells/mm2 in the periphery to 40 cells/mm2 in more central regions (mean cell density about 25 cells/mm2). The relatively large cell bodies are located exclusively in the innermost part of the inner nuclear layer. The dendrites form a dense plexus at the border between the inner plexiform layer and the inner nuclear layer. The finer dendritic processes contain many varicosities and form characteristic dendritic "rings" like those seen in other mammals. TH-immunoreactive processes also run between cell bodies in the vitread inner nuclear layer; a few extend into the sclerad inner nuclear layer and occasionally reach the outer plexiform layer (possible interplexiform cells). A few TH-immunoreactive processes are seen in the middle of the inner plexiform layer. Electron microscopy of TH-immunoreactive processes revealed conventional synapses onto somata and processes of unlabelled amacrine cells.     

Synapses from bipolar cells onto dopaminergic amacrine cells in cat and rabbit retinas

Dopaminergic amacrine cells in the vertebrate retina have long been characterized as 'interamacrine' as they were only found to be pre- and postsynaptic to other amacrine cells. Immunohistochemistry with antibodies directed against tyrosine hydroxylase (TH) revealed synapses from bipolar cell axon terminals to TH-containing neuronal processes at ribbon synapses in the rhesus monkey retina. This finding challenged the notion of the dopaminergic amacrine cell phenotype as 'interamacrine'. In order to determine if the finding of synapses from bipolar cells to dopaminergic amacrine cells could be generalized to other species, we studied the synaptic organization of dopaminergic amacrine cells in the retinas of cats and rabbits with electron microscopy of TH immunoreactivity. In both species, TH-immunoreactive processes were found to be postsynaptic to bipolar axon terminals at ribbon synapses demonstrating that the original finding in the primate may be a significant feature in the retinas of many other vertebrates as well.     

Dopaminergic and indoleamine-accumulating amacrine cells express GABA-like immunoreactivity in the cat retina

Colocalization of indoleamine uptake and GABA-like immunoreactivity was studied in the cat retina. Consecutive, semithin sections were incubated in antisera to either 5-HT (5-hydroxytryptamine) or GABA. More than 90% of all 5-HT-accumulating amacrine cells expressed GABA-like antigens. With the same approach, the colocalization of 5-HT uptake and GABA-like immunoreactivity was studied in rabbit and 75-80% of the 5-HT-accumulating amacrine cells expressed GABA-like immunoreactivity, thus confirming a previous study (Osborne and Beaton, 1986). Since, in both cat and rabbit, endogenous 5-HT could not be found by immunocytochemistry, one must consider the possibility that some GABAergic amacrine cells take up indoleamines. In the cat retina, antibodies against tyrosine hydroxylase (TH) label dopaminergic amacrine cells (Oyster et al., 1985). By incubating consecutive, semithin sections in antisera to either TH or GABA, it was found that 84% of the dopaminergic amacrine cells also expressed GABA-like immunoreactivity. GABA-like immunoreactivity and 3H-muscimol uptake were found to be colocalized in more than 90% of the amacrine cells labeled. However, dopaminergic amacrine cells did not accumulate 3H-muscimol. Evidence is presented from colocalization studies for 2 types of interplexiform cell in the cat retina. One is stained by GABA-like immunocytochemistry and by 3H-muscimol uptake. The other is the dopaminergic amacrine cell, which also expresses GABA-like immunoreactivity, but does not accumulate 3H-muscimol.     

Shapes and distributions of the catecholamine-accumulating neurons in the rabbit retina

Rabbit retinas were fixed with mixed aldehydes and examined for the fluorescence of catecholamines. Labeled cell bodies were present in the layer of the amacrine cells. A band of fluorescent processes was present in layer 1 of the inner plexiform layer. Weaker labeling was present in two deeper strata, one near the middle of the inner plexiform layer (presumably layer 3) and one at the juction of layers 4 and 5. Immunohistochemistry showed tyrosine hydroxylase (TH) to be present in the same cells and the same strata of the inner plexiform layer as the endogenous catecholamines. Exposing the retina to exogenous dopamine or norepinephrine resulted in stronger labeling in the middle and deep levels of the inner plexiform layer. At the same time a second population of amacrine cell bodies became visible. Catecholamine fluorescence contained in the amacrine cell bodies was used as a guide to their injection with Lucifer yellow CH. The filled dendritic arbors revealed two main types of cells. The type 1 cells are monostratified at the most distal level of the inner plexiform layer. They have relatively uncomplicated, radially branching dendritic trees. They are the cells densely stained by immunohistochemistry with antibodies against. TH. The type 2 cells are tristratified, with minor branching in layer 1 of the inner plexiform layer and major branching in the two deeper sublayers. The descending dendrites follow a complicated course, and it is not uncommon for intermediate dendrites to cross between strata more than once. The relationship of the cells to their dendritic plexuses was further studied in retinas in which the aldehyde-induced fluorescence of catecholamines was photoconverted to a diaminobenzidine product. The type 1 cells were found to dominate the plexus of dendrites in layer 1 of the inner plexiform layer. The catecholaminergic plexuses in the middle and deep levels of the inner plexiform layer are formedf by dendrites of the type 2 cells. The position of every type 1 cell was mapped in retinas stained with antibodies directed against TH. In one retina we counted 5,613 type 1 cells, distributed evenly across the retina. In another retina, all of the catecholamine-accumulating cells were counted. There were 9,058 with a distribution that peaks in the visual streak. The type 1 cells appear to be the dopaminergic cells previously studied by others and thought to regulate the flow of information from rod bipolar cells to ganglion cells. The low density and wide spread of type 2 cells suggests that they, too, perform a generalized control function, presumably a novel one that dictates their intricate, tristratified shape.     

The dopaminergic amacrine cell

The detailed morphology of the dopaminergic amacrine cell type has been characterized in the macaque monkey retina by intracellular injection of horseradish peroxidase (HRP). This cell type was recognized by its large soma in an in vitro, wholemount preparation of the retina stained with the fluorescent dye, acridine orange. HRP-fills revealed a large, sparsely branching, spiny dendritic tree and a number of extremely thin, axon-like processes that arose from the soma and proximal dendrites. The axon-like processes were studded with distinct varicosities and were traced for up to 3 mm beyond the dendritic tree. The true lengths of the axon-like processes were greater than 3 mm, however, because the HRP reaction product consistently diminished before an endpoint was reached. Both the dendrites and the axon-like processes were narrowly stratified close to the outer border of the inner plexiform layer, although in a few cases single axon-like processes projected into the outer nuclear and outer plexiform layers. The HRP-filled amacrines appeared equivalent to a subpopulation of neurons that are intensely immunoreactive for tyrosine hydroxylase (TH). TH-immunoreactive cells showed a nearly identical soma size and dendritic field size range, the same pattern of dendritic branching and spiny morphology, and also gave rise to distinct axon-like processes from both the soma and proximal dendrites. To test this correspondence more directly, the large acridine stained cells were injected with Lucifer Yellow and the retina was subsequently processed for TH immunoreactivity using diaminobenzidine as the chromagen. In all cases Lucifer Yellow injected cells also showed intense TH immunoreactivity. Spatial densities of the TH amacrine cells were therefore used to calculate coverage factors for the dendritic trees and for the axon-like components of the HRP-filled cells. The axon-like processes showed a coverage factor of at least 300, about 100 times that of the dendritic fields. This great overlap could be directly observed in TH-immunoreacted retinal wholemounts as a dense plexus of fine, varicose processes. The density of the TH plexus is greater than the density predicted from the lengths (1-3 mm) of the HRP-filled axon-like processes however, and suggests that the axon-like processes have an actual length of about 4-5 mm.(ABSTRACT TRUNCATED AT 400 WORDS)     

AII amacrine cells limit scotopic acuity in central macaque retina: A confocal analysis of calretinin labeling.

We have used calretinin antibodies to label selectively the mosaic of AII amacrine cells in the macaque retina. Confocal analysis of double-labeled material indicated that AII dendrites spiral down around descending rod bipolar axons before enveloping the synaptic terminals. Processes from a previously observed dopaminergic plexus in the inner nuclear layer were observed to contact the somata of calretinin-positive AII somata. Intracellular neurobiotin injection revealed that AII amacrine cells are tracer coupled to other AII amacrine cells and to some unidentified cone bipolar cells. An analysis of the retinal distribution of macaque AII amacrine cells, including an area in and around the fovea, showed a peak density of approximately 5,000 cells/mm(2) at an eccentricity of 1.5 mm. Staining of AII amacrine cells in central retina with antibodies to calretinin was confirmed by confocal microscopy. These results indicate that calretinin antibodies can be used to label the AII amacrine cell population selectively and that primate AII amacrine cells share many of the features of previously described mammalian AII amacrine cells. The peak AII cell density closely matched the peak sampling rate of scotopic visual acuity. Calculations suggest that, in central macaque retina, where midget ganglion cells are more numerous, AII amacrine cells form the limit of scotopic visual acuity (W?ssle et al. [1995] J. Comp. Neurol. 361:537-551). As the ganglion cell density falls rapidly away from the fovea, there is a cross-over point at around 15 degrees eccentricity that matches the inflection point in a psychophysically derived plot of scotopic visual acuity versus eccentricity (Lennie and Fairchild [1994] Vision Res. 34:477-482). The correspondence between the anatomic and psychophysical data supports our interpretation that the anatomic sampling rate of AII amacrine cells limits central scotopic acuity.     

Postnatal development of tyrosine hydroxylase immunoreactive amacrine cells in the rabbit retina: II. Quantitative analysis.

Tyrosine hydroxylase (TH)-immunoreactive (IR) amacrine cells of the rabbit retina mature during the first four postnatal weeks, and their cellular development is described in the preceding paper (Casini, G., and N.C. Brecha, J. Comp. Neurol. 326:283-301, 1992). The present investigation is a quantitative analysis of the postnatal development of the TH-IR amacrine cell population. TH-IR amacrine cells gradually increase in size from birth (soma area of 44.7 +/- 12.4 microns2, mean +/- standard deviation) to adulthood (144.2 +/- 28.0 microns2). Cell density slightly increases from postnatal day (PND) 0 (41.9 +/- 9.5 cells/mm2) to PND 6 (47.2 +/- 7.2 cells/mm2), then markedly decreases from PND 6 to adulthood (17.8 +/- 5.3 cells/mm2) as a consequence of retinal growth. TH-IR cell number almost doubles from PND 0 (about 4,100 cells/retina) to adulthood (about 7,850 cells/retina). The increase in the total number of TH-IR amacrine cells can be explained by the generation of new TH-IR cells in the inner nuclear layer, a delay in the expression of the TH phenotype after neurogenesis by cells committed to be dopaminergic, or the acquisition of this dopaminergic phenotype by uncommitted cells. The development of the TH-IR amacrine cell mosaic was assessed by an evaluation of the distribution of nearest neighbor distances of TH-IR cells. There is a poor correlation between this distribution and a theoretical nonrandom distribution before PND 12. After this age, the nearest neighbor distance distribution shifts towards a nonrandom distribution, and is similar to that of the TH-IR amacrine cell population in the adult retina. The establishment of the TH-IR amacrine cell population mosaic is likely to be achieved through different interacting events, including intrinsic (e.g., genetic) factors, environmental influences, and nonuniform retinal growth. Overall, the population parameters analyzed in the present study approach adult values about the time of eye opening (PND 12) and they reach adult values by PND 26.     

Amacrine cells in the tiger salamander retina: morphology, physiology, and neurotransmitter identification.

Amacrine cells of the vertebrate retina comprise multiple neurochemical types. Yet details of their electrophysiological and morphology properties as they relate to neurotransmitter content are limited. This issue of relating light responsiveness, dendritic projection, and neurotransmitter content has been addressed in the retinal slice preparation of the tiger salamander. Amacrine cells were whole-cell clamped and stained with Lucifer yellow (LY), then processed to determine their immunoreactivity (IR) to GABA, glycine, dopamine or tyrosine hydroxylase (TOH), and glucagon antisera. Widefield, ON-OFF amacrine cells were glycine-IR. The processes of these cells extended laterally in the inner plexiform layer (IPL) from 250-600 microns. They were either multistratified in the IPL or monostratified near the IPL midline. Three multistratified ON-OFF narrowfield glycine-IR cells also were found. Four types of ON amacrine cells were found to be GABA-IR; all types had their processes concentrated in the proximal IPL (sublamina b). Type I cells were narrowfield (approximately 100 microns) with a compact projection. Type II cells were widefield (220-300 microns) with a sparse projection. Type III cells had an asymmetrical projection and varicose processes. Type IV cells were pyriform and monostratified in sublamina b. One narrowfield ON-OFF amacrine cell, with processes broadly distributed in the middle of the IPL, was GABA-IR. This cell appeared similar to an ON-OFF cell that was glycine-IR and may comprise a type in which GABA and glycine colocalize. Another class of amacrine cell, with processes forming a major plexus along the distal border of the IPL and a lesser plexus in the proximal IPL, produced slow responses at light ON and OFF; these cells were dopamine/TOH-IR. A narrowfield class of transient ON-OFF amacrine cell, with processes ramifying throughout both sublaminae a and b of the IPL, were glucagon-IR; these cells appeared to be dye-coupled at the soma. We have shown that, with respect to GABA, glycine, dopamine, and glucagon, salamander amacrine cells fall into rather discrete groups on the basis of ramification patterns in the IPL and responses to photic stimulation. The physiological, structural, and neurochemical diversity of amacrine cells is indicative of multiple and complex roles in retinal processing.     

Neuronal markers in rat retinal grafts

Rat E15 retina was grafted to the retina of adult rat hosts. After varying survival times (1 week-6 months), grafts were stained by immunohistochemistry for neurofilament 160 kDa (NF), HPC-1 (an amacrine cell marker), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and somatostatin-28 (SS-28). The first differentiating graft amacrine cells (cholinergic and dopaminergic) could be seen 1 week after transplantation (corresponding to postnatal day 1 = P1). The inner plexiform layer of the graft started to differentiate at 2 weeks (corresponding to P8) seen by HPC-1 and GAD staining. ChAT, TH and SS-28 immunostaining revealed an abnormal lamination pattern in the graft inner plexiform layer. Also by 2 weeks, the outer plexiform layers of the graft contained NF-immunoreactive horizontal cells. No NF-stained retinal ganglion cells could be observed in the graft. Five and 7 weeks after grafting, the transplants had obtained the same staining intensity with different markers as the host retina.     

Characterization of small-field bistratified amacrine cells in macaque retina labeled by antibodies against synaptotagmin-2

Macaque retinae were immunostained with monoclonal antibodies directed against the protein synaptotagmin‐2 (Syt2). Syt2 was localized in a population of small‐field amacrine cells, whose cell bodies formed a regular mosaic within the inner nuclear layer, indicating they represent a single amacrine cell type. The labeled amacrine cells had a bistratified appearance with a dense dendritic plexus in the OFF‐layer and only a few lobular processes extending into the ON‐layer of the inner plexiform layer, similar to A8 amacrine cells described in cat and human retina. Syt2‐labeled cells were immunoreactive for glycine but lacked immunoreactivity for γ‐aminobutyric acid (GABA), suggesting they use glycine as their neurotransmitter. The density of these cells increases from ～200/mm² in peripheral retina to ～1,400/mm² in central retina. Their bipolar cell input was studied by immunolabeling experiments using various bipolar cell markers combined with CtBP2, a marker of presynaptic ribbons. Our data show that Syt2‐labeled amacrine cells receive input from both OFF and ON cone bipolar cells, as well as from rod bipolar cells. The OFF input is dominated by the diffuse bipolar cell DB1 (44%) and the OFF midget bipolar cell (38%). Here we describe a population of bistratified small‐field amacrine cells closely resembling A8 amacrine cells and their cone‐dominated bipolar cell input. J. Comp. Neurol. 521:709–724, 2013. © 2012 Wiley Periodicals, Inc.     

Type 2 wide‐field amacrine cells in TH::GFP mice show a homogenous synapse distribution and contact small ganglion cells

In vertebrate retinas, wide‐field amacrine cells represent a diverse class of interneurons, important for the extraction of selective features, like motion or objects, from the visual scene. Most types of wide‐field amacrine cells lack dedicated output processes, whereas some types spatially segregate outputs from inputs. In the tyrosine hydroxylase (TH)::green fluorescent protein (GFP) mouse line, two types of GFP‐expressing wide‐field amacrine cells have been described: dopaminergic type 1 and γ‐aminobutyric acid‐ergic type 2 cells (TH2). TH2 cells possess short and long radial processes stratifying in the middle of the inner plexiform layer, where they collect excitatory and inhibitory inputs from bipolar cells and other amacrine cells, respectively. Although it was shown that these inputs lead to ON–OFF light responses, their spatial distribution along TH2 cell processes is unknown. Also, the postsynaptic targets of TH2 cells have not been identified so far. Here, we analysed the synapse distribution of these cells in TH::GFP mice and show that they form a weakly coupled network. Electrical synapses (made of connexin36) and chemical (excitatory and inhibitory) synapses are uniformly distributed along TH2 dendrites, independent of dendrite length or distance from soma. Moreover, we reveal that TH2 cells contact at least two types of small ganglion cells; one of them is the W3 cell, a ganglion cell sensitive to object motion. Contacts were often associated with markers of inhibitory synapses. Thus, TH2 wide‐field amacrine cells likely provide postsynaptic inhibition to W3 ganglion cells and may contribute to object‐motion detection in the mouse retina.     

Dopaminergic amacrine cells in the retina of Japanese dace

The morphological and physiological identification of the cells in the inner retina of Japanese dace was made by means of intracellular single cell recording and dye injection techniques. Our flat mounts and physiological classification revealed that 97 out of 102 sample cells were amacrine cells except for 3 transient neurons possibly being ganglion cells. Only two cells were identified as interplexiform cells. The double-labeling histochemical technique showed that 17 cells including the two interplexiform cells are dopaminergic neurons. Therefore, 15 of 97 amacrine cells in dace retina were dopaminergic cells, a finding which is different from the previously published data.     

Brain-derived neurotrophic factor/neurotrophin-4 receptor TrkB is localized on ganglion cells and dopaminergic amacrine cells in the vertebrate retina

The tyrosine kinase TrkB is a receptor for the neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). Retinal ganglion cells are responsive to BDNF, and TrkB has been localized in ganglion cells as well as in a subpopulation of amacrine cells in the retina of the chicken and the rat. In the present paper, we analyzed the distribution of TrkB immunoreactivity in the retina of marmoset monkeys, ferrets, rabbits, rats, mice, chickens, pigeons, barn owls, Pseudemys turtles, Xenopus frogs, goldfishes, and carps. TrkB antibodies gave a positive reaction in all of these vertebrates. TrkB immunoreactivity was detected in the majority of retinal ganglion cells. Some amacrine cells also contained TrkB immunoreactivity; they were located mainly at the vitreal border of the inner nuclear layer, and their relative abundance varied in the different species. Until now, no information has been available concerning the neurochemical identity of the amacrine neurons containing TrkB. In some species (marmoset monkeys, rats, pigeons), we observed that the morphology and location of TrkB-immunoreactive amacrine cells was reminiscent of that of the well-described dopaminergic cells. To determine whether dopaminergic amacrine cells contained TrkB immunoreactivity, we therefore performed double-labelling immunohistochemistry by using tyrosine hydroxylase (TH) antibodies in combination with TrkB antibodies in marmoset monkeys, rats, pigeons, Pseudemys turtles, and goldfishes. The most novel finding of the present paper is that, in all of these species, the majority of dopaminergic neurons were found to contain TrkB immunoreactivity. Dopaminergic neurons, on the other hand, represented only a fraction of the TrkB⁺ amacrine cells. Our data suggest that BDNF and/or NT-4 might modulate expression of TH in the retina and may therefore influence the retinal dopaminergic system. Whatever the action of TrkB ligands on the retinal dopaminergic system, it was conserved during vertebrate evolution. J. Comp. Neurol. 386:149–160, 1997. © 1997 Wiley-Liss, Inc.     

Morphological impairments in retinal neurons of the scotopic visual pathway in a monkey model of Parkinson's disease

Physiological abnormalities resulting from death of dopaminergic neurons of the central nervous system in Parkinson's disease also extend to the retina, resulting in impaired visual functions. In both parkinsonian patients and animal models, low levels of dopamine and loss of dopaminergic cells in the retina have been reported. However, the morphology and connectivity of their postsynaptic neurons, the amacrine cells, have not been analyzed. Here we report, with macaques chronically treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model of Parkinson's disease, that morphological impairments in dopaminergic retinal neurons and their plexus in the inner retina are accompanied by an immunoreactivity decrease in gamma-aminobutyric acidergic and glycinergic amacrine cells. Especially deteriorated were AII amacrine cells, the main neuronal subtype postsynaptic to dopaminergic cells, which exhibited a marked loss of lobular appendages and dendritic processes. Concomitantly, electrical synapses among AII cells, as well as chemical synapses between these and rod bipolar cells, were highly deteriorated in parkinsonian monkeys. These results highlight that the scotopic visual pathway is severely impaired in the parkinsonian condition and provide a morphological basis for a number of abnormalities found in electrophysiological and psychophysical trials in Parkinson's disease patients and animal models.     

Identification and characterization of an aquaporin 1 immunoreactive amacrine-type cell of the mouse retina

Using immunocytochemistry, a type of amacrine cell that is immunoreactive for aquaporin 1 was identified in the mouse retina. AQP1 immunoreactivity was found in photoreceptor cells of the outer nuclear layer (ONL) and in a distinct type of amacrine cells of the inner nuclear layer (INL). AQP1-immunoreactive (IR) amacrine cell somata were located in the INL and their processes extended through strata 3 and 4 of the inner plexiform layer (IPL) with thin varicosities. The density of the AQP1-IR amacrine cells increased from 100/mm(2) in the peripheral retina to 350/mm(2) in the central retina. The AQP1-IR amacrine cells comprise 0.5% of the total amacrine cells. The AQP1-IR amacrine cell bodies formed a regular mosaic, which suggested that they represent a single type of amacrine cell. Double labeling with AQP1 and glycine, gamma-aminobutyric acid (GABA) or GAD(65) antiserum demonstrated that the AQP1-IR amacrine cells expressed GABA or GAD(65) but not glycine. Their synaptic input was primarily from other amacrine cell processes. They also received synaptic inputs from a few cone bipolar cells. The primary synaptic targets were ganglion cells, followed by other amacrine cells and cone bipolar cells. In addition, gap junctions between an AQP1-IR amacrine process and another amacrine process were rarely observed. In summary, a GABAergic amacrine cell type labeled by an antibody against AQP1 was identified in the mouse retina and was found to play a possible role in transferring a certain type of visual information from other amacrine or a few cone bipolar cells primarily to ganglion cells.     

AII amacrine cell population in the rabbit retina: Identification by parvalbumin immunoreactivity

Parvalbumin (PV) is a calcium-binding protein localized to selected neurons in the nervous system, including the retina. This investigation evaluated the distribution of PV immunoreactivity in the rabbit retina using immunohistochemistry with a monoclonal antibody directed to carp PV. In the inner nuclear layer (INL), PV immunoreactivity was present in horizontal and amacrine cells. In the ganglion cell layer, PV immunostaining was confined to somata that are likely to be both displaced amacrine cells and ganglion cells. PV-immunoreactive (IR) amacrine cells were positioned in the proximal INL adjacent to the inner plexiform layer (IPL). These cells usually gave rise to a single primary process, which arborized into two distinct bands in the IPL. In sublamina a, the processes were thin and had large, irregular endings. In sublamina b, multiple processes branched from the primary process and were characterized by varicosities and spines. PV-IR amacrine cell bodies measured from 8 to 10 μm in diameter. Their density was highest in the visual streak and lowest in the periphery of the superior retina. The average number of PV-IR amacrine cells was 464,045 cells per retina (N = 3), and the average regularity index of the PV-IR cell mosaic was 3.23. PV-IR amacrine cells were further characterized by double-label immunofluorescence experiments using antibodies to PV and tyrosine hydroxylase (TH). Varicose TH-IR processes were in close apposition to many PV-IR amacrine cells and often formed “ring structures” around them. Together, these morphological, quantitative, and histochemical observations indicate that PV immunoreactivity in the INL is localized predominantly to AII amacrine cells, and therefore it is a valuable marker for the identification of this cell type. © 1995 Wiley-Liss, Inc.     

Rod bipolar cells in the macaque monkey retina: immunoreactivity and connectivity.

Rod bipolar cells in the macaque monkey retina were labeled by three antibodies: an antibody against the alpha- and beta-subspecies of protein kinase C (PKC), a polyclonal antiserum against the L7 protein from mouse cerebellum, and a monoclonal antibody against rabbit olfactory bulb (MAb 115A 10). The MAb 115A10 antibody also labeled some cone bipolar and some amacrine cells. The antibody against PKC was used to study the synaptic connectivity of rod bipolar cells. Reconstructions of 28 rod spherules showed that usually two and up to four rod bipolar processes invaginate each rod spherule. Six rod bipolar axons in the inner plexiform layer were reconstructed; they all showed the same pattern of connectivity. Synaptic output at rod bipolar dyads usually was onto two amacrine cell profiles: one that resembled the All amacrine cell and another that frequently made a reciprocal synapse. Rod bipolar cells did not contact ganglion cells. Synaptic input to rod bipolar cells came from reciprocal amacrine cells at dyads and other amacrine cells. In these respects, the rod pathway in the monkey is very similar to that described in cat and rabbit. The density of rod bipolar cells was determined and compared with the density of rods. There is a maximum of 15,000-20,000 rod bipolar cells/mm2 at 1-3 mm eccentricity, close to where rod density is maximum. Rod density is 10 times higher than rod bipolar cell density within 2 mm of the fovea, and 30 times higher at 15 mm eccentricity. This change in relative density is compensated by an increase in the number of rods contacted by individual rod bipolar cells (seen in Golgi-stained whole-mount retina) so that the number of rod bipolar terminal boutons in each rod photoreceptor remains relatively constant with changing eccentricity. We estimate that each rod bipolar cell is contacted by about 20 rods at 2-4 mm eccentricity and about 60 rods at 6-7 mm eccentricity.     

Immunohistochemical Localization of Dopamine D Receptors in Rat Retina

We have localized the dopamine D1 receptor in rat retina using a subtype-specific monoclonal antibody. Immunolabelling can be detected in the inner and outer plexiform layers and in a number of cells in the inner nuclear layer. In the inner plexiform layer, labelled processes form four distinct horizontal bands and a series of patches. In order further to characterize the labelling pattern of the D₁ receptor antibody, double-labelling experiments were performed with antibodies against population-specific neuronal markers in the retina. Antibodies against tyrosine hydroxylase. choline acetyltransferase, calretinin, calbindin, the glutamate transporter GLT-I, protein kinase C, recoverin and parvalbumin were co-applied with the D, receptor antibody. With these cell markers we demonstrate that horizontal cells, at least three types of cone bipolar cells and a small number of amacrine cells are immunolabelled for the D1 receptor. In the inner plexiform layer, processes labelled by the D1 receptor antibody are co-stratified with processes labelled by the GLT-1 antibody. D₁ receptor-labelled processes are not co-localized with the processes of amacrine cells and ganglion cells labelled by antibodies against tyrosine hydroxylase, choline acetyltransferase or calretinin. Our results indicate that dopamine Dl receptors are localized predominantly to horizontal cells and cone bipolar cells. Furthermore, the spatial disparity between dopaminergic processes and the site of the majority of D1 receptors supports the idea that in the retina dopamine acts as a neuromodulator that diffuses through extracellular space. The localization of D1 receptors to a number of identified cell types enables future physiological work to be directed towards specific synaptic circuits 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.