Extrasynaptic release of GABA and dopamine by retinal dopaminergic neurons

In the mouse retina, dopaminergic amacrine (DA) cells synthesize both dopamine and GABA. Both transmitters are released extrasynaptically and act on neighbouring and distant retinal neurons by volume transmission. In simultaneous recordings of dopamine and GABA release from isolated perikarya of DA cells, a proportion of the events of dopamine and GABA exocytosis were simultaneous, suggesting co-release. In addition, DA cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod bipolar signals to cone bipolars. GABA_A but not dopamine receptors are clustered in the postsynaptic membrane. Therefore, dopamine, irrespective of its site of release—synaptic or extrasynaptic—exclusively acts by volume transmission. Dopamine is released upon illumination and sets the gain of retinal neurons for vision in bright light. The GABA released at DA cells'' synapses probably prevents signals from the saturated rods from entering the cone pathway when the dark-adapted retina is exposed to bright illumination. The GABA released extrasynaptically by DA and other amacrine cells may set a ‘GABAergic tone’ in the inner plexiform layer and thus counteract the effects of a spillover of glutamate released at the bipolar cell synapses of adjacent OFF and ON strata, thus preserving segregation of signals between ON and OFF pathways.     

Double-labeling techniques demonstrate that rod bipolar cells are under GABAergic control in the inner plexiform layer of the rat retina

The synaptic connectivity between rod bipolar cells and GABAergic neurons in the inner plexiform layer (IPL) of the rat retina was studied using two immunocytochemical markers. Rod bipolar cells were stained with an antibody specific for protein kinase C (PKC, α isoenzyme), and GABAergic neurons were stained with an antiserum specific for glutamic-acid decarboxylase (GAD). Some amacrine cells were also labeled with the anti-PKC antiserum. All PKC-labeled amacrine cells examined showed GABA immunoreactivity, indicating that PKC-labeled amacrine cells constitute a subpopulation of GABAergic amacrine cells in the rat retina. A total of 150 ribbon synapses established by rod bipolar cells were observed in the IPL. One member of the postsynaptic dyads was always an unlabeled AII amacrine cell process, and the other belonged to an amacrine-cell process showing GAD immunoreactivity. The majority (n=92) (61.3%) of these processes made reciprocal synapses back to the axon terminals of rod bipolar cells. In addition, 78 conventional synapses onto rod bipolar axons were observed, and among them 52 (66.7%) were GAD-immunoreactive. Thus GABA provides the major inhibitory input to rod bipolar cells.     

The mismatch problem for GABAergic amacrine cells in goldfish retina: Resolution and other issues

GABAergic neurons in the vertebrate retina have received intensive study. Yet there are several notable examples of a mismatch among the cytochemical markers used to identify GABAergic neurons. The mismatch between [³H]GABA uptake autoradiography and all other indicators of GABAergic neurons as they pertain to amacrine cells in goldfish retina is examined in this overview. The discrepancies can be accounted for largely by barriers to diffusion presented by significant GABA uptake sinks at the inner and outer margins of the retina and by the differential subcellular distribution of the various markers for GABAergic neurons. Also, conditions producing a redistribution of [³H]-GABA and endogenous GABA stores within the retina are described and discussed.Special issue dedicated to Dr. Eugene Roberts     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.     

Seizure-Related Gene 6 (Sez-6) in Amacrine Cells of the Rodent Retina and the Consequence of Gene Deletion

Background

Seizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development.

Methodology/Principal Findings

The distribution pattern of Sez-6 in the retina was studied using a polyclonal antibody that detects the multiple isoforms of Sez-6. Prominent immunostaining was detected in GABAergic, but not in AII glycinergic, amacrine cell subpopulations of the rat and mouse retina. Amacrine cell somata displayed a distinct staining pattern with the Sez-6 antibody: a discrete, often roughly triangular-shaped bright spot positioned between the nucleus and the apical dendrite superimposed over weaker general cytoplasmic staining. Displaced amacrines in the ganglion cell layer were also positive for Sez-6 and weaker staining was occasionally observed in neurons with the morphology of alpha ganglion cells. Two distinct Sez-6 positive strata were present in the inner plexiform layer in addition to generalized punctate staining. Certain inner nuclear layer cells, including bipolar cells, stained more weakly and diffusely than amacrine cells, although some bipolar cells exhibited a perinuclear “bright spot” similar to amacrine cells. In order to assess the role of Sez-6 in the retina, we analyzed the morphology of the Sez-6 knockout mouse retina with immunohistochemical markers and compared ganglion cell dendritic arbor patterning in Sez-6 null retinae with controls. The functional importance of Sez-6 was assessed by dark-adapted paired-flash electroretinography (ERG).

Conclusions

In summary, we have reported the detailed expression pattern of a novel retinal marker with broad cell specificity, useful for retinal characterization in rodent experimental models. Retinal morphology, ganglion cell dendritic branching and ERG waveforms appeared normal in the Sez-6 knockout mouse suggesting that, in spite of widespread expression of Sez-6, retinal function in the absence of Sez-6 is not affected.     

Morphological analysis of CD15-immunoreactive neurons in the guinea pig retina

Using immunocytochemistry, morphometry and electron microscopy, we have investigated the distribution and characteristics of CD15-immunoreactive (IR) neurons in the guinea pig retina. In the present study, two types of amacrine cells, including interplexiform cells in the inner nuclear layer (INL) and some cells in the ganglion cell layer (GCL), were labeled with anti-CD15 antisera. Type 1 amacrine cells had large somata located in the INL, with long and branched processes ramifying mainly in strata 4 and 5 of the inner plexiform layer (IPL). Somata of type 2 cells had smaller diameters, and were also located in the INL. Their processes stratified in stratum 1. The densities of type I and type 2 amacrine cells increased from 152.8+/-36.7/mm2 and 160.6+/-61.7/mm2 in the peripheral retina, to 404.3+/-41.5/mm2 and 552.2+/-72.2/mm2 in the central retina, respectively. Cells in the GCL exhibiting CD15 immunoreactivity were rarely observed. Colocalization experiments, using consecutive semi-thin sections, demonstrated that these CD15-IR amacrine cells exhibited gamma-aminobutyric acid (GABA) immunoreactivity. In addition, the processes of the type 1 cells formed one member of the postsynaptic dyads that are formed in the axon terminals of rod bipolar cells. Most of these processes made reciprocal synapses back to the axon terminals of the rod bipolar cells. Thus, CD15-IR amacrine cells constitute a subpopulation of GABAergic amacrine cells in the guinea pig retina, and the type 1 cells among them provide the inhibitory input to rod bipolar cells.     

Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double-labeling experiments, most metabotropic glutamate receptor 1-positive cell bodies in the inner nuclear layer also label with anti-GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca(2+) elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca(2+) elevations were enhanced by metabotropic glutamate receptor 1-selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca(2+) signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells.     

Response Properties of a Newly Identified Tristratified Narrow Field Amacrine Cell in the Mouse Retina

Amacrine cells were targeted for whole cell recording using two-photon fluorescence microscopy in a transgenic mouse line in which the promoter for dopamine receptor 2 drove expression of green fluorescent protein in a narrow field tristratified amacrine cell (TNAC) that had not been studied previously. Light evoked a multiphasic response that was the sum of hyperpolarizing and depolarization synaptic inputs consistent with distinct dendritic ramifications in the off and on sublamina of the inner plexiform layer. The amplitude and waveform of the response, which consisted of an initial brief hyperpolarization at light onset followed by recovery to a plateau potential close to dark resting potential and a hyperpolarizing response at the light offset varied little over an intensity range from 0.4 to ~10^6 Rh*/rod/s. This suggests that the cell functions as a differentiator that generates an output signal (a transient reduction in inhibitory input to downstream retina neurons) that is proportional to the derivative of light input independent of its intensity. The underlying circuitry appears to consist of rod and cone driven on and off bipolar cells that provide direct excitatory input to the cell as well as to GABAergic amacrine cells that are synaptically coupled to TNAC. Canonical reagents that blocked excitatory (glutamatergic) and inhibitory (GABA and glycine) synaptic transmission had effects on responses to scotopic stimuli consistent with the rod driven component of the proposed circuit. However, responses evoked by photopic stimuli were paradoxical and could not be interpreted on the basis of conventional thinking about the neuropharmacology of synaptic interactions in the retina.     

Novel expression patterns of metabotropic glutamate receptor 6 in the zebrafish nervous system

The metabotropic glutamate receptor 6 (mGluR6 or GRM6) belongs to the class III of the metabotropic glutamate receptor family. It is the only known mGluR that mediates direct synaptic transmission in the nervous system and is thought to mediate the ON-response in the ON-pathway of the vertebrate retina. Phylogenetic and gene structure analysis indicated that the zebrafish genome harbours two mglur6 paralogs, mglur6a and mglur6b. Besides expression in the inner nuclear layer and distinct regions in the brain, both mglur6 paralogs are expressed in ganglion cells of the retina, an expression pattern which can also be observed in the downstream effector molecules gnaoa and gnaob. This unexpected expression pattern is consistent with immunohistological labeling using a peptide antibody specific for the mGluR6b paralog. These expression patterns contradict the existing view that mGluR6 is solely located on ON-bipolar cells where it functions in signal transmission. Consistent with expression in ON-bipolar cells, we report a decreased b-wave amplitude in the electroretinogram after morpholino-based downregulation of mGluR6b, showing a function in the ON response. Our data suggest more widespread functions of mGluR6 mediated signaling in the central nervous system, possibly including sign reversing synapses in the inner retina.     

The oscillation-like activity in bullfrog ON–OFF retinal ganglion cell

Oscillatory activity of retinal ganglion cell (RGC) has been observed in various species. It was reported such oscillatory activity is raised within large neural network and involved in retinal information coding. In the present research, we found an oscillation-like activity in ON–OFF RGC of bullfrog retina, and studied the mechanisms underlying the ON and OFF activities respectively. Pharmacological experiments revealed that the oscillation-like activity patterns in both ON and OFF pathways were abolished by GABA receptor antagonists, indicating GABAergic inhibition is essential for generating them. At the meantime, such activities in the ON and OFF pathways showed different responses to several other applied drugs. The oscillation-like pattern in the OFF pathway was abolished by glycine receptor antagonist or gap junction blocker, whereas that in the ON pathway was not affected. Furthermore, the blockade of the ON pathway by metabotropic glutamate receptor agonist led to suppression of the oscillation-like pattern in the OFF pathway. These results suggest that the ON pathway has modulatory effect on the oscillation-like activity in the OFF pathway. Therefore, the mechanisms underlying the oscillation-like activities in the ON and OFF pathways are different: the oscillation-like activity in the ON pathway is likely caused by GABAergic amacrine cell network, while that in the OFF pathway needs the contributions of GABAergic and glycinergic amacrine cell network, as well as gap junction connections.     

Localization of CD15 immunoreactivity in the rat retina

Using immunocytochemistry, we have investigated the localization of CD15 in the rat retina. In the present study, two types of amacrine cell in the inner nuclear layer (INL) and some cells in the ganglion cell layer were labeled with anti-CD15 antisera. Type 1 amacrine cells have large somata located in the INL, with long and branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). Type 2 cells have a smaller soma and processes branching in stratum 1 of the IPL. A third population showing CD15 immunoreactivity was a class of displaced amacrine cells in the ganglion cell layer. The densities of type 1 and type 2 amacrine cells were 166/mm(2) and 190/mm(2) in the central retina, respectively. The density of displaced amacrine cells was 195/mm(2). Colocalization experiments demonstrated that these CD15-immunoreactive cells exhibit gamma-aminobutyric acid and neuronal nitric oxide synthase (nNOS) immunoreactivities. Thus, the same cells of the rat retina are labeled by anti-CD15 and anti-nNOS antisera and these cells constitute a subpopulation of GABAergic amacrine cells.     

Immunohistochemical localization of GABAA receptors in the scotopic pathway of the cat retina

The distribution of GABA_A receptors in the inner plexiform layer of cat retina was studied using monoclonal antibodies against the 2/3 subunits. A dense band of receptor labeling was found in the inner region of the inner plexiform layer where the rod bipolar axons terminate. Three forms of evidence indicate that the GABA_A receptor labeling is on the indoleamine-accumulating, GABAergic amacrine cell that is synaptically interconnected with the rod bipolar cell terminal. (1) Electron microscopy showed that the anti-GABA_A receptor antibody (62-3G1) labeled profiles that were postsynaptic to rod bipolar axons and made reciprocal synapses. (2) Indoleamine uptake (and the subsequent autofluorescence) combined with GABA_A receptor immunohistochemistry showed co-localization of the two markers in half of the receptor-positive amacrine cells. (3) Double labeling demonstrated that half of the receptor-positive somata also contained GABA. These results indicate that a GABAergic amacrine cell interconnected with the rod bipolar cell, most likely the so-called A17 amacrine cell, itself bears GABA_A receptors.     

Long-term plasticity mediated by mGluR1 at a retinal reciprocal synapse

The flow of information across the retina is controlled by reciprocal synapses between bipolar cell terminals and amacrine cells. However, the synaptic delays and properties of plasticity at these synapses are not known. Here we report that glutamate release from goldfish Mb-type bipolar cell terminals can trigger fast (delay of 2-3 ms) and transient GABA(A) IPSCs and a much slower and more sustained GABA(C) feedback. Synaptically released glutamate activated mGluR1 receptors on amacrine cells and, depending on the strength of presynaptic activity, potentiated subsequent feedback. This poststimulus enhancement of GABAergic feedback lasted for up to 10 min. This form of mGluR1-mediated long-term synaptic plasticity may provide retinal reciprocal synapses with adaptive capabilities.     

GABA release induced by aspartate-mediated activation of NMDA receptors is modulated by dopamine in a selective subpopulation of amacrine cells

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS, including the retina. In the chick retina, GABA is located in horizontal and amacrine cells and in some cells in the ganglion cell layer. It has been shown that glutamate and its agonists, NMDA, kainate, and aspartate, promote the release of GABA from isolated retina and from cultured retinal cells. Dopamine, the major catecholamine in the retina, inhibits the induction of GABA release by NMDA. Two to seven-day-old intact chicken retinas were stimulated with different glutamatergic agonists and the GABA remaining in the tissue was detected by immunohistochemical procedures. The exposure of retinas to 100 μ M NMDA for 30 minutes resulted in 50% reduction in the number of GABA-immunoreactive amacrine cells. Aspartate (100 μ M) treatment also resulted in 60% decrease in the number of GABA-immunoreactive amacrine cells. The number of GABA-immunoreactive horizontal cells was not affected by either NMDA or aspartate. In addition, dopamine reversed by 50% the reduction of the number of GABA-immunoreactive amacrine cells exposed to NMDA or aspartate. Kainate stimulation promoted a 50% reduction in the number of both GABA-immunoreactive amacrine and horizontal cells. Dopamine did not interfere with the kainate effect. While in control and in non-stimulated retinas a continuous and homogeneous immunolabeling was observed throughout the inner plexiform layer, retinas exposed to NMDA, kainate and aspartate displayed only a faint punctate labeling in the inner plexiform layer. It is concluded that, under our experimental conditions, both NMDA and aspartate induce the release of GABA exclusively from amacrine cells, and that the release is modulated by dopamine. On the other hand, kainate stimulates GABA release from both amacrine and horizontal cells with no interference of dopamine.     

The neurons of the ground squirrel retina as revealed by immunostains for calcium binding proteins and neurotransmitters

Ground squirrel retinas were immunostained with antibodies against calcium binding proteins (CBPs) and classical neurotransmitters in order to describe neuronal phenotypes in a diurnal mammalian retina and to then compare these neurons with those of more commonly studied nocturnal retinas like cats' and rabbits'. Double immunostained tissue was examined by confocal microscopy using antibodies against the following: rhodopsin and the CBPs, calbindin, calretinin, parvalbumin, calmodulin and recoverin (CB, CR, PV, CM, RV), glycine, GABA, choline acetyltransferase (CHAT) and tyrosine hydroxylase (TOH). In ground squirrel retina, the traditional cholinergic mirror symmetric amacrine cells colocalize CHAT with PV and GABA and faintly with glycine. A second cholinergic amacrine cell type colocalizes glycine alone. CR is found in at least 3 different amacrine cell types. The CR-immunoreactive (IR) cell population is a mixture of glycinergic and GABAergic types. The dopamine cell type IR to tyrosine hydroxylase has the typical morphology of a wide field cell with dendrites in S1 but the “rings” seen in cat or rabbit retina are not as numerous. TOH-IR amacrine cells send large club-shaped processes to the outer plexiform layer. CB and CR are in bipolar cells, A- and B-type horizontal cells and several amacrine cell types. Anti-rhodopsin labels the low density rod photoreceptor population in this species. Anti-recoverin labels cones and some bipolar cells while PKC is found in several different bipolar cell types. One ganglion cell with dendritic branching in S3 is strongly CR-IR. We find no evidence for an AII amacrine cell in the ground squirrel, with either anti-CR or anti-PV. An amacrine cell with similarity to the DAP1-3 cell of rabbit is CR-IR and glycine-IR. We discuss this labeling pattern in relationship to other mammalian species. The differences in staining patterns and phenotypes revealed suggest a functional diversity in the populations of amacrine cells according to whether the retinas are rod or cone dominated.     

GABA and GAD-like immunoreactivity in the primate retina

GABA immunoreactivity was studied and compared with GAD immunoreactivity in the retinae of baboon, cynomolgus monkey and man. The central and peripheral parts of the retinae were investigated separately in cynomolgus monkey and in man. The same kinds of structures were stained with both antisera. Cells with a position corresponding to amacrine cells were stained, as well as processes in the inner plexiform layer and some cells in the ganglion cell layer. The outer plexiform layer and some cells with the position and configuration of horizontal cells also appeared immunoreactive. Staining was also observed in bipolar-like cells, in man most clearly when using the GABA antiserum in sections from the central parts of the retina. It is possible that horizontal cells, as well as bipolar-like cells, may play a previously unsuspected role in GABAergic transmission in the primate retina.     

GABA and GAD-like immunoreactivity in the primate retina

Summary GABA immunoreactivity was studied and compared with GAD immunoreactivity in the retinae of baboon, cynomolgus monkey and man. The central and peripheral parts of the retinae were investigated separately in cynomolgus monkey and in man. The same kinds of structures were stained with both antisera. Cells with a position corresponding to amacrine cells were stained, as well as processes in the inner plexiform layer and some cells in the ganglion cell layer. The outer plexiform layer and some cells with the position and configuration of horizontal cells also appeared immunoreactive. Staining was also observed in bipolar-like cells, in man most clearly when using the GABA antiserum in sections from the central parts of the retina. It is possible that horizontal cells, as well as bipolar-like cells, may play a previously unsuspected role in GABAergic transmission in the primate retina.     

GABA receptor rho1 subunit interacts with a novel splice variant of the glycine transporter, GLYT-1

Ionotropic gamma-aminobutyric acid (GABA(A) and GABA(C)) receptors mediate fast synaptic inhibition in the central nervous system. GABA(C) receptors are expressed predominantly in the retina on bipolar cell axon terminals, and are thought to mediate feedback inhibition from GABAergic amacrine cells. Utilizing the yeast two-hybrid system, we previously identified MAP1B as a binding partner of the GABA(C) receptor rho1 subunit. Here we describe the isolation of an additional rho1 interacting protein: a novel C-terminal variant of the glycine transporter GLYT-1. We show that GLYT-1 exists as four alternatively spliced mRNAs which encode proteins expressing one of two possible intracellullar N- and C-terminal domains. Variants containing the novel C terminus efficiently transport glycine when expressed in COS cells, but with unusual kinetics. We have confirmed the interaction between the novel C terminus and rho1 subunit and demonstrated binding in heterologous cells. This interaction may be crucial for the integration of GABAergic and glycinergic neurotransmission in the retina.     

Serum TRPM1 Autoantibodies from Melanoma Associated Retinopathy Patients Enter Retinal ON-Bipolar Cells and Attenuate the Electroretinogram in Mice

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma and the presence of autoantibodies that label neurons in the inner retina. The visual symptoms and electroretinogram (ERG) phenotype characteristic of MAR resemble the congenital visual disease caused by mutations in TRPM1, a cation channel expressed by both melanocytes and retinal bipolar cells. Four serum samples from MAR patients were identified as TRPM1 immunoreactive by 1. Labeling of ON-bipolar cells in TRPM1+/+ but not TRPM1−/− mouse retina, 2. Labeling of TRPM1-transfected CHO cells; and 3. Attenuation of the ERG b-wave following intravitreal injection of TRPM1-positive MAR IgG into wild-type mouse eyes, and the appearance of the IgG in the retinal bipolar cells at the conclusion of the experiment. Furthermore, the epitope targeted by the MAR autoantibodies was localized within the amino-terminal cytoplasmic domain of TRPM1. Incubation of live retinal neurons with TRPM1-positive MAR serum resulted in the selective accumulation of IgG in ON-bipolar cells from TRPM1+/+ mice, but not TRPM1−/− mice, suggesting that the visual deficits in MAR are caused by the uptake of TRPM1 autoantibodies into ON-bipolar cells, where they bind to an intracellular epitope of the channel and reduce the ON-bipolar cell response to light.     

Changes in retinal neuronal populations in the DBA/2J mouse

DBA/2J (D2) mice develop a form of progressive pigmentary glaucoma with increasing age. We have compared retinal cell populations of D2 mice with those in control C57BL/6J mice to provide information on retinal histopathology in the D2 mouse. The D2 mouse retina is characterized by a reduction in retinal thickness caused mainly by a thinning of the inner retinal layers. Immunocytochemical staining for specific inner retinal neuronal markers, viz., calbindin for horizontal cells; protein kinase C (PKC) and recoverin for bipolar cells, glycine, -aminobutyric acid (GABA), choline acetyltransferase (ChAT), and nitric oxide synthase (NOS) for amacrine cells, and osteopontin (OPN) for ganglion cells, was performed to detect preferentially affected neurons in the D2 mouse retina. Calbindin, PKC, and recoverin immunoreactivities were not significantly altered. Amacrine cells immunoreactive for GABA, ChAT, and OPN were markedly decreased in number, whereas NOS-immunoreactive amacrine cells increased in number. However, no changes were observed in the population of glycine-immunoreactive amacrine cells. These findings indicate a significant loss of retinal ganglion and some amacrine cells, whereas glycinergic amacrine cells, horizontal, and bipolar cells are almost unaffected in the D2 mouse. The reduction in amacrine cells appears to be attributable to a loss of GABAergic and particularly cholinergic amacrine cells. The increase in nitrergic neurons with the consequent increase in NOS and NO may be important in the changes in the retinal organization that lead to glaucomain D2 mice. Thus, the D2 mouse retina represents a useful model for studying the pathogenesis of glaucoma and mechanisms of retinal neuronal death and for evaluating neuroprotection strategies.Jung-Il Moon and In-Beom Kim contributed equally to this work.This work was supported by a Korea Research Foundation Grant (FP 0005) and by BK 21 in Korea.