Light- and electron-microscopic analysis of neuropeptide Y-immunoreactive amacrine cells in the guinea pig retina

We investigated the morphology and synaptic connections of neuropeptide Y (NPY)-containing neurons in the guinea pig retina by immunocytochemistry, using antisera against NPY. Specific NPY immunoreactivity was localized to a population of wide-field and regularly spaced amacrine cells with processes ramifying mainly in stratum 1 of the inner plexiform layer (IPL). Double-label immunohistochemistry demonstrated that all NPY-immunoreactive cells possessed glutamic acid decarboxylase 65 immunoreactivity. The synaptic connectivity of NPY-immunoreactive amacrine cells was identified in the IPL by electron microscopy. The NPY-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in stratum 1 of the IPL. The most frequent postsynaptic targets of NPY-immunoreactive amacrine cells were other amacrine cell processes. Synaptic outputs to bipolar cells were also observed in a small number of cases. This finding suggests that NPY-containing amacrine cells may influence inner retinal circuitry in stratum 1 of the IPL, thus mediating visual processing.     

iNOS-inhibitor driven neuroprotection in a porcine retina organ culture model

Nitrite oxide plays an important role in the pathogenesis of various retinal diseases, especially when hypoxic processes are involved. This degeneration can be simulated by incubating porcine retinal explants with CoCl₂. Here, the therapeutic potential of iNOS-inhibitor 1400W was evaluated. Degeneration through CoCl₂ and treatment with the 1400W were applied simultaneously to porcine retinae explants. Three groups were compared: control, CoCl₂, and CoCl₂ + iNOS-inhibitor (1400W). At days 4 and 8, retinal ganglion cells (RGCs), bipolar, and amacrine cells were analysed. Furthermore, the influence on the glia cells and different stress markers were evaluated. Treatment with CoCl₂ resulted in a significant loss of RGCs already after 4 days, which was counteracted by the iNOS-inhibitor. Expression of HIF-1α and its downstream targets confirmed the effective treatment with 1400W. After 8 days, the CoCl₂ group displayed a significant loss in amacrine cells and also a drastic reduction in bipolar cells was observed, which was prevented by 1400W. The decrease in microglia could not be prevented by the inhibitor. CoCl₂ induces strong degeneration in porcine retinae by mimicking hypoxia, damaging certain retinal cell types. Treatment with the iNOS-inhibitor counteracted these effects to some extent, by preventing loss of retinal ganglion and bipolar cells. Hence, this inhibitor seems to be a very promising treatment for retinal diseases.     

The distribution and developmental regulation of NMDA receptor subunit proteins in the outer and inner retina of the rat

In order to investigate whether N-methyl-D-aspartate (NMDA) receptors with distinct pharmacological properties are differentially distributed within the retinal layers, the spatial distribution and temporal regulation of all NMDA receptor subunits was analyzed in parallel on the protein level in the rat retina during development. Immunohistochemistry was performed on retinal sections at different developmental ages between embryonic (E) days 20/21 and the adult stage using specific antibodies against NMDA subunits (NR1, NR2A-D). All NMDA subunits were expressed in the rat retina postnatally but showed different spatial patterns. In particular, and in contrast to previous in situ hybridization studies, labeling of NR2 subunits was observed in horizontal cell bodies and in the outer plexiform layer, indicating that functional NMDA receptors are expressed in this retinal cell type in the rat. Expression of NR2D was restricted to the inner retina and seemed to be involved in neurotransmission within the rod pathway. In the inner plexiform layer (IPL), distinct patterns of labeling were observed for different NMDA subunits. NR1 was found in two bands which can be related to the off- and on-signal pathways, whereas NR2A and NR2B were located in two bands within the off-sublaminae of the IPL. The antibody against NR2C was distributed throughout the whole IPL, and NR2D was expressed exclusively in the innermost part of the IPL where rod bipolar cell terminals terminate. Distinct bands of immunoreactivity in the IPL were observed only from P14 on. In conclusion, there are clear differences in the spatial distribution and temporal expression of NMDA receptor subtypes in the rodent retina. This indicates that specific retinal cells selectively express glutamate receptors composed of different subunit combinations and thus display different pharmacological and kinetic properties.     

N-Methyl-d-aspartate receptors in the retina

The vertebrate retina is a “genuine neural center” (Ramón y Cajal), in which glutamate is a major excitatory neurotransmitter. Both N-methyl-d-aspartate (NMDA) and non-NMDA receptors are expressed in the retina. Although non-NMDA receptors and/or metabotropic glutamate receptors are generally thought to be responsible for mediating the transfer of visual signals in the outer retina, there is recent evidence suggesting that NMDA receptors are also expressed in photoreceptors, as well as horizontal and bipolar cells. In the inner retina, NMDA receptors, in addition to other glutamate receptor subtypes, are abundantly expressed to mediate visual signal transmission from bipolar cells to amacrine and ganglion cells, and could be involved in modulation of inhibitory feedback from amacrine cells to bipolar cells. NMDA receptors are extrasynaptically expressed in ganglion cells (and probably amacrine cells) and may play physiological roles in a special mode. Activity of NMDA receptors may be modulated by neuromodulators, such as d-serine and others. This article discusses retinal excitotoxicity mediated by NMDA receptors.     

Expression of alpha 7 nicotinic acetylcholine receptors by bipolar, amacrine, and ganglion cells of the rabbit retina.

Cholinergic agents affect the light responses of many ganglion cells (GCs) in the mammalian retina by activating nicotinic acetylcholine receptors (nAChRs). Whereas retinal neurons that express beta2 subunit-containing nAChRs have been characterized in the rabbit retina, expression patterns of other nAChR subtypes remain unclear. Therefore, we evaluated the expression of alpha7 nAChRs in retinal neurons by means of single-, double-, and triple-label immunohistochemistry. Our data demonstrate that, in the rabbit retina, several types of bipolar cells, amacrine cells, and cells in the GC layer express alpha7 nAChRs. At least three different populations of cone bipolar cells exhibited alpha7 labeling, whereas glycine-immunoreactive amacrine cells comprised the majority of alpha7-positive amacrine cells. Some GABAergic amacrine cells also displayed alpha7 immunoreactivity; alpha7 labeling was never detected in rod bipolar cells or rod amacrine cells (AII amacrine cells). Our data suggest that activation of alpha7 nAChRs by acetylcholine (ACh) or choline may affect glutamate release from several types of cone bipolar cells, modulating GC responses. ACh-induced excitation of inhibitory amacrine cells might cause either inhibition or disinhibition of other amacrine and GC circuits. Finally, ACh may act on alpha7 nAChRs expressed by GCs themselves.     

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.     

Prolonged expression of puma in cholinergic amacrine cells during the development of rat retina

During development of the nervous system, large numbers of neurons are overproduced and then eliminated by programmed cell death. Puma is a BH3-only protein that is reported to be involved in the initiation of developmental programmed cell death in rodent retinal neurons. The expression and cellular localization of Puma in retinal tissues during development are not, however, well known. Here the authors report the expression pattern of Puma during retinal development in the rat. During the period of programmed cell death in the retina, Puma was expressed in some members of each retinal neuron, including retinal ganglion cells, amacrine cells, bipolar cells, horizontal cells, and photoreceptor cells. Although the developmental programmed cell death of cholinergic amacrine cells is known to be independent of Puma, this protein was expressed in almost all their dendrites and somata of cholinergic amacrine cells at postnatal age 2 to 3 weeks, and it continued to be detected in cholinergic dendrites in the inner plexiform layer for up to 8 weeks after birth. These results suggest that Puma has some significant roles in retinal neurons after eye opening, especially that of cholinergic amacrine cells, in addition to programmed cell death of retinal neurons before eye opening.     

Birth of Cone Bipolar Cells,but Not Rod Bipolar Cells,Is Associated with Existing RGCs

Retinal ganglion cells (RGCs) play important roles in retinogenesis. They are required for normal retinal histogenesis and retinal cell number balance. Developmental RGC loss is typically characterized by initial retinal neuronal number imbalance and subsequent loss of retinal neurons. However, it is not clear whether loss of a specific non-RGC cell type in the RGC-depleted retina is due to reduced cell production or subsequent degeneration. Taking advantage of three knockout mice with varying degrees of RGC depletion, we re-examined bipolar cell production in these retinas from various aspects. Results show that generation of the cone bipolar cells is correlated with the existing number of RGCs. However, generation of the rod bipolar cells is unaffected by RGC shortage. Results report the first observation that RGCs selectively influence the genesis of subsequent retinal cell types.     

A glycine site‐specific NMDA receptor antagonist protects retina ganglion cells from ischemic injury by modulating apoptotic cascades

Glutamate neurotoxicity is one of the causative factors leading to neural degeneration including retina. Inhibition of NMDA receptors has been shown neuroprotective effects. However, specifically inhibition of glycine subunit in NMDA receptors and its effects on retina neural protection has not been tested. In this study, using a glycine site‐specific NMDA receptor antagonist, we investigated its neuroprotective effects on rat retinal ganglion cells (RGCs) from a transient ischemic injury and its possible underlying mechanisms. Following an ischemia/reperfusion injury the structural damages of rat retinas were assessed by an immunofluorescence method and the apoptosis of retinal neural cells was evaluated by using a terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) method. The survived RGCs were labeled by retrograde manner and counted on whole‐mounted retinas. In the presence of glycine site‐specific NMDA receptor antagonist, the thickness of retina was sustained, especially in the inner nuclear layers compared with mock controls. While a significantly higher numbers of TUNEL‐positive apoptotic cells and fewer of RGCs were observed in the retina without the glycine antagonist, indicating its strong protective roles. Some apoptotic factors such as Bax, Bcl‐2, CAMK II, COX1, COX4, Caspase‐3, and GRIN1 gene have been tested from retinal samples with or without the glycine antagonist. A significantly lower of expressions of Bax, CAMK II, COX1, COX4, Caspase‐3, and GRIN1 have been shown in the retinas with the antagonist. Bcl‐2/Bax ratio was significantly higher with the antagonist, suggested that the glycine site‐specific NMDA receptor antagonist protecting RGC death might through inhibition of apoptotic signaling. J. Cell. Physiol. 223:819–826, 2010. © 2010 Wiley‐Liss, Inc.     

Morphology and synaptic connectivity of nitric oxide synthase-immunoreactive neurons in the guinea pig retina

Immunocytochemical methods with an antiserum against neuronal nitric oxide synthase (NOS) were applied to identify the morphology and synaptic connectivity of NOS-like immunoreactive neurons in the guinea pig retina. In the present study, two types of amacrine cells were labeled with anti-NOS antisera. Type 1 cells had large somata located in the inner nuclear layer (INL) with long, sparsely branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). The somata of type 2 cells (smaller diameters) were located in the INL. Some displaced amacrine cells in the ganglion cell layer were labeled. The soma size of the displaced amacrine cells was similar to that of the type 2 amacrine cells. However, processes originating from type 2 amacrine cells and displaced amacrine cells stratified mainly in strata 1 and 5, respectively. Some cone bipolar cells were weakly NOS-immunoreactive. The synaptic connectivity of NOS-like immunoreactive amacrine cells was identified in the IPL by electron microscopy. NOS-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in all strata of the IPL. The most frequent postsynaptic targets of NOS-immunoreactive amacrine cells were other amacrine cell processes. Cone bipolar cells were postsynaptic to NOS-labeled amacrine cells in all strata of the IPL. Labeled amacrine cells synapsing onto ganglion cells were found only in sublamina b. A few synaptic contacts were observed between labeled cell processes. In the outer plexiform layer, dendrites of labeled bipolar cells made basal contact with cone pedicles or formed a synaptic triad opposed to a synaptic ribbon of cone pedicles.     

Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1–like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.     

Brain-derived neurotrophic factor modulates the dopaminergic network in the rat retina after axotomy

Dopaminergic cells in the retina express the receptor for brain-derived neurotrophic factor (BDNF), which is the neurotrophic factor that influences the plasticity of synapses in the central nervous system. We sought to determine whether BDNF influences the network of dopaminergic amacrine cells in the axotomized rat retina, by immunocytochemistry with an anti-tyrosine hydroxylase (TH) antiserum. In the control retina, we found two types of TH-immunoreactive amacrine cells, type I and type II, in the inner nuclear layer adjacent to the inner plexiform layer (IPL). The type I amacrine cell varicosities formed ring-like structures in contact with AII amacrine cell somata in stratum 1 of the IPL. In the axotomized retinas, TH-labeled processes formed loose networks of fibers, unlike the dense networks in the control retina, and the ring-like structures were disrupted. In the axotomized retinas treated with BDNF, strong TH-immunoreactive varicosities were present in stratum 1 of the IPL and formed ring-like structures. Our data suggest that BDNF affects the expression of TH immunoreactivity in the axotomized rat retina and may therefore influence the retinal dopaminergic system. E.-J. Lee and M.-C. Song contributed equally to this work. This work was supported by Korea Research Foundation (grant no. E00004, 2004).     

The expression analysis of Sfrs10 and Celf4 during mouse retinal development

Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the genes in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 expression was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation.     

Electron microscopy of the indoleamine-accumulating neurons in the retina of the rabbit

Summary The recently discovered indoleamine-accumulating retinal neurons were studied electron microscopically after destruction of the dopaminergic retinal neurons and subsequent labeling with 5,6-dihydroxytryptamine. These observations confirm earlier fluorescence microscopical studies on the distribution of the indoleamine-accumulating neurons in the rabbit retina. Their perikarya are known to be located in the inner nuclear layer (INL) among the amacrine cell bodies. Their processes are found only in the inner plexiform layer (IPL), most of them in the innermost third part of that layer. The indoleamine-accumulating terminals are pre- and postsynaptic to bipolar neurons in the innermost sublayer of the IPL. Reciprocal synapses are probably the rule. The synaptic vesicles of indoleamine-accumulating synapses onto bipolar cells are arranged in globular clusters around a central electron dense, round body. A number of synapses formed by unlabeled amacrine neurons with postsynaptic indoleamine-accumulating elements were also detected. These synapses were mainly found in the outermost third of the IPL. Synaptic contacts between presynaptic indoleamine-accumulating neurons and postsynaptic unlabeled processes of amacrine cells are very rare.     

Synaptic connections of calbindin-immunoreactive cone bipolar cells in the inner plexiform layer of rabbit retina

In the mammalian retina, information concerning various aspects of an image is transferred in parallel, and cone bipolar cells are thought to play a major role in this parallel processing. We have examined the synaptic connections of calbindin-immunoreactive (IR) ON cone bipolar cells in the inner plexiform layer (IPL) of rabbit retina and have compared these synaptic connections with those that we have previously described for neurokinin 1 (NK1) receptor-IR cone bipolar cells. A total of 325 synapses made by calbindin-IR bipolar axon terminals have been identified in sublamina b of the IPL. The axons of calbindin-IR bipolar cells receive synaptic inputs from amacrine cells through conventional synapses and are coupled to putative AII amacrine cells via gap junctions. The major output from calbindin-IR bipolar cells is to amacrine cell processes. These data resemble our findings for NK1 receptor-IR bipolar cells. However, the incidences of output synapses to ganglion cell dendrites of calbindin-IR bipolar cells are higher compared with the NK1-receptor-IR bipolar cells. On the basis of stratification level and synaptic connections, calbindin-IR ON cone bipolar cells might thus play an important role in the processing of various visual aspects, such as contrast, orientation, and approach sensing, and in transferring rod signals to the ON cone pathway.     

NMDA receptor activation modulates programmed cell death during early post-natal retinal development: a BDNF-dependent mechanism

Glutamate is a classical excitotoxin of the central nervous system (CNS), but extensive work demonstrates neuroprotective roles of this neurotransmitter in developing CNS. Mechanisms of glutamate-mediated neuroprotection are still under scrutiny. In this study, we investigated mediators of glutamate-induced neuroprotection, and tested whether this neurotransmitter controls programmed cell death in the developing retina. The protective effect of N-methyl-d-aspartate (NMDA) upon differentiating cells of retinal explants was completely blocked by a neutralizing antibody to brain-derived neurotrophic factor (BDNF), but not by an antibody to neurotrophin-4 (NT-4). Consistently, chronic activation of NMDA receptor increased the expression of BDNF and trkB mRNA, as well as BDNF protein content, but did not change the content of NT-4 mRNA in retinal tissue. Furthermore, we showed that in vivo inactivation of NMDA receptor by intraperitoneal injections of MK-801 increased natural cell death of specific cell populations of the post-natal retina. Our results show that chronic activation of NMDA receptors in vitro induces a BDNF-dependent neuroprotective state in differentiating retinal cells, and that NMDA receptor activation controls programmed cell death of developing retinal neurons in vivo.