Functional Analysis and Immunochemical Analyses of Ca²⁺ Homeostasis-Related Proteins Expression of Glaucoma-Induced Retinal Degeneration in Rats

The retinal degeneration resulting from elevated intraocular pressure was evaluated through functional and morphological analyses, for better understanding of the pathophysiology of glaucoma. Ocular hypertension was induced via unilateral episcleral venous cauterization in rats. Experimental time was set at 1 and 3 days, and 1, 2, 4, and 8 weeks post-operation. Retinal function was analyzed using electroretinography. For morphological analysis, retinal tissues were processed for immunochemistry by using antibodies against the calcium-sensing receptor and calcium-binding proteins. Apoptosis was analyzed using the TUNEL method and electron microscopy. Amplitudes of a- and b-wave in scotopic and photopic responses were found to be reduced in all glaucomatous retinas. Photopic negative response for ganglion cell function significantly reduced from 1-day and more significantly reduced in 2-week glaucoma. Calcium-sensing receptor immunoreactivity in ganglion cells remarkably reduced at 8 weeks; conversely, protein amounts increased significantly. Calcium-binding proteins immunoreactivity in amacrine cells clearly reduced at 8 weeks, despite of uneven changes in protein amounts. Apoptosis appeared in both photoreceptors and ganglion cells in 8-week glaucomatous retina. Apoptotic feature of photoreceptors was typical, whereas that of ganglion cells was necrotic in nature. These findings suggest that elevated intraocular pressure affects the sensitivity of photoreceptors and retinal ganglion cells, and leads to apoptotic death. The calcium-sensing receptor may be a useful detector for alteration of extracellular calcium levels surrounding the ganglion cells.

The Transient Intermediate Plexiform Layer, a Plexiform Layer-like Structure Temporarily Existing in the Inner Nuclear Layer in Developing Rat Retina

The retina is a highly specialised part of the brain responsible for visual processing. It is well-laminated; three layers containing five different types of neurons are compartmentalised by two synaptic layers. Among the retinal layers, the inner nuclear layer (INL) is composed of horizontal, bipolar, and amacrine cell types. Bipolar cells form one sublayer in the distal half of the IPL, while amacrine cells form another sublayer in the proximal half, without any border-like structure. Here, we report that a plexiform layer-like structure exists temporarily in the border between the bipolar and amacrine sublayers in the INL in the rat retina during retinal development. This transient intermediate plexiform layer (TIPL) appeared at postnatal day (PD) 7 and then disappeared around PD 12. Most apoptotic cells in the INL were found near the TIPL. These results suggest that the TIPL may contribute to the formation of sublayers and the cell number limit in the INL.

Reexamination of Dopaminergic Amacrine Cells in the Rabbit Retina: Confocal Analysis with Double- and Triple-labeling Immunohistochemistry

Dopaminergic amacrine cells (DACs) are among the most well-characterized neurons in the mammalian retina, and their connections to AII amacrine cells have been described in detail. However, the stratification of DAC dendrites differs based on their location in the inner plexiform layer (IPL), raising the question of whether all AII lobules are modulated by dopamine release from DACs. The present study aimed to clarify the relationship between DACs and AII amacrine cells, and to further elucidate the role of dopamine at synapses with AII amacrine cell. In the rabbit retina, DAC dendrites were observed in strata 1, 3, and 5 of the IPL. In stratum 1, most DAC dendritic varicosities—the presumed sites of neurotransmitter release—made contact with the somata and lobular appendages of AII amacrine cells. However, most lobular appendages of AII amacrine cells localized within stratum 2 of the IPL exhibited little contact with DAC varicosities. In addition, double- or triple-labeling experiments revealed that DACs did not express the GABAergic neuronal markers anti-GABA, vesicular GABA transporter, or glutamic acid decarboxylase. These findings suggest that the lobular appendages of AII amacrine cells are involved in at least two different circuits. We speculate that the circuit associated with stratum 1 of the IPL is modulated by DACs, while that associated with stratum 2 is modulated by unknown amacrine cells expressing a different neuroactive substance. Our findings further indicate that DACs in the rabbit retina do not use GABA as a neurotransmitter, in contrast to those in other mammals.

Immunochemical changes of calbindin, calretinin and SMI32 in ischemic retinas induced by increase of intraocular pressure and by middle cerebral artery occlusion / 대한해부학회지

The reaction of neuroactive substances to ischemic conditions in the rat retina evoked by different methods was immunochemically evaluated in adult Sprague-Dawley rats. Ocular ischemic conditions were unilaterally produced by elevating intraocular pressure (EIOP) or by middle cerebral artery occlusion (MCAO). Two EF-hand calcium binding proteins, calbindin D28K (CB) and calretinin (CR), in the normal retina showed similar immunolocalization, such as the amacrine and displaced amacrine cells, the ganglion cells, and their processes, particularly CB in horizontal cells. CB immunoreactive neurons in the ganglion cell layer in both types of ischemic retinas were more reduced in number than CR neurons compared to those in a normal retina. The CB protein level in both ischemic retinas was reduced to 60-80% of normal. The CR protein level in MCAO retinas was reduced to about 80% of normal but increased gradually to the normal value, whereas that in the EIOP showed a gradual reduction and a slight recovery. SMI32 immunoreactivity, which detects a dephosphorylated epitope of neurofilaments-M and -H, appeared in the axon bundles of ganglion cells in the innermost nerve fiber layer of normal retinas. The reactivity in the nerve fiber bundles appeared to only increase slightly in EIOP retinas, whereas a moderate increase occurred in MCAO retinas. The SMI32 protein level in MCAO retinas showed a gradual increasing tendency, whereas that in the EIOP showed a slight fluctuation. Interestingly, the MCAO retinas showed additional SMI32 immunoreactivity in the cell soma of presumed ganglion cells, whereas that of EIOP appeared in the Muller proximal radial fibers. Glial fibrillary acidic protein (GFAP) immunoreactivity appeared in the astrocytes located in the nerve fiber layer of normal retinas. Additional GFAP immunoreactivity appeared in the Muller glial fibers deep in EIOP retinas and at the proximal end in MCAO retinas. These findings suggest that the neurons in the ganglion cell layer undergo degenerative changes in response to ischemia, although EIOP retinas represented a remarkable Muller glial reaction, whereas MCAO retinas had only a small-scaled axonal transport disturbance.

Changes in transcript and protein levels of calbindin D28k, calretinin and parvalbumin, and numbers of neuronal populations expressing these proteins in an ischemia model of rat retina / 대한해부학회지

Excessive calcium is thought to be a critical step in various neurodegenerative processes including ischemia. Calbindin D28k (CB), calretinin (CR), and parvalbumin (PV), members of the EF-hand calcium-binding protein family, are thought to play a neuroprotective role in various pathologic conditions by serving as a buffer against excessive calcium. The expression of CB, PV and CR in the ischemic rat retina induced by increasing intraocular pressure was investigated at the transcript and protein levels, by means of the quantitative real-time reverse transcription-polymerase chain reaction, western blot and immunohistochemistry. The transcript and protein levels of CB, which is strongly expressed in the horizontal cells in both normal and affected retinas, were not changed significantly and the number of CB-expressing horizontal cells remained unchanged throughout the experimental period 8 weeks after ischemia/reperfusion injury. At both the transcript and protein levels, however, CR, which is strongly expressed in several types of amacrine, ganglion, and displaced amacrine cells in both normal and affected retinas, was decreased. CR-expressing ganglion cell number was particularly decreased in ischemic retinas. Similar to the CR, PV transcript and protein levels, and PV-expressing AII amacrine cell number were decreased. Interestingly, in ischemic retinas PV was transiently expressed in putative cone bipolar cell types possibly those that connect with AII amacrine cells via gap junctions. These results suggest that these three calcium binding proteins may play different neuroprotective roles in ischemic insult by their ability to buffer calcium in the rat retina.

The Calretinin Immunoreactive Ganglion Cell Postsynaptic to the ON-Cholinergic Amacrine Cell in the Guinea Pig

PURPOSE: To demonstrate the characterization calretinin-immunoreactive displaced amacrine cells in the ganglion cell layer using immunohistochemistry and electron microscopy. METHODS: For immunohistochemistry, sections from guinea pig retina were incubated with mouse monoclonal antibody directed against calretinin. For double label studies, sections were incuated in mixture of mouse monoclonal anti-calretinin or rabbit polyclonal anti-calretinin with following antibodies: goat polyclonal anti-ChAT, rabbit polyclonal anti-GABA, mouse monoclonal anti-GABAA receptor alpha1, beta2/3. Sections were analyzed using Bio-rad Radiance Plus confocal scanning microscope. Stained sections from three guinea pig were observed with transmission electron microscope. RESULTS: Calretinin immunoreactivity was present in displaced amacrine cells and ganglion cells gaving rise to processes ramified in the inner part of the inner plexiform layer in stratum 4. The same stratum was also occupied by the dendrites of ON-cholinergic amacrine cells. Double-labeling demonstrated that dendrites and cell bodies of displaced amacrine cells colocalized with ON-cholinergic amacrine cells and dendrites of ganglion cells directly overlapped with dendrites of ON-cholinergic amacrine cells. The synaptic connectivity was identified by electron microscopy. Ganglion cell dendrites received synaptic input from ON-cholinergic amacrine cell. GABAA receptor beta2/3 subunit bands cofaciculates the dendrites of displaced amacrine cell and ganglion cell that are juxtapose to the alpha1 subunit of GABAA receptor. CONCLUSIONS: These results indicate that ON-cholinergic amacrine cells modulate calretinin-labeled ganglion cell via GABAA receptor beta2/3 in the guinea pig retina.

Pre-synaptic Neuronal Changes of AII Amacrine Cells in the Streptozotocin-induced Diabetic Rat Retina / 대한해부학회지

It has been previously reported that parvalbumin expression was downregulated in AII amacrine cells, while upregulated in a subset of cone bipolar cells electrically synapse with AII amacrine cell in the streptozotocin-induced diabetic rat retina. In the present study, we aimed to trace biochemical changes of pre-synaptic neurons to AII amacrine cells in rat retina following diabetic injury. Diabetic condition was induced by streptozotocin injection into Sprague-Dawley rats aged of 8 weeks. The experimental term of induced diabetes was set at 1, 4, 12 and 24 weeks. Changes of pre-synaptic neurons were evaluated by immunohistochemistry and Western blot analysis with anti-protein kinase C (PKC)-alpha and anti-tyrosine hydroxylase (TH) antibodies. Rod bipolar cells immunolocalized with PKC-alpha antibody extended their enlarged axon terminals into stratum 5 of the inner plexiform layer. In later diabetes, the axon was shorten and its terminals of rod bipolar cell are slightly enlarged. The protein levels of PKC-alpha were slightly increased along with the duration of diabetes. TH immunoreactive neurons are morphologically classified into two subtypes of amacrine cells in the inner nuclear layer: one (type 1) has large soma with long and primary dendrites, classified with dopaminergic, and the other (type 2) has small soma with dendritic arborization. In the outermost inner plexiform layer, ring-like structures being composed of type 1 cell processes were densely distributed. In diabetic retina, the intensity of TH immunoreactivity in type 1 neurons was reduced. In accordance with morphological changes, the protein levels of TH were reduced during diabetes. These results demonstrate that TH immunoreactive dopaminergic amacrine cells are more susceptible to diabetic injury than the rod bipolar cells in the rat retina and may suggest that downregulation of parvalbumin expression in AII amacrine cells of diabetic retina is mainly due to dysfunction of pre-synaptic dopaminergic amacrine cells.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 µg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 µg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 ± 393 cells/mm²) compared to control (1886 ± 892 cells/mm²; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 ± 56 cells/mm² (2 µg/g) and 845 ± 82 cells/mm² (6 µg/g), also lower than control (1312 ± 31 cells/mm²; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 µg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Altered Expression of Parvalbumin in Neural Retina of the Insulin-dependent Diabetic Rat / 대한해부학회지

Calcium-binding proteins in the nervous system are functioned in Ca2+ buffering and Ca2+ transport and regulation of various enzyme systems. They potentially have a number of different effects on cells includingaltering the duration of action potentials, promoting neuronal bursting activity and protecting cells against the damaging effects of excessive calcium influx. The present study has been designed to clarify the differential responding patterns of parvalbumin immunoreactive neurons in the rat retina following diabetic injury, for better understandings of role of parvalbumin in the retinal circuitry and in calcium homeostasis. Experimental diabetes was induced by a single intravenous injection of streptozotocin in a dose of 60 mg/kg body weight. Diabetic rats showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8, 12 and 24 weeks, respectively. The retinas at each time point were processed for immunohistochemistry and Western blotting using antiparvalbumin antibody. In the rat retina at normal, parvalbumin immunoreactivity appeared in AII amacrine cells, amacrine cells of a widefield type and displaced amacrine cells. A few bipolar cells are also showed the reactivity. During diabetes, the intensity of parvalbumin immunoreactivity is decreased especially in the AII amacrine cells. The cell number of parvalbumin immunoreactive neurons has showed no large changes throughout the diabetes, except that of bipolar cells. That population of parv immunoreactive of bipolar cells has increased remarkably at later diabetic periods. The protein levels of parvalbumin have showed transiently a slight increase at earlier diabetic periods, and then decreased to lower than that of normal. Parvalbumin immunoreactive bipolar cells at diabetes are co-localized not with PKC-alpha or recoverin, but with glutamate transporter Glt-1b. AII amacrine cell processes were joined with each other and with axon terminals of presumed cone bipolar cells by gap junction. These results suggest that the calcium buffering activity of parvalbumin is shifted from AII amacrine cells to a certain type of cone bipolar cells, in response to diabetes. This event may be occurred through electrically coupled gap junction in between these cell processes.

Expression of Substance P in the Experimental Diabetic Rat Retina / 대한해부학회지

Substance P (Sub P) being composed of 11 amino acids sequence is a kind of tachykinin family peptides. It has been known that this substance plays a role of neurotransmitter and/or neuromodulator and is a very potent vascular growth factor in the nervous system. This study has been investigated expression pattern of Sub P in the rat retina at normal and alteration of Sub P expression following diabetic injury using immunohistochemistry. Diabetic condition was induced by a single injection of streptozotocin in Sprague-Dawley rats aged 8 weeks. The animals showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8 and 12 weeks, respectively. The whole-mounted or sectional preparations of the retinas were used for Sub P immunohistochemistry. Sub P immunoreactivity has been localized in subsets of amacrine cells in the inner nuclear layer (INL) and displaced amacrine cells in the ganglion cell layer (GCL) in the normal retina. The dendrites from amacrine cells in the INL were ramified with strata 1 and 3, and those from displaced amacrine cells in the GCL with strata 5 of the inner plexiform layer. Sub P immunoreactive neurons in both the INL and the GCL were more densely populated in the superior half of the retina. During diabetes, the cell number of Sub P immunoreactive neurons was decreased to one third of the normal value at 4 weeks of diabetes and then slightly increased to half of the normal value at 12 weeks of diabetes. In addition, Sub P mRNA levels were reduced at 4 weeks but reincreased at 12 weeks. These results suggest that Sub P in the rat retina at normal state may function differentially in the superior or the inferior halves and Sub P synthetic pathway in the retinal neurons maybe irradiated in earlier stages of diabetic retinopathy.

Alterations of Tyrosine Hydroxylase and Choline Acetyltransferase in the Retina of the Diabetic Rat / 대한해부학회지

To investigate the effect of hyperglycemia on the visual system, we investigated the retinal dopaminergic and cholinergic systems using tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the rat retinas of streptozotocin (STZ)-induced diabetes. Diabetes was induced by a single intraperitoneal injection of STZ (50 mg/kg) to Sprague-Dawley rats (250~300 g). We first analyzed morphologic thickness changes in the several retinal layers of 6-week-old control and STZ-diabetic rats after H & E staining. To confirm whether TH and ChAT protein expressions changed, we carried out immunohistochemistry analysis and Western blotting. After induction of diabetes, significant changes were not shown in the retinal thickness at 6 weeks. TH and ChAT immunoreactivities were clearly detected in amacrine cells and sublaminas in the inner retina of both control and diabetic rats, showing continuously reduced positive amacrine cells in the retinas during diabetes. In addition, the decline in TH and ChAT protein expression was already present to a significant extent in the retina at 6 weeks in early diabetes. Our present study demonstrates the possibility that the observed alterations in TH and ChAT in the diabetic retina may cause the visual system changes in the retinal pathophysiology associated with diabetes mellitus.

Expression of Nitric Oxide Synthase Isoforms in a Rat Model of Chronic Glaucoma

PURPOSE: This study investigated the expression of three isoforms of nitric oxide synthase (NOS): neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) in an experimental rat model of chronic glaucoma. The aim was to research the role of nitric oxide (NO) as a neurotoxic molecule in connection with damage to and the degeneration of retinal ganglion cells in glaucoma. METHODS: Retinal tissues were obtained after inducing chronic elevation of intraocular pressure by cauterization of episcleral vessels. We then performed quantification and localization of NOS isoforms through western blot and immuno-fluorescence staining of the tissues. RESULTS: The expression of nNOS and iNOS increased significantly but that of eNOS did not. nNOS expressed in the amacrine and displaced amacrine cell of the normal retinal tissue, as well as in retinal ganglion cells in the experimental group. iNOS that expressed in the microglia of the normal retinal tissue was also expressed in the cell thought to be an astrocyte or Muller cell end-feet in the experimental group. Administration of L-NAME (NG-nitro-L-arginine-methyl-esther), a non-specific NOS inhibitor, tended to reduce retinal ganglion cell loss, but this result was without statistical significance. CONCLUSIONS: These results showed that the cytotoxicity of excessive NO took part in retinal ganglion cell loss in glaucoma, and the expression of nNOS in retinal ganglion cells suggests that it may play an important role in the selective death of the retinal ganglion cell.

Morphological Changes of the Dopaminergic Network in the Rat Retina after Axotomy / 대한해부학회지

In the retina, dopaminergic cells express the receptor for brain-derived neurotrophic factor (BDNF), which is known to be retrogradely transported from higher center to the retina. This study was conducted to identify the effect of optic nerve transaction on the dopaminergic cells in the rat retina by immunocytochemistry using antityrosine 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 (INL) 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. Our data suggest that retrogradely transported neurotrophic factor affects the expression of TH immunoreactivity in the axotomized rat retina and may therefore influence the retinal dopaminergic system.

Changes in Retinal Neuronal Population in DBA/2J Mice with Topical Intraocular Pressure Lowering Drugs

PURPOSE: The DBA/2J (D2) mouse is a transgenic mouse with pigmentary glaucoma. In a previous study, we found a reduction of inner retinal thickness in D2 mice. We attempted to discover the effect of eye drops on the retina of D2 mice. METHODS: Ten-month-old D2 mouse eyes were treated with Timoptic XE(R), Cosopt(R), and Xalacom(R) eye drops for a 1-month period. Immunohistochemical staining was performed on the mouse eye sections for analysis. RESULTS: In the control group, GABA and OPN immunoreactivity were markedly decreased and NOS immunoreactivity was increased. In all experimental group, GABA and OPN immunoreactivity were increased, and OPN immunoreactivity was markedly increased especially in the Cosopt(R) group. NOS immunoreactivity was decreased in all experimental groups. There was no difference in glycine immunoreactivity between the control and experimental groups. CONCLUSIONS: Combination anti-glaucoma eye-drops to the D2 mouse changed the retinal neuronal population and these drugs might play an important role in the mechanisms of retinal neuronal death; potential strategies for neuroprotection should therefore be evaluated.

Maturation of the Neurokinin 1 (NK1) Receptor Immunoreactive Amacrine Cells in the Rat Retina during Postnatal Development / 대한해부학회지

We examined the morphological maturation of amacrine cells expressing neurokinin 1 (NK1) receptor, whose ligand is substance P, in the rat retina, focusing on the period from postnatal day 5 (P5) when the outer plexiform layer is formed, to postnatal day 13 (P13) when the eyes open, with immunohistochemistry using a specific antiserum against NK1 receptor, and we compared maturing NK1 receptor-immunoreactive (NK1 receptor-IR) amacrine cells with adult one. In the adult retina, numerous NK1 receptor-IR amacrine cells were located in the inner part of the inner nuclear layer (INL) adjacent to the inner plexiform layer (IPL), and their processes emerging from the somata branched and stratified at 1, 2, and 5 strata of within the IPL. NK1 receptor-IR amacrine cells were already observed at P5. The cell bodies were located in the inner INL away from the IPL and their processes branched and formed two distinct bands in the IPL. Afterwards, somata of NK1 receptor-IR amacrine cells moved toward the inner part of the INL, and thus, were located in the INL adjacent to the IPL. Their processes formed three distinct bands at P10 and then, at P13, three bands occupied the same strata as those of the adult, which were posed at 1, 2, and 5 strata of the IPL. During the postnatal development, most of NK1 receptor-IR amacrine cells directly extended one or a few primary dendrites toward the IPL and formed the strata. However, some of the labeled cells located at the outermost row had horizontal processes emerging from their primary dendrites, and these horizontal processes branched and formed plexuses in the INL. The NK1 receptor-IR amacrine cells with horizontal processes were frequently observed at P7, rarely at P10, and not at P13 and in the adult. These results indicate that the NK1 receptor-IR amacrine cells of the rat retina morphologically mature by way of migration of their somata within the INL and formation of distinct processes during postnatal development, and suggest that they morphologically and functionally complete the maturation process about the time of P13.

Altered Expression of Metabotropic Glutamate Receptors mGluR1, mGluR5, and mGluR2/3 in the Diabetic Rat Retina / 대한해부학회지

An excitatory neurotransmitter glutamate is engaged in slow transmission by activating the secondary signal transduction pathway through metabotropic receptors of the target cells. The present study has been investigated the localization of group I (mGluR1 and mGluR5) and II (mGluR2/3) metabotropic glutamate receptors in the retina and their altered expression patterns following long-term diabetes using immunohistochemistry, in order to clarify the involvement of the slow transmission of glutamate in diabetic retinopathy. Insulin-dependent diabetes was induced by a single intravenous injection of streptozotocin. Experimental periods were set at 1, 4, 12 and 24 weeks after the onset of diabetes. MGluR1 and mGluR5 were expressed in the outer plexiform layer, 1, 3, and 5 strata of the inner plexiform layer, and the photoreceptor layer in the retina at normal state. In the early periods of diabetes, the expression pattern of group I receptors was no large change. The expression level in the photoreceptor layer of 12 and 24 weeks diabetic retinas was increased, while that in the IPL was decreased. MGluR2/3 was expressed in the amacrine cells, in the displaced amacrine cells, and in two bands in the inner plexiform layer at normal retina. In later diabetic periods, the expression level of mGluR2/3 was increased in the two bands especially. These results demonstrate that diabetes induces the activation of I and II mGluRs in the retina, and may suggest the involvement of slow transmission of glutamate via metabotropic receptors in progression of diabetic retinopathy.

Morphology and Distribution of the Vasoactive Intestinal Polypeptide (VIP)-Immunoreactive Amacrine Cells in the Mouse Retina / 대한해부학회지

Vasoactive intestinal polypeptide (VIP) is a neuroactive substance that is widely expressed in both non-mammalian and mammalian retinas. In this study, we immunocytochemically identified and investigated the VIP-containing neurons in the mouse retina, which has become an important model for the study of the structure and function of the mammalian retina, mainly because of the wide availability of transgenic animals. VIP immunoreactivity was observed in the somata of the amacrine cells in the inner nuclear layer (INL) and their varicose processes ramifying in strata 1 and 3 of the inner plexifrom layer (IPL). The distribution of VIP-immunoreactive (IR) amacrine cells showed a peak of 430 cells/mm2 in the central retina and minimum values of 50 cells/mm2 in the peripheral one. Double-label experiments demonstrated that all VIP-IR amacrine cells possessed GABA immunoreactivity. These results demonstrate that VIP-IR amacrine cells of the mouse retina make up a neurochemically and morphologically distinct subpopulation of the GABAergic amacrine cell population.

Immunocytochemical Study on the Development of the Rod Pathway in the Rat Retina / 대한해부학회지

Rod bipolar cells constitute the second-order neuron in the rod pathway. Previous investigations of the rat retina have evaluated the development of other components of the rod pathway namely the AII amacrine cell and GABAergic amacrine cell populations. To gain further insights into the maturation of this retinal circuitry, we studied the development of rod bipolar cells, immunocytochemistry with antibodies directed to the protein kinase C (PKC), in the rat retina. PKC immunoreactivity first appeared in postnatal day 9 (P9), faint PKC immunoreactivity was observed in the cell bodies located at the distal inner nuclear layer (INL), dendrites in the outer plexiform layer (OPL) and immunoreactive bands in the proximal inner plexiform layer (IPL). PKC immunoreactive cells and terminal bulbs at P10 show stronger immunostaining. At P15, the time of eye opening, PKC immunoreactive cells display stronger immunostaining than those of P10 and more mature characteristics like in the adult retina. Double fluorescence immunocytochemistry using an antiserum against parvalbumin, a marker for the AII amacrine cells, or GABA revealed that PKC immunoreactive rod bipolar cell terminals make contact with AII amacrine cells and GABAergic neurons in the proximal IPL from P9. Given these results, the different components of the rod pathway follow a similar pattern of maturation, presumably allowing the rod pathway to function at the early developmental stage of retina.

Immunohistochemical Localization of Guanine Aminohydrolase, a Protein Identical with Novel Protein p51-nedasin, and SAP 102 in the Rat Retina / 대한해부학회지

Guanine aminohydrolase (GAH), one of purine metabolizing enzymes rich in the nervous system was proved to have identical amino acid sequence to a recently identified novel protein p51-nedasin, NE-dlg/SAP102-associated protein. Nedasin has been reported to localize at neuronal cell bodies and binds to SAP102, so it might have a role in modulating NMDA receptor 2B clustering of SAP102 or synaptic organization in neuronal cells. In this study, we localize GAH and SAP102 in rat retina using immunohistochemical method. Immunoreactivities are detected for both GAH and SAP102 in ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer and pigment layer. They seemed to be colocalized in ganglion cells, amacrine cells, horizontal cells and pigment cells. The staining profile for SAP102 is almost identical with NMDA receptor 2B mainly in fibrous elements in both the inner and outer plexiform layer. Our results support the possibility of close structural relationship between GAH and SAP102 in specific retinal cells and GAH involvement in synaptic organization association with SAP102 in the rat retina.

Immunocytochemical Localization of GABA Transporter-3 in the Guinea Pig Retina / 대한해부학회지

The cellular localization of the GABA transporter-3 (GAT-3) was examined in the guinea pig retina by immunocytochemistry, using antisera against GAT-3. GAT-3 immunoreactivity was localized to cell bodies in the inner nuclear layer, and labeled processes were densely distributed in the inner plexiform layer (IPL) close to the ganglion cell layer. All GAT-3 labeled cells exhibited GAD65 immunoreactivity. In addition, 67% of GAT-3 labeled amacrine cells showed carbohydrate epitope CD15 immunoreactivity. These results indicate that GAT-3 is involved in modulating the rod pathway in the IPL of the guinea pig retina via presumptive A17 amacrine cells.