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.
Amacrine Cells ; Animals ; Apoptosis ; Brain ; Cell Count ; Neurons ; Rats ; Retina ; Retinaldehyde

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.
Amacrine Cells ; Animals ; Antibodies ; Apoptosis ; Calcium ; Calcium-Binding Proteins ; Cautery ; Electroretinography ; Ganglion Cysts ; Glaucoma ; Immunochemistry ; In Situ Nick-End Labeling ; Intraocular Pressure ; Methods ; Microscopy, Electron ; Ocular Hypertension ; Rats ; Receptors, Calcium-Sensing ; Retina ; Retinal Degeneration ; Retinal Ganglion Cells ; Retinaldehyde

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.
Amacrine Cells* ; Dendrites ; Dopamine ; GABAergic Neurons ; gamma-Aminobutyric Acid ; Glutamate Decarboxylase ; Immunohistochemistry* ; Mammals ; Neurons ; Neurotransmitter Agents ; Retina* ; Synapses

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.
Adult ; Amacrine Cells ; Animals ; Astrocytes ; Axonal Transport ; Axons ; Calcium-Binding Protein, Vitamin D-Dependent ; Calcium-Binding Proteins ; Carisoprodol ; Ganglion Cysts ; Glial Fibrillary Acidic Protein ; Humans ; Infarction, Middle Cerebral Artery ; Intraocular Pressure ; Ischemia ; Middle Cerebral Artery ; Nerve Fibers ; Neurons ; Rats ; Rats, Sprague-Dawley ; Reference Values ; Retina

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.
Amacrine Cells ; Animals ; Blotting, Western ; Calcium ; Calcium-Binding Protein, Vitamin D-Dependent ; Calcium-Binding Proteins ; Cell Count ; Ganglion Cysts ; Gap Junctions ; Humans ; Immunohistochemistry ; Intraocular Pressure ; Ischemia ; Neurons ; Proteins ; Rats ; Retina

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.
Amacrine Cells ; Animals ; Calcium-Binding Protein, Vitamin D-Dependent ; Dendrites ; Electrons ; Ganglion Cysts ; Goats ; Guinea ; Guinea Pigs ; Immunohistochemistry ; Mice ; Microscopy, Electron ; Retina

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.
Amacrine Cells* ; Animals ; Antibodies ; Axons ; Blotting, Western ; Carisoprodol ; Dendrites ; Down-Regulation ; Immunohistochemistry ; Neurons* ; Phosphotransferases ; Presynaptic Terminals ; Rats* ; Rats, Sprague-Dawley ; Retina* ; Streptozocin ; Synapses

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.
Amacrine Cells ; Animals ; Astrocytes ; Blotting, Western ; Cautery ; Glaucoma* ; Intraocular Pressure ; Microglia ; Models, Animal* ; Neurons ; NG-Nitroarginine Methyl Ester ; Nitric Oxide Synthase* ; Nitric Oxide* ; Protein Isoforms ; Rats* ; Retinal Ganglion Cells ; Retinaldehyde

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.
Amacrine Cells ; Animals ; Blotting, Western ; Choline O-Acetyltransferase* ; Choline* ; Diabetes Mellitus ; Hyperglycemia ; Immunohistochemistry ; Injections, Intraperitoneal ; Rats* ; Rats, Sprague-Dawley ; Retina* ; Retinaldehyde ; Streptozocin ; Tyrosine 3-Monooxygenase* ; Tyrosine*

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.
Amacrine Cells ; Amino Acids ; Animals ; Blood Glucose ; Cell Count ; Dendrites ; Diabetic Retinopathy ; Ganglion Cysts ; Humans ; Immunohistochemistry ; Nervous System ; Neurons ; Neuropeptides ; Neurotransmitter Agents ; Peptides ; Rats* ; Rats, Sprague-Dawley ; Reference Values ; Retina* ; Retinal Neurons ; RNA, Messenger ; Streptozocin ; Substance P* ; Tachykinins