Immunohistochemical localization of protein kinase C-alpha in the retina of pigs during postnatal development

The cellular localization and protein expression level of protein kinase C (PKC)-alpha was examined in pig retina at different ages. Western blot analysis detected PKC-alpha in the retinas of 3-day-old piglets and indicated significantly increased expression in 6-month-old young adult and 2-year-old adult pigs. Immunohistochemistry of 3-day-old retinas revealed intense PKC-alpha reactivity in the inner plexiform and inner nuclear cell layers, weak reactivity in the ganglion cell layer, and few positive cells in the outer nuclear cell layer. The cellular localization of PKC-alpha in the adult retina was similar, with staining more intense than that in neonates. PKC-alpha was co-localized in some glial fibrillary acidic protein-positive cells and glutamine synthetase-positive cells in the retina. This study demonstrates that the protein level of retinal PKC-alpha is increased with maturation and suggests that PKC-alpha plays a role in signal transduction pathways for postnatal development in porcine retina.     

Immunohistochemical localization of heat shock protein 27 in the retina of pigs

The expression of heat shock protein 27 (HSP27) was examined in the retinas of pigs. Western blot analysis detected the expression of HSP27 in the retinas of 1-day-old piglets and showed that it was enhanced in the retinas of 6-month-old adult pigs. Immunohistochemically, HSP27 immunostaining was seen mainly in ganglion cell bodies in the ganglion cell layer, and in some processes of astrocytes in the innermost nerve fiber layer. In 1-day-old piglets, HSP27 was detected weakly in the inner plexiform, inner nuclear cell, outer plexiform, and rod and cone layers. The HSP27 immunoreactivity across the retinal layers was enhanced in the retinas of 6-month-old pigs compared with newborn piglets. The HSP27 immunoreactivity in the radial processes of Müller cells was particularly prominent in adult pig retinas. In summary, this finding suggests that HSP27 plays an important role in signal transduction of glial cells and neuronal cells in the retina.     

Localization of substance P-like immunoreactivity in the adult and developing goldfish retina

Substance P-like immunoreactivity was localized to amacrine cells in both adult and developing goldfish retina using immunohistochemical techniques. These studies utilized a well-characterized monoclonal antiserum directed to substance P. Specificity was established by absorption of the anti-serum with 10 μm synthetic substance P. Specific substance P-like immunoreactivity was localized within a seemingly distinct population of unistratified amacrine cells which were distributed in both central and peripheral retinal regions. The immunoreactive somata were located at the border of the inner nuclear layer and inner plexiform layer and were characterized by a round or ovoid somata which measured about 9μm in diameter. These immunoreactive amacrine cells typically had a single process which descended to and ramified within lamina 3 of the inner plexiform layer.Specific substance P-like immunoreactivity first appeared 60 h after hatching (stage 27) within both somata and processes located in differentiated retinal regions. No substance P-like immunoreactive somata or processes were observed in undifferentiated retinal regions. In retinas from stage 27 to 14 days after hatching, the immunoreactive somata were characterized by an ellipsoidal soma and a large nucleus devoid of immunoreactivity. These immunoreactive cells were also characterized by a single process that descended to and ramified within lamina 3 of the differentiated inner plexiform layer. At 30 days after hatching, the substance P-containing cells were identical in appearance to these same cell types observed within the adult retina.     

Immunohistochemical evidence for epinephrine-containing retinal amacrine cells

The enzyme for the synthesis of epinephrine, phenylethanolamine-N-methyltransferase, has been localized, by an indirect immunofluorescent staining method, to a subpopulation of amacrine cells in the rat retina. The immunoreactive cells are located primarily in the inner nuclear layer and send a single process to the inner plexiform layer. Most of the immunoreactivity is found in the center of the inner plexiform layer. A small percentage of immunoreactive cell bodies were found in the inner plexiform layer and occasionally cells were observed in the ganglion cell layer. These epinephrine-containing amacrine cells are morphologically distinct from the dopamine-containing amacrine cells previously described by formaldehyde fluorescence and we speculate from reports in the literature that epinephrine-containing amacrine cells may play a role in modulating the activity of dopamine-containing amacrine cells.     

Immunohistochemical localization of phospholipase D1 in the retina of pigs

The expression of phospholipase D1 (PLD1) was examined in the retinas of pigs. Western blot analysis detected the expression of PLD1 in the retinas of 1-day-old piglets and showed that it was enhanced in the retinas of 2 years old adult pigs. Immunohistochemically, PLD1 was mainly immunostained in ganglion cell bodies in the ganglion cell layer, in some radial processes of Muller cells in the retinal layer and in the inner and outer segments of the rod and cone layer in newborn and adult pigs, but not in astrocytic bundles in nerve fiber layers. The immunoreactivity of PLD1 in the radial processes of Muller cells across the retinal layers was enhanced in adult pig retinas compared to those of newborn piglets. This was the first demonstration to show that PLD1 is constitutively expressed in the retina of pigs, implying that retinal PLD1 expression is enhanced in radial fibers of Muller cells with age. This finding suggests that PLD1 plays an important role in signal transduction of glial cells and neuronal cells in the retina.     

The concentration of enkephalin-like material in the chick retina is light dependent

The enkephalin-like immunoreactivity in the retina of chicks has been studied using immunohistochemical and radioimmunoassay techniques. The histochemical experiments showed that the immunoreactivity was confined to a subpopulation of amacrine cells in the inner nuclear layer which projected processes into sublaminae 1 and 3-5 of the inner plexiform layer. The distribution of the immunoreactivity was markedly influenced by the ambient lighting conditions: it was reduced in the dark and restored by a period in the light. The reactivity was lost from both cell soma in the inner nuclear layer and from the processes. Radioimmunoassays showed that the quantity of enkephalin-like material was reduced by more than 60% after 12 h in the dark. Attempts to entrain a rhythm by keeping chicks on 12/12 h light/dark cycles for up to 4 days were largely unsuccessful. A rhythm may have been partially entrainable, but the major factor involved was light. These results highlight the lability of the neuropeptide in the retina and the need for controlled lighting conditions in studies of this kind. They also indicate that this system may be a fruitful model to explore two important issues: (i) it could allow studies of neuropeptide metabolism in a physiologically intact system; (ii) the role of particular amacrine cells in visual processing could be determined by depleting them of their neurotransmitter/neuromodulator.     

Localization of primary cilia in mouse retina

The primary cilia are considered as “cellular antennae” that sense and interchange information with the extracellular environment. Nearly all mammalian cells have a single primary cilium. In the retina, the outer segment of the photoreceptor is known to be a specialized form of primary cilium, but studies on cilia in other layers of the retina are scarce. In this study, we investigated the expression of primary cilia in the whole thickness of the mouse retina using immunofluorescence with three different ciliary markers: Arl13b, acetylated α-tubulin and adenylyl cyclase III. Our results show positive reactions in the photoreceptor layer, outer plexiform layer and ganglion cell layer, which might suggest the possible presence of primary cilia in these areas, but we could not directly prove the strand-like shape of cilia in those areas. In the outer plexiform layer, all three markers showed intense staining along the neuronal synapses, which suggests that the neuronal processes themselves might share the features of cilia.     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer     

Evidence for an amacrine cell system in the ganglion cell layer of the rat retina

In the ganglion cell layer of the rat retina approx 50% of the cells with the Nissl morphology of neurons survive optic nerve section in infant and adult rats and cannot be retrogradely labelled with horseradish peroxidase. The number of neurons which can be retrogradely labelled with horseradish peroxidase from subcortical visual centres is similar to the number of axons in the optic nerve, and it is concluded that the small neurons do not send an axon into the optic nerve. The dendritic tree of the cells which have axons was demonstrated by filling the cells with horseradish peroxidase from the optic nerve. The dendritic structure of the cells which survive optic nerve section was shown by injecting horseradish peroxidase into the retina or impregnating with the Golgi method the cells which survive optic nerve section. A variety of amacrine cells were found in the ganglion cell layer which form branches in the lower part of the inner plexiform layer.It can be concluded that amacrine cells form a substantial number of the neurons in the ganglion cell layer.     

Expression and cell localization of brain-derived neurotrophic factor and TrkB during zebrafish retinal development

Brain‐derived neurotrophic factor (BDNF) signaling through TrkB regulates different aspects of neuronal development, including survival, axonal and dendritic growth, and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF and TrkB in the retina, the cell types in the retina that express BDNF and TrkB, and the variations in their levels of expression during development, remain poorly defined. The goal of the present study is to determine the age‐dependent changes in the levels of expression and localization of BDNF and TrkB in the zebrafish retina. Zebrafish retinas from 10 days post‐fertilization (dpf) to 180 dpf were used to perform PCR, Western blot and immunohistochemistry. Both BDNF and TrkB mRNAs, and BDNF and full‐length TrkB proteins were detected at all ages sampled. The localization of these proteins in the retina was very similar at all time points studied. BDNF immunoreactivity was found in the outer nuclear layer, the outer plexiform layer and the inner plexiform layer, whereas TrkB immunoreactivity was observed in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer. These results demonstrate that the pattern of expression of BDNF and TrkB in the retina of zebrafish remains unchanged during postembryonic development and adult life. Because TrkB expression in retina did not change with age, cells expressing TrkB may potentially be able to respond during the entire lifespan of zebrafish to BDNF either exogenously administered or endogenously produced, acting through paracrine mechanisms.     

Neurotensin-like and somatostatin-like immunoreactivity within amacrine cells of the retina

Neurotensin-like and somatostatin-like immunoreactivity was demonstrated in the pigeon retina, using both immunohistochemical and radioimmunoassay techniques.Immunohistochemical studies utilized both the indirect immunofluorescence and immunoperoxidase procedures with two well-characterized antisera to neurotensin and three well-characterized antisera to somatostatin. Specific immunoreactivity of each antiserum was established by absorption with either 10 μM synthetic neurotensin, somatostatin or leu⁵-enkephalin. Specific immunohistochemical staining for neurotensin and for somatostatin was observed within separate populations of multistratified amacrine cells. Neurotensin-like and somatostatin-like immunoreactivity were observed within somata located in the inner nuclear layer and within varicose processes ramifying in laminae 1, 3 and 4 of the inner plexiform layer. Immunoreactive somata and processes were observed throughout the retina and their density appeared to be greatest within central retinal regions. The somata-containing neurotensin-like and somatostatin-like immunoreactivity measured about 7 μm in diameter. The cell to cell spacing of neurotensin-like immunoreactive somata was approximately 30 μm and the cell to cell spacing of somatostatin-like immunoreactive somata was approximately 27 μm in central retinal regions. Within more peripheral retinal regions, immunoreactive cells were spaced farther apart.Radioimmunoassays utilizing well-characterized antisera to neurotensin and somatostatin demonstrate specific neurotensin-like and somatostatin-like immunoreactivity in acetic acid extracts of the retina. The concentration of immunoreactive neurotensin is 59 ± 7 fmoles per whole retina (mean ± S.E.M.) or 15.4 ± 2 fmoles per mg protein. The concentration of immunoreactive somatostatin is 2209 ± 440 fmoles per whole retina or 527 ± 76 fmoles per mg protein.These results demonstrate the existence of two additional neuropeptides within selected populations of retinal amacrine cells. The localization of several different neuropeptides within the retina suggests that neuropeptides play a specific role in retinal function.     

NR2C and NR2D subunits of NMDA receptors in frog and turtle retina

Glutamate NMDA (N-methyl-d-aspartate) receptors are widely distributed in the central nervous system where they are involved in cognitive processes, motor control and many other functions. They are also well studied in the retina, which may be regarded as a biological model of the nervous system. However, little is known about NR2C and NR2D subunits of NMDA receptors, which have some specific features as compared to other subunits. Consequently the aim of the present study was to investigate their distribution in frog (Rana ridibunda) and turtle (Emys orbicularis) retinas which possess mixed and cone types of retina respectively. The experiments were performed using an indirect immunofluorescence method. Four antibodies directed to NR2C and NR2D subunits of NMDA receptor, as well as three antibodies directed to different splice variants of NR1 subunit, which is known to be obligatory for proper functioning of the receptor, were applied. All antibodies caused well expressed labeling in frog and turtle retinas. The NR2C and NR2D subunits were localized in glial Müller cells, while the NR1 subunit had both neuronal and glial localization. Our results show that glial NMDA receptors differ from neuronal ones in their subunit composition. The functional significance of the NMDA receptors and their NR2C and NR2D subunits, in particular for the neuron-glia interactions, is discussed.     

Abnormal innervation patterns in the anorectum of ETU-induced fetal rats with anorectal malformations

Valproate (VPA) is commonly used in the treatment of bipolar disorder and epilepsy. The mechanism underlying its clinical efficacy is complicated, including its ability to inhibit histone deacetylase (HDAC). Here, we show that VPA promoted endoplasmic reticulum (ER) chaperone expression and attenuated ER-induced apoptosis after ischemia/reperfusion (I/R) injury in retina. Male Wistar rats were randomly divided into four groups: sham (group A), sham + VPA (group B), I/R + vehicle (group C), and I/R + VPA (group D). VPA was administered subcutaneously at 300 mg/kg twice daily before insult. Morphological changes were analyzed on stained histological sections and flat-mounted retinas labeled by Fluoro-gold. Western blot analysis was used to determine protein levels of GRP78, CHOP, caspase-12 and acetylation of histone H3 in each group. In group C, the severe retinal damage was shown in histological sections, however, the damage was reduced by VPA in group D. Significant loss of retinal ganglion cells (RGCs) was observed in group C, whereas, the density of RGCs was significantly higher in group D at 7 days post-insult. VPA increased GRP78 expression and acetylation of histone H3, attenuated upregulation of CHOP and activation of caspase-12 in group D. Our results suggest that VPA can protect ischemic retinas from ER stress-induced apoptosis by mechanisms that may involve HDAC inhibition.     

信号转导子与转录激活子3蛋白在金黄地鼠视网膜生后早期发育过程中的表达

目的 观察信号转导子与转录激活子3(STA13)蛋白在金黄地鼠视网膜生后早期发育过程中的表达。方法 STA13蛋白免疫细胞化学染色和Westem免疫印迹方法结果生后发育早期整个视网膜神经层均有STA13阳性产物分布,以视网膜节细胞层(GcL)、内网层(IPL)和成神经细胞层(NBL)的内层更明显,随着发育进程,其阳性产物逐渐局限于视网膜节细胞的胞浆和胞核;蛋白半定量结果显示,生后1周内STA13蛋白表达水平较高,之后逐渐降低,成年后最低。结论 STA13蛋白的表达及变化可能与视网膜生后早期发育密切相关。     

Roller organ cultures of the retina from postnatal rats

Whole retinas of 2–14-day-old rats were cultured in a roller device for 2–14 days. Floating retinas of 7–14-day-old rats formed hole spheroid structures (spheroids) with the wall completely retaining the linear structure and layer-by-layer cellular and fibrous architecture, including the outer nuclear, outer plexiform, inner nuclear, inner plexiform layers, layers of ganglion cells and nerve fibers. The retina obtained at earlier terms of development often formed folds, with pyknotic nuclei of dead neurons in their deep compartments. In organ cultures of the retina isolated from rats at early postnatal periods, rosettes were formed in sites of local injury to the outer nuclear layer and pigmented epithelium. Roller organ cultures can be used for in vitro studies of the development and experimental diseases of the retina. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 10, pp. 471–474, October, 2006     

急性高眼压后大鼠视网膜谷氨酰胺合成酶的表达变化

为了探索谷氨酰胺合成酶在青光眼视网膜中的表达变化及其可能作用,本实验用急性高眼压模型,结合免疫组织化学染色和Western blot检测了急性高眼压后大鼠视网膜中谷氨酰胺合成酶的表达。结果显示:正常视网膜中,谷氨酰胺合成酶免疫组织化学染色主要见于Muller细胞胞体;0 d组中,Muller细胞胞体染色稍淡,而内网层中表达增加,且呈明显的点状分布; 1d组、3 d组中Muller细胞胞体染色进一步变淡,但内网层中呈现弥散染色。至再灌第7 d、14 d,Muller细胞胞体又出现浓的染色。平均灰度值显示:与正常组相比,0 d组中谷氨酰胺合成酶表达有增加但差异无显著性;1 d组中表达显著增加,3 d组、7 d组表达逐渐减少,至第14 d时基本恢复正常。Western blot显示谷氨酰胺合成酶为一分子量约为45 kD的单一蛋白带,与其它组相比,1 d组中表达显著增加。提示:急性高眼压导致的视网膜缺血再灌早期,Muller细胞中谷氨酰胺合成酶的快速重新分布和表达上调可能加速了胞外谷氨酸的代谢,对缺血再灌条件下的视网膜特别是节细胞起到保护作用。     

Ionotropic purinergic receptors P2X in frog and turtle retina: Glial and neuronal localization

Purinergic signaling is represented in both the peripheral and central nervous system (CNS), and in particular in the retina, which may be regarded as a part of the CNS. While purigenic signaling is relatively well studied in mammalian retinas, little is known about it in retinas of lower vertebrates. The aim of present study was to investigate, using immunocytochemistry, the distribution of purinoreceptors P2X in retinas of frog and turtle, which are appropriate models of the brain neuron-to-glia interactions. The results showed widespread expression of all seven ionotropic purinoreceptors (P2X1–P2X7) in both frog and turtle retinas. They were predominantly expressed in Müller cells, the principal glial cells in the retina. All structures typical of Müller cells: the outer and the inner limiting membranes, the cells bodies in the inner nuclear layer, the radial processes in the inner plexiform layer (IPL), and the so called endfeet (frog) or the orthogonal arrays of particles (turtle) in the ganglion cells layer were immunostained. Colocalizations between P2X1–P2X7 and the glial cell marker Vimentin proved that the immunostaining was in the Müller cells. In addition to the glial staining, neuronal staining was also seen as fine puncta in the inner plexiform layer and by small dots and patches in the outer plexiform layer. Some cell bodies of horizontal, amacrine and ganglion cells were also stained. The results obtained imply that the purinergic P2X receptors may significantly contribute to the neuron-to-glia signaling in retinas of the lower vertebrates.