Immunocytochemical description of five bipolar cell types of the mouse retina

With the ever-growing number of transgenic mice being used in vision research, a precise knowledge of the cellular organization of the mouse retina is required. As with the cat, rabbit, rat, and primate retinae, as many as 10 cone bipolar types and one rod bipolar type can be expected to exist in the mouse retina; however, they still have to be defined. In the current study, several immunocytochemical markers were applied to sections of mouse retina, and the labeling of bipolar cells was studied using confocal microscopy and electron microscopy. By using antibodies against the neurokinin-3 receptor NK3R; the plasma membrane calcium ATPase1 (PMCA1); and the calcium (Ca)-binding proteins CaB1, CaB5, caldendrin, and recoverin, three different OFF-cone bipolar cells could be identified. One type of ON-cone bipolar cell was identified through its immunoreactivity for CaB5 and PMCA1. Rod bipolar cells, comparable in morphology to those of other mammalian retinae, expressed protein kinase Calpha and CaB5. It was also shown that putative OFF-cone bipolar cells receive light signals through flat contacts at the cone pedicle base, whereas ON-cone bipolar signaling involves invaginating contacts. The distribution of the kainate receptor subunit GluR5 was studied by confocal and electron microscopy. GluR5 was expressed at flat bipolar cell contacts; however, it appears to be involved with only certain types of OFF-cone bipolar cells. This suggests that different bipolar cell types receive their light signals through different sets of glutamate receptors.     

Expression of janusin (J1–160/180) in the retina and optic nerve of the developing and adult mouse

We have analyzed the expression of the oligodendrocyte-derived extracellular matrix molecule janusin (previously termed J1–160/180) in the retina and optic nerve ofdeveloping and adult mice using indirect light and electron microscopic immunocytochemistry, immunoblot analysis, and enzyme-linked immunosorbent assay. In the optic nerve, janusin is not detectable in neonatal and only weakly detectable in 7-day-old animals. Expression is at a peak in 2- or 3-week-old animals and subsequently decreases with inceasing age. In the retina, expression inceases until the third postnatal week and then remains at a constant level. In immunocytochemical investigations at the light microscopic level, janusin was found in the myelinated regions of the nerve with spots of increased immunoreactivity possibly corresponding to an accumulation of the light microscopic level, janusin was found in the myelinated regions of the nerve with sports of increased immunoreactivity possibly corresponding to an accumulation of the molecule at the nodes of Ranvier. At the electron microscopic level, contact sites between unmyelinated axons, between axons, and glial cells, and between axons and processes of myelinating oligodendrocytes were immunoreactive. Cell surfaces of astrocytes at the periphery of the nerve and forming the glial-limiting membrane, in contrast, were only weakly immunopositive or negative. In cell cultures of young postnatal mouse or rat optic nerves, oligodendrocytes and type-2 astrocytes, but not type-1 astrocytes were stained by janusin antibodies. In the oligodendrocyte-free retina, janusin was detectable in association with neuronal cell surfaces, but not with cell surfaces of Muller cells or retinal astrocytes. Our observations indicate that expression of janusin in the optic nerve and in the retina is developmentally differentially regulated and that other cell types, in addition to oligodendrocytes, express the molecule. Since the time course of janusin expression in the optic nerve coincides with the appearance of oligodendrocytes and myelin and since janusin is associated with cell surfaces of oligodendrocytes and outer aspects of myelin sheaths and is concentrated at nodes of Ranvier, we suggest that janusin is functionally involved in the process of myelination. © 1993 Wiley-Liss, Inc.     

Photic regulation of c-fos expression in neural components governing the entrainment of circadian rhythms.

The rapid and transient induction of the proto-oncogene c-fos in mature neurons within the brain occurs in response to a variety of extracellular stimuli. To determine whether lighting conditions influence c-fos gene expression in the primary neural structures mediating the photoentrainment and generation of mammalian circadian rhythms, the expression of the c-fos protein (Fos) and related proteins in the retina and suprachiasmatic nuclei (SCN) of the anterior hypothalamus was examined immunohistochemically in rats exposed to a light-dark cycle of 12 h of light and 12 h of darkness (LD 12:12), constant light (LL), or constant dark (DD). The retina exhibited clear light-dark differences in the expression of Fos protein(s), such that immunopositive nuclei were readily evident during exposure to light (i.e., during the day of diurnal lighting or in LL), but were absent during exposure to darkness. In the SCN, the distribution of Fos immunoreactivity within specific subfields was differentially affected by photic conditions. Following exposure to light, a dense population of Fos-immunopositive cells was found in close association with the immunohistochemically distinct cell and fiber populations distinguishing the ventrolateral subfield of the SCN. In dark-exposed animals, Fos-immuno-reactive profiles were distributed throughout the SCN in areas coextensive with the immunohistochemical localization of peptidergic neural elements in both the ventrolateral and dorsomedial subfields. As a consequence of this light-dark difference in the distribution of Fos immunoreactivity, the density of labeled cells was increased within the ventrolateral SCN, but was decreased within the dorsomedial subfield, as a result of exposure to light versus darkness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic expression of ganglion cell markers in retinal progenitors during the terminal cell cycle

The vertebrate neural retina contains seven major cell types, which arise from a common multipotent progenitor pool. During neurogenesis, these cells stop cycling, commit to a single fate, and differentiate. The mechanism and order of these steps remain unclear. The first-born type of retinal neurons, ganglion cells (RGCs), develop through the actions of Math5 (Atoh7), Brn3b (Pou4f2) and Islet1 (Isl1) factors, whereas inhibitory amacrine and horizontal precursors require Ptf1a for differentiation. We have examined the link between these markers, and the timing of their expression during the terminal cell cycle, by nucleoside pulse-chase analysis in the mouse retina. We show that G2 phase lasts 1-2 h at embryonic (E) 13.5 and E15.5 stages. Surprisingly, we found that cells expressing Brn3b and/or Isl1 were frequently co-labeled with EdU after a short chase (<1 h) in early embryos (E15), Brn3b and Isl1 were exclusively expressed in post-mitotic cells, even as new RGCs are still generated. In contrast, Ptf1a and amacrine marker AP2α were detected only after terminal mitosis, at all developmental stages. Using a retroviral tracer in embryonic retinal explants (E12-E13), we identified two-cell clones containing paired ganglion cells, consistent with RGC fate commitment prior to terminal mitosis. Thus, although cell cycle exit and fate determination are temporally correlated during retinal neurogenesis, the order of these events varies according to developmental stage and final cell type.     

Dopaminergic and indoleamine-accumulating amacrine cells express GABA-like immunoreactivity in the cat retina

Colocalization of indoleamine uptake and GABA-like immunoreactivity was studied in the cat retina. Consecutive, semithin sections were incubated in antisera to either 5-HT (5-hydroxytryptamine) or GABA. More than 90% of all 5-HT-accumulating amacrine cells expressed GABA-like antigens. With the same approach, the colocalization of 5-HT uptake and GABA-like immunoreactivity was studied in rabbit and 75-80% of the 5-HT-accumulating amacrine cells expressed GABA-like immunoreactivity, thus confirming a previous study (Osborne and Beaton, 1986). Since, in both cat and rabbit, endogenous 5-HT could not be found by immunocytochemistry, one must consider the possibility that some GABAergic amacrine cells take up indoleamines. In the cat retina, antibodies against tyrosine hydroxylase (TH) label dopaminergic amacrine cells (Oyster et al., 1985). By incubating consecutive, semithin sections in antisera to either TH or GABA, it was found that 84% of the dopaminergic amacrine cells also expressed GABA-like immunoreactivity. GABA-like immunoreactivity and 3H-muscimol uptake were found to be colocalized in more than 90% of the amacrine cells labeled. However, dopaminergic amacrine cells did not accumulate 3H-muscimol. Evidence is presented from colocalization studies for 2 types of interplexiform cell in the cat retina. One is stained by GABA-like immunocytochemistry and by 3H-muscimol uptake. The other is the dopaminergic amacrine cell, which also expresses GABA-like immunoreactivity, but does not accumulate 3H-muscimol.     

Photic regulation of c-fos expression in neural components governing the entrainment of circadian rhythms

The rapid and transient induction of the proto-oncogene c-fos in mature neurons within the brain occurs in response to a variety of extracellular stimuli. To determine whether lighting conditions influence c-fos gene expression in the primary neural structures mediating the photoentrainment and generation of mammalian circadian rhythms, the expression of the c-fos protein (Fos) and related proteins in the retina and suprachiasmatic nuclei (SCN) of the anterior hypothalmus was examined immunohistochemically in rats exposed to a light-dark cycle of 12 h of light and 12 h of darkness (LD 12:12), constant light (LL), or constant dark (DD). The retina exhibited clear light-dark differences in the expression of Fos protein(s), such that immunopositive nuclei were readily evident during exposure to light (i.e., during the day of diurnal lighting or in LL), but were absent during exposure to darkness. In the SCN, the distribution of Fos immunoreactivity within specific subfields was differentially affected by photic conditions. Following exposure to light, a dense population of Fos-immunopositive cells was found in close association with the immunohistochemically distinct cell and fiber populations distinguishing the ventrolateral subfield of the SCN. In dark-exposed animals, Fos-immunoreactive profiles were distributed throughout the SCN in areas coextensive with the immunohistochemical localization of peptidergic neural elements in both the ventrolateral and dorsomedial subfields. As a consequence of this light-dark difference in the distribution of Fos immunoreactivity, the density of labeled cells was increased within the ventrolateral SCN, but was decreased within the dorsomedial subfield, as a result of exposure to light versus darkness. In the absence of photic time cues, temporal variation in the pattern of Fos immunostaining in the SCN, or within specific subfields of the nucleus, was evident only within dorsomedial SCN during exposure to LL, such that the density of immunopositive cells was greater during the subjective day than during the subjective night. These data demonstrate that light stimulation causes an increase in the expression of Fos protein(s) in the retina and within the ventrolateral, but not the dorsomedial, subfield of the SCN. The inductive effect of light on Fos expression within the retina and the ventrolateral or retinorecipient subfield of the SCN suggests that Fos protein(s) may play a role in the transduction of light signals by the primary neural components governing the generation and photoentrainment of circadian rhythms in mammals.     

Ontogeny of somatostatin immunoreactivity in the cat retina.

In the ganglion cell layer of the adult cat retina, subgroups of displaced amacrine cells and alpha ganglion cells are immunoreactive for somatostatin or a somatostatinlike substance. Both types of immunoreactive cells are found preferentially in inferior retina. We studied the development of somatostatin immunoreactivity in the prenatal and postnatal cat retina to determine how such unusual distributions of immunoreactive cells arise. Somatostatin-immunoreactive profiles were first observed at embryonic day (E) 30, within the inner retina in a central region that included the optic disk and the area centralis. By E36, immunoreactivity had virtually disappeared from the central retina but was present throughout the periphery. The immunoreactive profiles could not be classified morphologically because of their immaturity but were most likely retinal ganglion cells, the earliest born cells of the inner retina. Of the two types of immunoreactive cell observed in the adult, the first to be recognized morphologically was the displaced amacrine cell, at E45. These cells were virtually adultlike in morphology and number by E51, two weeks before birth. In contrast, immunoreactive alpha ganglion cells were not apparent until five days after birth and did not achieve their mature numbers and immunoreactive staining characteristics until more than a month later. From the time they could initially be recognized, both immunoreactive displaced amacrine cells and alpha cells were distributed mainly in the inferior retina. A third type of somatostatin-immunoreactive cell was transiently observed in the superior and inferior retina during prenatal and early postnatal development. These cells were characterized by granular staining in irregular shapes and few, if any, faintly stained processes. Injections of retrograde tracers into retinorecipient targets revealed that many of these cells were retinal ganglion cells. They disappeared by postnatal day 38. Our results indicate that somatostatin immunoreactivity initially follows a central-to-peripheral pattern of development, as is typical of other developmental events in the mammalian retina. They also indicate that the two types of somatostatin-immunoreactive neurons present in the adult cat retina (displaced amacrine and alpha ganglion cells) attain their mature immunocytochemical properties with very different timecourses. Finally, the observation that somatostatin immunoreactivity appears transiently in the granular-staining ganglion cells, distributed throughout the superior and inferior retina, suggests that the peptide may play a regulatory role in the development of the retina and/or retinofugal pathways.     

Distinct Temporal Patterns of Expression of Sodium Channel-like Immunoreactivity During the Prenatal Development of the Monkey and Cat Retina

Polyclonal and monoclonal antibodies prepared against the α-subunit of the voltage-gated sodium channel (αNaCh) were used to examine the distribution of sodium channel-like immunoreactivity during the prenatal development of the cat and rhesus monkey ( Macaca mulatta ) retina. At all prenatal ages studied, beginning on embryonic day 29 (E29) in the cat and E52 in the monkey, both antibodies labelled optic axons. With the polyclonal antibodies, the appearance of positive cells largely mirrored the onset of their morphological maturation. Immunoreactivity appeared first in the somata of ganglion cells, and subsequently the inner plexiform layer could be distinguished by its intense immunolabelling. A few weeks later horizontal cells displayed immunolabelling that extended to their dendrites in the developing outer plexiform layer. This was followed by immunoreactive cones, with bipolar cells labelled only postnatally. By contrast, with the monoclonal antibody some cells were found to be immunoreactive while their somata were still in the ventricular layer (E33 in cat and E52 in monkey). Many of these cells appeared to migrate to the outer portion of the prospective inner nuclear layer, where they gradually acquired the morphological appearance of bipolar cells. Transient expression of immunolabelling with monoclonal sodium channel antibody was found in the cones of the cat and cones and rods of the monkey. These results indicate that different types of αNaCh-like proteins are expressed in the mammalian retina at distinct developmental periods. Their presence at very early stages during development suggests that these proteins could play a specific role in the commitment and/or differentiation of specific retinal cell types.     

Early postnatal expression of L1 by retinal fibers in the optic tract and synaptic targets of the Syrian hamster

Previous immunohistochemical studies in mouse, rat, and chick have reported that the expression of the glycoprotein and cell adhesion molecule L1, a member of the immunoglobulin superfamily, shows regulation during development of retina and optic nerve. To extend our understanding of the role of L1 in developing neural circuitry, we have examined L1 expression in the optic tract and thalamic and midbrain synaptic targets of retinal fibers in the early postnatal Syrian hamster, a well-characterized developmental model of the primary visual projection. Metabolic labeling studies reveal that a synaptically targeted, sulfated, and glycosylated form of L1 undergoes rapid axonal transport from the retina. Retinofugal transport of L1 decreases commensurate with the decline in immunoreactivity of retinal fibers in the visual pathway. Retinal ganglion cell axons show intense L1 immunoreactivity as they navigate in highly fasciculated bundles in the optic tract overlying the lateral geniculate body and in the superior colliculus. We found no evidence of L1 immunoreactivity on retinal axon collaterals as they defasciculate from the optic tract and branch into target neuropils. L1 immunoreactivity wanes in optic tract as axon terminal arbors are elaborated in the lateral geniculate body and superior colliculus and as myelination in the visual pathway commences. This pattern of L1 expression suggests that, in the early postnatal period, L1 may support fasciculation of retinal fibers, maintaining them within the optic tract, and that subsequent down-regulation of L1 may facilitate their terminal arborization and myelination.     

Horizontal Cells in the Monkey Retina: Cone connections and dendritic network

Horizontal cells of the macaque monkey retina were quantified and the number of cones converging onto an individual horizontal cell as well as the number of horizontal cells contacting a single cone were determined. This was done by combining data from individual horizontal cells stained by the Golgi method with the results of immunocytochemical staining described in the preceding paper (Röhrenbeck et al., 1989). The observation (Boycott et al., 1987) that all horizontal cells contact all cones in their dendritic field irrespective of cone type was confirmed. The particular cones contacted by the terminal aggregates of each horizontal cell were found. The dendritic fields of H1 and H2 cells increase with increasing eccentricity; close to the fovea H1 cells are smaller than H2 cells, at 6 mm eccentricity they are about the same size and in peripheral retina H1 cells are much larger than H2 cells. The density gradients of the two cell types balance their denritic field changes so that throughout the retina each and every cone synapses with 3–5 horizontal cells of each type. Horizontal cells of both cat (Wässle et al., 1978) and monkey retina follow the general rule that all cones in the dendritic fields are contacted, their perikarya form a regular mosaic and the boundaries of their dendritic fields are marked by the perikarya of their homologous neighbours.     

Substance P-like immunoreactive amacrine cells in the cat retina

Substance P-like immunoreactivity was localized by immunocytochemical techniques to two subpopulations of amacrine cells in the cat retina. One cell was a unistratified amacrine with processes ramifying within stratum 4 of the inner plexiform layer. The other cell type was a bistratified cell with processes in both stratum 1 (s1) and stratum 4 (s4). Both cell types were seen with their somas displaced to the ganglion cell layer as well as in the conventional amacrine location in the inner nuclear layer. Substance P cells were present in the greatest density within the area centralis and decreased in number toward the periphery. The ratio of amacrine to displaced amacrine cells also decreased peripherally. However, the coverage by immunoreactive fibers in s4 remained three times that seen in s1. Computer-assisted analysis confirmed the location of substance P-containing processes at 5-15% (s1) and 50-70% (s4) depth levels in the inner plexiform layer. A comparison of substance P-like immunoreactivity in light- and dark-adapted cat retinas showed no apparent differences in the distribution of immunoreactivity due to lighting conditions.     

Localization of the calcium‐binding protein secretagogin in cone bipolar cells of the mammalian retina

Secretagogin, a recently cloned member of the EF‐hand family of calcium binding proteins, was localized in the mouse, rat, and rabbit retina using immunofluorescence immunohistochemistry. Secretagogin is expressed in subpopulations of ON and OFF cone bipolar cells; however, no immunoreactivity was observed in rod bipolar cells in any of these species. Using subtype‐specific markers and mice expressing green fluorescent protein (GFP) within specific cell classes, we found that secretagogin is expressed in Types 2, 3, 4, 5, 6 and possibly Type 8 cone bipolar cells in the mouse retina. The expression pattern in the rat retina differs slightly with expression in cone bipolar cell Types 2, 5, 6, 7, and 8. Evaluation of secretagogin in the developing mouse retina revealed expression as early as postnatal day 6, with OFF cone bipolar cells showing secretagogin expression prior to the ON cone bipolar cells. Secretagogin is a useful marker of cone bipolar cells for studying alterations in bipolar cell morphology during development and degeneration. Further work will be necessary to elucidate the functional role of this protein in bipolar cells. J. Comp. Neurol. 518:513–525, 2010. © 2009 Wiley‐Liss, Inc.     

AII amacrine cells in the mammalian retina show disabled-1 immunoreactivity

Disabled 1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by Reelin, which controls cell positioning in the developing brain. It has been localized to AII amacrine cells in the mouse and guinea pig retinas. This study was conducted to identify whether Dab1 is commonly localized to AII amacrine cells in the retinas of other mammals. We investigated Dab1-labeled cells in human, rat, rabbit, and cat retinas in detail by immunocytochemistry with antisera against Dab1. Dab1 immunoreactivity was found in certain populations of amacrine cells, with lobular appendages in the outer half of the inner plexiform layer (IPL) and a bushy, smooth dendritic tree in the inner half of the IPL. Double-labeling experiments demonstrated that all Dab1-immunoreactive amacrine cells were immunoreactive to antisera against calretinin or parvalbumin (i.e., other markers for AII amacrine cells in the mammalian retina) and that they made contacts with the axon terminals of the rod bipolar cells in the IPL close to the ganglion cell layer. Furthermore, all Dab1-labeled amacrine cells showed glycine transporter-1 immunoreactivity, indicating that they are glycinergic. The peak density was relatively high in the human and rat retinas, moderate in the cat retina, and low in the rabbit retina. Together, these morphological and histochemical observations clearly indicate that Dab1 is commonly localized to AII amacrine cells and that antiserum against Dab1 is a reliable and specific marker for AII amacrine cells of diverse mammals.     

Spatial density and immunoreactivity of bipolar cells in the macaque monkey retina.

The anatomical substrates of spatial and color vision in the primate retina are investigated by measuring the immunoreactivity and spatial density of bipolar, amacrine and horizontal cells in the inner nuclear layer of the macaque monkey retina. Bipolar cells can be distinguished from amacrine and horizontal cells by their differential immunoreactivity to antisera against glutamate, glycine, GABA, parvalbumin, calbindin (CaBP D-28K), and the L7 protein from mouse cerebellum. The spatial density of bipolar cells is compared to the densities of photoreceptors and ganglion cells at different retinal eccentricities. In the centralmost 2 mm, cone bipolar cells outnumber ganglion cells by about 1.4:1. The density of cone bipolar cells is thus high enough to allow for input to different (parasol and midget) ganglion cell classes by different (diffuse and midget) bipolar cell classes. The density gradient of cone bipolar cells follows closely that of ganglion cells in central retina but falls less steeply in peripheral retina. This suggests that the convergence of cone signals to the receptive fields of ganglion cells in the peripheral retina occurs in the inner plexiform layer. The density of cone bipolar cells is 2.5-4 times that of cones at all eccentricities studied, implying that cone connectivity to bipolar cells remains constant throughout the retina. Different subgroups of bipolar cells are distinguished by their relative immunoreactivity to the different antisera. All rod and cone bipolar cells show moderate to strong glutamate-like immunoreactivity. The bipolar cells that show weak to moderate GABA-like immunoreactivity are also labeled with the antiserum to the L7 protein and are thus identified as rod bipolar cells. Nearly half of all cone bipolar cells showed glycine-like immunoreactivity. The results suggest that the inhibitory neurotransmitter candidates GABA and glycine are segregated respectively in rod and cone bipolar cell pathways. A diffuse, cone bipolar cell type can be identified by the anti-parvalbumin and the anti-calbindin antisera. All horizontal cells show parvalbumin-like immunoreactivity. Nearly all amacrine cells show GABA-like or glycine-like immunoreactivity; a variety of subpopulations also show immunoreactivity to one or more of the other markers used.     

Differential expression and cellular localization of doublecortin in the developing rat retina

Doublecortin is 40 kDa microtubule-associated phosphoprotein required for neuronal migration and differentiation in various regions of the developing central nervous system. We have investigated the expression and cellular localization of doublecortin in the developing rat retina using immunocytochemistry and Western blot analysis. The expression of doublecortin was high from embryonic day 18 (E18) until E20 and was low during the postnatal period. The doublecortin immunoreactivity first appeared in a few radially orientated cells in the mantle zone of the primitive retina at E15. From E16 onward, the immunoreactivity appeared in two different regions: the inner part of the retina and middle of the neuroblastic layer. In the inner part, the somata of cells in the ganglion cell layer, in the distal row of the neuroblastic layer and profiles in the inner plexiform layer showed doublecortin immunoreactivity up to postnatal day 1 (P1). Afterwards, the doublecortin immunoreactivity persisted in the inner plexiform layer until P15, although the intensity decreased gradually with the maturation of the retina. In the middle of the neuroblastic layer, doublecortin immunoreactivity appeared in the radially orientated cells. These cells transformed into horizontal cells. The doublecortin immunoreactivity persisted in these cells up to P21. Given these results, doublecortin may play an important role in the migration and differentiation of specific neuronal populations in developmental stages of the rat retina.     

Choline acetyltransferase is found in terminals of horizontal cells that label with GABA, nitric oxide synthase and calcium binding proteins in the turtle retina

In this study, we discriminated the various types of horizontal cell in the turtle retina on their content of neuroactive substances. Double label immunocytochemistry was performed on sectioned and wholemount retina using antisera to neural- and endothelial-nitric oxide synthase (nNOS, and eNOS), calretinin (CR), calbindin (CB), gamma-aminobutyric acid (GABA) and choline acetyltransferase (ChAT). H1 cells and their axon terminals label with CR, CB and GABA. Only H1 axon terminals label with eNOS. H2 cells contain CB, CR, nNOS and GABA maybe in their dendrites. H3 cells label only with nNOS. The localization of nNOS in the H2 and H3 cells is a novel finding. None of these antibodies labels H4 cells. The photoreceptor subtypes have been differentiated by different intensity of labeling with CB. The accessory member of the double cone is less intensely labeled with CB than the principal member and rods and blue cones do not appear to label at all. ChAT-IR is located in terminal boutons of H1 and H2 horizontal cells and H1 axon terminals and these boutons contact rods and all spectral types of cones. Clearly, GABA is present in H1 horizontal cells and may be used in neurotransmission between horizontal cells and possibly for feedback pathways to photoreceptors. The evidence of nNOS immunoreactivity in H2 and H3 horizontal cells, combined with available physiological evidence, suggests that NO may be involved in electrical coupling and/or modulation of synaptic input to these types of cells. Furthermore, our results raise the possibility that cholinergic synaptic transmission may occur from horizontal cell processes to photoreceptors in the outer plexiform layer of the turtle retina.     

Induction of heat shock (stress) protein 70 and its mRNA in the normal and light-damaged rat retina after whole body hyperthermia

In situ hybridization and immunocytochemistry were used to investigate the distribution of the 70 kDa heat shock or stress protein (hsp70) and its mRNA in specific layers of the retina of adult rats at 0, 4, 18, and 48 or 50 hr after a brief whole body hyperthermic treatment. Induction of hsp70 mRNA was noted in the photoreceptor layer of the retina within 4 hr after hyperthermia. Pronounced accumulation of inducible hsp70 immunoreactivity was observed in cytoplasmic extensions of the photoreceptor cells, especially the inner segment zone which attained peak levels at the 18 hr time point. Selective destruction of photoreceptors by light damage prior to hyperthermia inhibited the post-hyperthermic rise in newly synthesized retinal hsp70. Our results suggest that the photoreceptor cell layer is the primary site of synthesis of hsp70 in the rat retina and that the greatest increase in hsp70 immunoreactivity following such a hyperthermic stress occurs in that layer. This stress response of the photoreceptors is discussed in relation to their location and function in the retina. © 1994 Wiley-Liss, Inc.     

Expression of the high-affinity glutamate transporter EAAT4 in mammalian cerebral cortex

RT-PCR, immunocytochemistry and Western blotting were used to study the expression of the glutamate transporter EAAT4 in the cerebral cortex of cat and mouse. By means of RT-PCR we were able to detect EAAT4 mRNA in the cerebral cortex of both species. Sequencing ensured the specificity of the amplified fragment. Immunocytochemistry and Western blotting enabled us to localize EAAT4 protein in cat and mouse cerebral cortex. Intense EAAT4 immunoreactivity was found in the soma and dendrites of neurons mainly of layers II, III and V. For both species, the signal in the cerebellum was very intense and confined to the molecular and Purkinje cell layer.