Electrophysiological responses of the mouse retina to 12C ions

Phosphenes ("light flashes") have been reported by most astronauts on space missions and by healthy subjects whose eyes were exposed to ionizing radiation in early experiments in particle accelerators. The conditions of occurrence suggested retinal effects of heavy ions. To develop an in vivo animal model, we irradiated the eyes of anesthetized wild-type mice with repeated bursts of 12C ions delivered under controlled conditions in accelerator. 12C ions evoked electrophysiological retinal mass responses and activated the visual system as indicated by responses recorded from the visual cortex. No retinal immunohistological damage was detected. Mice proved a suitable animal model to study radiation-induced phosphenes in vivo and our findings are consistent with an origin of phosphenes in radiation activating 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.     

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.     

The area centralis of the retina in the cat and other mammals: focal point for function and development of the visual system

In many mammals, particularly species with frontalised eyes, a small region o retina is strongly specialised for high resolution, binocular vision. The region is typically located near the centre of the retina, a few millimetres temporal to the optic disc, and is termed the "area centralis" or, in some primates in which the specialisation is particularly well developed, the "fovea centralis". Where the specialisation is well developed, the area or fovea centralis dominates the organisation of the adult visual system. Studies of the histogenesis of the retina of the cat indicate that the process of retinal maturation is centred on the area centralis, which thus seems to be an organising focus in the ontogeny as well as the adult function of the visual system.     

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.     

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.     

Distinct neuronal localization of microtubule-associated protein 4 in the mammalian brain

Although recent studies have suggested the role of microtubule-associated protein (MAP) 4 in some neuron-specific events, there are no reports that directly observed its neuronal localization. Here we show the detailed expression of MAP4 in the mammalian brain. Immunoblotting revealed the presence of MAP4 in all neuronal tissues. The site-specific localization of MAP4 was observed in sagittal brain sections: MAP4 was rich in brain-specific cells, cerebellum Purkinje cells and hippocampus pyramidal cells. When primary cultures of cortical neurons were immunostained, MAP4 was detected in the cell bodies and processes with patchy staining pattern. These results suggested that MAP4 play some roles in the central nervous system, such as the dynamic cytoskeletal reorganization and regulation of the microtubule-dependent long-range transport.     

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.     

Gaseous transmitters in human retinogenesis

Endogenous gaseous transmitters (nitric oxide, carbon monoxide, and hydrogen sulphide) form a special neuromodulation system mediating the development and modification of nerve centers. Here, we examined the localization of key gaseous transmitter enzymes: cystathionine β-synthetase (CBS), cystathionine γ-lyase (CSE), heme oxygenase 2 (HO-2), and constitutive NO synthase (nNOS) in the fetal human retina at different stages of development. The number of CBS- and CSE-positive photoreceptors and intermediate retinal neurons was high in trimester I and gradually decreased to the end of trimester III. The number of HO-2-positive cells followed the same trend. The number of nNOS-positive intermediate retinal neurons and neurons within the ganglion cell layer showed the opposite dynamics with the peak in trimester III. The results are interpreted in terms of the role of gaseous transmitters in retinogenesis and cytoprotection.     

Immunohistochemical localization and ontogenic development of prolactin-releasing peptide in the brain of the ovoviviparous fish species Poecilia reticulata (guppy)

Immunohistochemical localization and ontogenic development of prolactin-releasing peptide (PrRP) in the brain of the ovoviviparous fish species Poecilia reticulata (guppy) were examined to gain a better understanding of this hormone in teleost fish. In adult guppies, PrRP-immunoreactive (ir) cell bodies were detected in the posterior part of the hypothalamus. In the pituitary, a small number of PrRP-ir fibers were observed adjacent to the prolactin cells, whereas numerous PrRP-ir fibers were detected not only in the hypothalamus but also widely throughout the brain. PrRP-ir cell bodies and prolactin cells were already detected on the birth day in the hypothalamus and pituitary, respectively. The number of PrRP-ir fibers in the brain increased as the fish developed. These results suggest that PrRP is involved in neuromodulation in the brain and that PrRP plays some physiological roles in the early development of the guppy.     

Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RIα2C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation

Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RIα, RIIα, Cα and Cβ, barely detectable levels of RIβ mRNA, and no detectable RIIβ or Cγ mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RIα and RIIα, but no RIβ or RIIβ. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[³²P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RIβ. Immunofluorescence staining of RIα and RIIα demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RIα, whereas the RIIα subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RIα subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RIIα subunits remains unchanged.     

Intracerebral injection of oil cyst content of human craniopharyngioma (oil machinery fluid) as a toxic model in the rat brain

Craniopharyngiomas (CPs) are benign epithelial cystic tumors of the sellar and suprasellar region with a high survival rate and high recurrence in children. CPs contain dense oily fluid, but little is known yet about this content and its contribution to tissue damage and tumoral growth. In this study, we developed a simple experimental model produced by intracortical injection to rats of the cyst fluid content collected from human CPs to explore its possible contribution to brain tissue damage. The cyst fluid of the CPs (“oil machinery fluid”) was collected during surgical removal, briefly preserved and further tested in rats through intracortical infusion. The group receiving “oil machinery fluid” presented increased reactive oxygen species formation, oxidative damage to lipids and reactive gliosis accompanied by augmented immunoreactivity to peroxiredoxin and thioredoxin reductase 1 at 15, 30 and 45 days post-injection. Other markers of inflammation and cell damage were stimulated at all post-lesion days tested. There was also a body weight gain. The persistence of tissue damage and oxidative stress suggests that “oil machinery fluid” exerts progressive alterations similar to those observed in patients with CPs, supporting the concept that some components of cyst fluid may contribute to brain tissue damage in these patients.     

The multi-faceted basis of vitamin B12 (cobalamin) neurotrophism in adult central nervous system: Lessons learned from its deficiency

Glial cells, myelin and the interstitium are the structures of the mammalian central nervous system (CNS) mainly affected by vitamin B₁₂ (cobalamin, Cbl) deficiency. Most of the response to the damage caused by Cbl deficiency seems to come from astrocytes and microglia, and is manifested as an increase in the number of cells positive for glial fibrillary acidic protein, the presence of ultrastructural signs of activation, and changes in cytokine and growth factor production and secretion. Myelin damage particularly affects the lamellae, which are disorganized by edema, as is the interstitium. Surprisingly, rat Schwann cells (myelin-forming cells of the peripheral nervous system) are fully activated but the few oligodendrocytes (myelin-forming cells of the CNS) are scarcely activated. The presence of intramyelin and interstitial edema raises questions about the integrity of the blood–brain barrier and blood–cerebrospinal fluid (CSF) barrier.     

Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS

Analysis of the interplay between cell proliferation and death has been greatly advantaged by the development of CNS slice preparations. In slices, interactions between neurons and neurons and the glial cells are fundamentally preserved in a fashion close to the in vivo situation. In parallel, these preparations offer the possibility of an easy experimental manipulation. Two main types of slices are currently in use: the acute slices, which are short living preparations where the major functions of the intact brain (including neurogenesis) are maintained, and the organotypic cultures, where the maturation and plasticity of neuronal circuitries in relation to naturally occurring neuronal death and/or experimental insults can be followed over several weeks in vitro.