Axonal Synapses Utilize Multiple Synaptic Ribbons in the Mammalian Retina

In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a, thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a. The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.     

The nature of surround-induced depolarizing responses in goldfish cones

Cones in the vertebrate retina project to horizontal and bipolar cells and the horizontal cells feedback negatively to cones. This organization forms the basis for the center/surround organization of the bipolar cells, a fundamental step in the visual signal processing. Although the surround responses of bipolar cells have been recorded on many occasions, surprisingly, the underlying surround-induced responses in cones are not easily detected. In this paper, the nature of the surround-induced responses in cones is studied. Horizontal cells feed back to cones by shifting the activation function of the calcium current in cones to more negative potentials. This shift increases the calcium influx, which increases the neurotransmitter release of the cone. In this paper, we will show that under certain conditions, in addition to this increase of neurotransmitter release, a calcium-dependent chloride current will be activated, which polarizes the cone membrane potential. The question is, whether the modulation of the calcium current or the polarization of the cone membrane potential is the major determinant for feedback-mediated responses in second-order neurons. Depolarizing light responses of biphasic horizontal cells are generated by feedback from monophasic horizontal cells to cones. It was found that niflumic acid blocks the feedback-induced depolarizing responses in cones, while the shift of the calcium current activation function and the depolarizing biphasic horizontal cell responses remain intact. This shows that horizontal cells can feed back to cones, without inducing major changes in the cone membrane potential. This makes the feedback synapse from horizontal cells to cones a unique synapse. Polarization of the presynaptic (horizontal) cell leads to calcium influx in the postsynaptic cell (cone), but due to the combined activity of the calcium current and the calcium-dependent chloride current, the membrane potential of the postsynaptic cell will be hardly modulated, whereas the output of the postsynaptic cell will be strongly modulated. Since no polarization of the postsynaptic cell is needed for these feedback-mediated responses, this mechanism of synaptic transmission can modulate the neurotransmitter release in single synaptic terminals without affecting the membrane potential of the entire cell.     

Regulation of retinal cone bipolar cell differentiation and photopic vision by the CVC homeobox gene Vsx1

Cone bipolar cells of the vertebrate retina connect photoreceptors with ganglion cells to mediate photopic vision. Despite this important role, the mechanisms that regulate cone bipolar cell differentiation are poorly understood. VSX1 is a CVC domain homeoprotein specifically expressed in cone bipolar cells. To determine the function of VSX1, we generated Vsx1 mutant mice and found that Vsx1 mutant retinal cells form but do not differentiate a mature cone bipolar cell phenotype. Electrophysiological studies demonstrated that Vsx1 mutant mice have defects in their cone visual pathway, whereas the rod visual pathway was unaffected. Thus, Vsx1 is required for cone bipolar cell differentiation and regulates photopic vision perception.     

The cone synapses of cone bipolar cells of primate retina

One each of bipolar cell types DB2 and DB4, together with a flat and an invaginating midget bipolar cell, were taken from a Golgi-stained rhesus macaque retina; then serially sectioned for EM examination of their synapses with cone pedicles. The cone input to the dendrites of the DB2 cell was exclusively at basal junctions; it had a characteristic distribution. Fifty per cent of the basal synapses were with cone pedicle membrane immediately adjacent to the dendrite of a bipolar cell invaginating to end opposite the ribbon of a cone triad (this, therefore, is called triad-associated). The remainder were one or more synapses distant from the triad-associated position (and, therefore, non-triad associated). The DB4 cell had both basal (predominantly in the triad-associated position) and ribbon-related synaptic input. But the basal to invaginating ratio differed from that of our previously published cell; 56% basal, 43% invaginating, as compared with 31% basal and 69% invaginating. Like foveal IMB cells the synapses of the mid-peripheral invaginating midget bipolar cell were exclusively invaginating; but were about 25% more numerous. The flat midget bipolar cell made exclusively basal synapses. These were 2.5 times more numerous than those of foveal flat midget bipolar cells, and 3.5 times the number of invaginating midget bipolar synapses at equivalent eccentricity. The synapses between cones and diffuse and midget bipolar cells are characteristic for each particular bipolar cell type, but the details depend on a cell’s distance from the fovea (eccentricity). A rather constant number of cone pedicle synaptic ribbons 38.6±2.5 (n±:60) was found across mid-peripheral macaque and vervet monkey retinae. The smaller mean number for vervet monkey, 27.4±3.5 (n ±:23), suggests there can also be generic differences in synaptic detail at cone bipolar cell synapses.     

Synapse formation and modification between distal retinal neurons in larval and juvenile Xenopus

A serial section analysis of photoreceptor synaptic bases was undertaken in the clawed frog Xenopus laevis. The developmental period from tadpole stage 48 through metamorphosis was studied. Horizontal cells contacted rod and cone photoreceptors at ribbon synapses; the number of such contacts per receptor base was constant for rods, but increased for cones as a function of developmental stage. In pre-metamorphic animals bipolar cells contacted receptors only through basal junctions; their number in cone bases increased dramatically during development but was unchanged in rod bases. A densitometric estimation of the cleft width of basal junctions showed that it ranged from 10 to 18 nm, but the junctions could not be divided reliably into the 'wide' and 'narrow' categories reported for other vertebrate species. Near metamorphic climax a new type of ribbon-related bipolar cell junction appeared. Gap junctions between horizontal cells and conventional synapses of horizontal cell onto bipolar cell processes were first seen in mid-larval developmental stages.     

Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c^w59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c^w59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c^w59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.     

Synaptic connections of calbindin-immunoreactive cone bipolar cells in the inner plexiform layer of rabbit retina

In the mammalian retina, information concerning various aspects of an image is transferred in parallel, and cone bipolar cells are thought to play a major role in this parallel processing. We have examined the synaptic connections of calbindin-immunoreactive (IR) ON cone bipolar cells in the inner plexiform layer (IPL) of rabbit retina and have compared these synaptic connections with those that we have previously described for neurokinin 1 (NK1) receptor-IR cone bipolar cells. A total of 325 synapses made by calbindin-IR bipolar axon terminals have been identified in sublamina b of the IPL. The axons of calbindin-IR bipolar cells receive synaptic inputs from amacrine cells through conventional synapses and are coupled to putative AII amacrine cells via gap junctions. The major output from calbindin-IR bipolar cells is to amacrine cell processes. These data resemble our findings for NK1 receptor-IR bipolar cells. However, the incidences of output synapses to ganglion cell dendrites of calbindin-IR bipolar cells are higher compared with the NK1-receptor-IR bipolar cells. On the basis of stratification level and synaptic connections, calbindin-IR ON cone bipolar cells might thus play an important role in the processing of various visual aspects, such as contrast, orientation, and approach sensing, and in transferring rod signals to the ON cone pathway.     

Cell death and tissue reorganization in Rhynchosciara americana (Sciaridae: Diptera) metamorphosis and their relation to molting hormone titers

Programmed cell death (PCD) is a focal topic for understanding processes underlying metamorphosis in insects, especially so in holometabolous orders. During adult morphogenesis it allows for the elimination of larva-specific tissues and the reorganization of others for their functionalities in adult life. In Rhynchosciara, this PCD process could be classified as autophagic cell death, yet the expression of apoptosis-related genes and certain morphological aspects suggest that processes, autophagy and apoptosis may be involved. Aiming to reveal the morphological changes that salivary gland and fat body cells undergo during metamorphosis we conducted microscopy analyses to detect chromatin condensation and fragmentation, as well as alterations in the cytoplasm of late pupal tissues of Rhynchosciara americana. Transmission electron microscopy and confocal microscopy revealed cells in variable stages of death. By analyzing the morphological structure of the salivary gland we observed the presence of cells with autophagic vacuoles and apoptotic bodies and DNA fragmentation was confirmed with the TUNEL assay in salivary gland. The reorganization of fat body occurs with discrete detection of cell death by TUNEL assay. However, both salivary gland histolysis and fat body reorganization occur under control of the hormone ecdysone.     

植物细胞程序死亡的机理及其与发育的关系

细胞程序死亡（PCD）是在植物体发育过程中普遍存在的,在发育的特定阶段发生的自然的细胞死亡过程,这一死亡过程是由某些特定基因编码的“死亡程序”控制的。PCD是细胞分化的最后阶段。细胞分化的临界期就处于死亡程序执行中的某个阶段。PCD包含启动期、效应期和清除期三个阶段,其间caspase家族起着重要作用。PCD在细胞和组织的平衡、特化,以及组织分化、器官建成和对病原体的反应等植物发育过程中起着重要作用。PCD中的形态学变化和生物化学变化都有着严格的时序性。植物的PCD和动物的PCD有许多共性,包括细胞形态和DNA降解等变化。也有一些不同,植物PCD的产物既可被其它细胞吸收利用;也可用于构建自身的次生细胞壁。     

植物细胞程序死亡的机理及其与发育的关系

细胞程序死亡（ＰＣＤ）是在植物体发育过程中普遍存在的,在发育的特定阶段发生的自然的细胞死亡过程,这一死亡过程是由某些特定基因编码的“死亡程序”控制的。ＰＣＤ的细胞分化的最后阶段。细胞分化的临界期就牌死亡程序执行中的某个阶段。ＰＣＤ包含启动期和清除期三个阶段,其间ＣＡＳＰＡＳＥ家族起着重要作用。ＰＣＤ在细胞和组织的平衡、特化,以及组织分化、器官建成和对病原体的反应等植物发育过程中起着重要作用。ＰＣＤ     

Impact of carbon and phosphate starvation on growth and programmed cell death of maritime pine suspension cells

Programmed cell death is a fundamental aspect of plant development and defense. In suspension cultures of maritime pine (Pinus pinaster Ait.), cell death was associated with the simultaneous depletion of sugar and phosphate. This present work suggests that sugar rather than phosphate deprivation induced programmed cell death events, including degradation of nuclear DNA and remobilization of phosphate. However, phosphate starvation may have a synergistic effect on programmed cell death mediated by the lack of carbon source. Sugar and phosphate analogs were used to evaluate the nature of signaling events, and results suggested that programmed cell death induction by sugar starvation occurs downstream of hexokinase-based sugar sensing mechanisms, and that the synergistic effect of lack of phosphate is independent of phosphate sensing.     

Winter wheat cells subjected to freezing temperature undergo death process with features of programmed cell death

Programmed cell death is a process defined as genetically regulated self-destruction or cell suicide. It can be activated by different internal and external factors, but few studies have investigated whether this process occurs under cold and freezing temperatures. In this study, a freezing treatment (?8 °C for 6 h) induced cell death with features of programmed cell death in suspension cultures of winter wheat (Triticum aestivum L.). This process occurred for 10 days after cold exposure. The death of cells in culture was slow and prolonged, and was accompanied by protoplast shrinkage, DNA fragmentation, and an increase in the level of reactive oxygen species. Other changes observed after the freezing treatment included an increase in the respiration rate, changes in mitochondrial transmembrane potential (?Ψ _m ), and the release of cytochrome c from mitochondria into the cytosol. These findings indicated that mitochondria are involved in the cell death process that occurs after a freezing treatment in cells of winter wheat.     

Eggs over easy: cell death in the Drosophila ovary

Programmed cell death is the most common fate of female germ cells in Drosophila and many animals. In Drosophila, oocytes form in individual egg chambers that are supported by germline nurse cells and surrounded by somatic follicle cells. As oogenesis proceeds, 15 nurse cells die for every oocyte that is produced. In addition to this developmentally regulated cell death, groups of germ cells or entire egg chambers may be induced to undergo apoptosis in response to starvation or other insults. Recent findings suggest that these different types of cell death involve distinct genetic pathways. This review focuses on progress towards elucidating the molecular mechanisms acting during programmed cell death in Drosophila oogenesis.     

Inhibition of palatal epithelial cell death by altered protein synthesis.

Programmed cell death occurs in a defined region of the secondary palatal epithelium (medial edge) during its development in vivo or in vitro. The purpose of this study was to examine the effects of various inhibitors of macromolecular synthesis, particularly glycoprotein synthesis, on the death of palatal medial-edge epithelial cells in vitro. Rat palatal shelves explanted on gestational Day 15 and cultured singly showed medial-edge cell degeneration after 48 hr, whereas in the presence of 6-diazo-5-oxo-l-norleucine (DON), 2-deoxyglucose (DOG), or cycloheximide, cell death was prevented. This was in contrast to the lack of inhibition of cell death in the presence of actionomycin D or cytosine arabinoside. Selectively adding metabolites which bypass the effect of DON on various synthetic pathways demonstrated that the inhibition of cell death by DON was most likely due to a block in glucosamine formation. The synthesis and activity of various lysosomal enzymes were not affected by DON, whereas a marked decrease was observed with both cycloheximide and DOG. These results suggest that palatal epithelial cell death is not a passive event, but an active process requiring the programmed synthesis of specific proteins.     

Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes

Programmed cell death can be divided into several categories including type I (apoptosis) and type II (autophagic death). The Bcl-2 family of proteins are well-characterized regulators of apoptosis, and the multidomain pro-apoptotic members of this family, such as Bax and Bak, act as a mitochondrial gateway where a variety of apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak double knockout mice are resistant to apoptosis, we found that these cells still underwent a non-apoptotic death after death stimulation. Electron microscopic and biochemical studies revealed that double knockout cell death was associated with autophagosomes/autolysosomes. This non-apoptotic death of double knockout cells was suppressed by inhibitors of autophagy, including 3-methyl adenine, was dependent on autophagic proteins APG5 and Beclin 1 (capable of binding to Bcl-2/Bcl-x(L)), and was also modulated by Bcl-x(L). These results indicate that the Bcl-2 family of proteins not only regulates apoptosis, but also controls non-apoptotic programmed cell death that depends on the autophagy genes.     

The WNT antagonist cSFRP2 modulates programmed cell death in the developing hindbrain

In the avian hindbrain, the loss of premigratory neural crest cells from rhombomeres 3 and 5 (r3, r5) through programmed cell death contributes to the patterning of emigrant crest cells into three discrete streams. Programmed cell death is induced by the upregulation of Bmp4 and Msx2 in r3 and r5. We show that cSFRP2, a WNT antagonist, is expressed in the even-numbered rhombomeres and that over-expression of cSfrp2 inhibits Bmp4 expression in r3 and r5, preventing programmed cell death. By contrast, depleting cSFRP2 function in r4 results in elevated levels of Msx2 expression and ectopic programmed cell death, as does overexpression of Wnt1. We propose that programmed cell death in the rhombencephalic neural crest is modulated by pre-patterned cSfrp2 expression and a WNT-BMP signalling loop.     

On the origin, evolution, and nature of programmed cell death: a timeline of four billion years

Programmed cell death is a genetically regulated process of cell suicide that is central to the development, homeostasis and integrity of multicellular organisms. Conversely, the dysregulation of mechanisms controlling cell suicide plays a role in the pathogenesis of a wide range of diseases. While great progress has been achieved in the unveiling of the molecular mechanisms of programmed cell death, a new level of complexity, with important therapeutic implications, has begun to emerge, suggesting (i) that several different self-destruction pathways may exist and operate in parallel in our cells, and (ii) that molecular effectors of cell suicide may also perform other functions unrelated to cell death induction and crucial to cell survival. In this review, I will argue that this new level of complexity, implying that there may be no such thing as a 'bona fide' genetic death program in our cells, might be better understood when considered in an evolutionary context. And a new view of the regulated cell suicide pathways emerges when one attempts to ask the question of when and how they may have become selected during evolution, at the level of ancestral single-celled organisms.