Carbon and nitrogen turnover in the Arctic deep sea : in situ benthic community response to diatom and coccolithophorid phytodetritus

In the Arctic Ocean, increased sea surface temperature and sea ice retreat have triggered shifts in phytoplankton communities. In Fram Strait, coccolithophorids have been occasionally observed to replace diatoms as the dominating taxon of spring blooms. Deep-sea benthic communities depend strongly on such blooms, but with a change in quality and quantity of primarily produced organic matter (OM) input, this may likely have implications for deep-sea life. We compared the in situ responses of Arctic deep-sea benthos to input of phytodetritus from a diatom (Thalassiosira sp.) and a coccolithophorid (Emiliania huxleyi) species. We traced the fate of C-13- and N-15-labelled phytodetritus into respiration, assimilation by bacteria and infauna in a 4-day and 14-day experiment. Bacteria were key assimilators in the Thalassiosira OM degradation, whereas Foraminifera and other infauna were at least as important as bacteria in the Emiliania OM assimilation. After 14 days, 5 times less carbon and 3.8 times less nitrogen of the Emiliania detritus was recycled compared to Thalassiosira detritus. This implies that the utilization of Emiliania OM may be less efficient than for Thalassiosira OM. Our results indicate that a shift from diatom-dominated input to a coccolithophorid-dominated pulse could entail a delay in OM cycling, which may affect benthopelagic coupling

Identification of a Common Non-Apoptotic Cell Death Mechanism in Hereditary Retinal Degeneration

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments

Localization of Retinal Ca2+/Calmodulin-Dependent Kinase II-β (CaMKII-β) at Bipolar Cell Gap Junctions and Cross-Reactivity of a Monoclonal Anti-CaMKII-β Antibody With Connexin36

Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas via phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-β at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-β. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-β-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-β. However, a monoclonal antibody (CB-β-1) recognized CaMKII-β but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-β expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-β also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-β influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-β-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-β is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-β with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-β may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs

Differential Distribution of Retinal Ca2+/Calmodulin-Dependent Kinase II (CaMKII) Isoforms Indicates CaMKII-β and -δ as Specific Elements of Electrical Synapses Made of Connexin36 (Cx36)

AII amacrine cells are essential interneurons of the primary rod pathway and transmit rod-driven signals to ON cone bipolar cells to enable scotopic vision. Gap junctions made of connexin36 (Cx36) mediate electrical coupling among AII cells and between AII cells and ON cone bipolar cells. These gap junctions underlie a remarkable degree of plasticity and are modulated by different signaling cascades. In particular, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized as an important regulator of Cx36, capable of potentiating electrical coupling in AII cells. However, it is unclear which CaMKII isoform mediates this effect. To obtain a more detailed understanding of the isoform composition of CaMKII at retinal gap junctions, we analyzed the retinal distribution of all four CaMKII isoforms using confocal microscopy. These experiments revealed a differential distribution of CaMKII isoforms: CaMKII-α was strongly expressed in starburst amacrine cells, which are known to lack electrical coupling. CaMKII-β was abundant in OFF bipolar cells, which form electrical synapses in the outer and the inner retina. CaMKII-γ was diffusely distributed across the entire retina and could not be assigned to a specific cell type. CaMKII-δ labeling was evident in bipolar and AII amacrine cells, which contain the majority of Cx36-immunoreactive puncta in the inner retina. We double-labeled retinas for Cx36 and the four CaMKII isoforms and revealed that the composition of the CaMKII enzyme differs between gap junctions in the outer and the inner retina: in the outer retina, only CaMKII-β colocalized with Cx36-containing gap junctions, whereas in the inner retina, CaMKII-β and -δ colocalized with Cx36. This finding suggests that gap junctions in the inner and the outer retina may be regulated differently although they both contain the same connexin. Taken together, our study identifies CaMKII-β and -δ as Cx36-specific regulators in the mouse retina with CaMKII-δ regulating the primary rod pathway

Connexin30.2:<i>In vitro</i> interaction with connexin36 in hela cells and expression in AII amacrine cells and intrinsically photosensitive ganglion cells in the mouse retina

Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36 – both expressed in AII amacrine cells – are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners

Synaptic Transmission from Horizontal Cells to Cones Is Impaired by Loss of Connexin Hemichannels

In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was strongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an important step in resolving a long-standing debate about the unusual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina

Control of the Nucleotide Cycle in Photoreceptor Cell Extracts by Retinal Degeneration Protein 3

Retinal degeneration protein 3 (RD3) is crucial for photoreceptor cell survival and linked to Leber Congenital Amaurosis type 12 (LCA12), a hereditary retinal disease in humans. RD3 inhibits photoreceptor guanylate cyclases GC-E and GC-F and is involved in transport of GCs from the inner to the outer segments. Otherwise, its role in photoreceptor physiology is poorly understood. Here, we describe a new function of RD3. Purified RD3 evoked an increase in guanylate kinase activity, an enzyme that is involved in the nucleotide cycle in photoreceptors. We demonstrate a direct interaction between guanylate kinase and RD3 using back-scattering interferometry and show by immunohistochemistry of mouse retina sections that RD3 and guanylate kinase co-localize in photoreceptor inner segments and to a lesser extent in the outer plexiform layer. Our findings point toward a more complex function of RD3 in photoreceptors. The RD3 – guanylate kinase interaction may also play a role in other cellular systems, while the GC – RD3 interaction is exclusive to photoreceptors

Connexin Expression in Renin-Producing Cells

Absence of connexin 40 (Cx40) leads to ectopic juxtaglomerular renin expression and abrogates recruitment of renin-expressing cells in the adult kidney but does not disturb renin expression during kidney development. To find an explanation for these observations, we aimed to analyze the expression pattern of major vascular Cxs in normal juxtaglomerular epithelioid cells, in recruited renin-expressing cells, and in fetal renin-expressing cells. We found that during kidney development, the appearance of renin-producing cells paralleled the expression of Cx40 and, to a lesser extent, Cx45 but not other Cxs. In the adult kidney, juxtaglomerular epithelioid cells expressed Cx40 and lesser amounts of Cx37 and Cx43 but not Cx45, which localized to arteriolar smooth muscle cells. Recruitment of renin-producing cells in adult kidneys in response to long-term salt deprivation of mice correlated with the reappearance of only Cx40. Cx40-null renin-producing cells did not express Cx37, Cx43, or Cx45. These findings suggest that Cx40 expression is a characteristic of renin-producing cells in the kidney, and it seems to be essential in the recruitment of renin-producing cells in the adult but not the fetal kidney