Intracellular recordings from gecko photoreceptors during light and dark adaptation

Intracellular recordings were obtained from rods in the Gekko gekko retina and the adaptation characteristics of their responses studied during light and dark adaptation. Steady background illumination induced graded and sustained hyperpolarizing potentials and compressed the incremental voltage range of the receptor. Steady backgrounds also shifted the receptor's voltage-intensity curve along the intensity axis, and bright backgrounds lowered the saturation potential of the receptor. Increment thresholds of single receptors followed Weber's law over a range of about 3.5 log units and then saturated. Most of the receptor sensitivity change in light derived from the shift of the voltage-intensity curve, only little from the voltage compression. Treatment of the eyecup with sodium aspartate at concentrations sufficient to eliminate the beta-wave of the electroretinogram (ERG) abolished initial transients in the receptor response, possibly indicating the removal of horizontal cell feedback. Aspartate treatment, however, did not significantly alter the adaptation characteristics of receptor responses, indicating that they derive from processes intrinsic to the receptors. Dark adaptation after a strongly adapting stimulus was similarly associated with temporary elevation of membrane potential, initial lowering of the saturation potential, and shift of the voltage-intensity curve. Under all conditions of adaptation studied, small amplitude responses were linear with light intensity. Further, there was no unique relation between sensitivity and membrane potential suggesting that receptor sensitivity is controlled at least in part by a step of visual transduction preceding the generation of membrane voltage change.     

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells

Purpose

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.

Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo

Here we describe an approach for making targeted patch-clamp recordings from single neurons in vivo, visualized by two-photon microscopy. A patch electrode is used to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus enabling the neuron to be visualized as a negative image ('shadow') and identified on the basis of its somatodendritic structure. The same electrode is then placed on the neuron under visual control to allow formation of a gigaseal ('shadowpatching'). We demonstrate the reliability and versatility of shadowpatching by performing whole-cell recordings from visually identified neurons in the neocortex and cerebellum of rat and mouse. We also show that the method can be used for targeted in vivo single-cell electroporation of plasmid DNA into identified cell types, leading to stable transgene expression. This approach facilitates the recording, labeling and genetic manipulation of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations.     

A single-cell Arabidopsis root atlas reveals developmental trajectories in wild-type and cell identity mutants

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Isolation and long-term culture of gallbladder epithelial cells from wild-type and CF mice

Summary Mice with targeted disruption of the cftr gene show pathophysiologic changes in the gallbladder, which correlate with hepatobiliary disease seen in cystic fibrosis patients. As gallbladder epithelium secretes mucin, and as this epithelium consists of a relatively homogenous cell type, study of CFTR function in these cells would be beneficial to delineate the complex cellular functions of this protein. The size and anatomic location of the murine gallbladder makes such studies difficult in vivo. Therefore, the need exists for in vitro models of gallbladder epithelium. We describe a method to isolate and culture murine gallbladder epithelium from wild-type and CF mice. Cells were grown in a monolayer on porous inserts over a feeder layer of fibroblasts. These nontransformed cells can be successively passaged and maintain a well-differentiated epithelial cell phenotype as shown by morphologic criteria, characterized by polarized columnar epithelial cells with prominent microvilli and intercellular junctions. Organotypic cultures showed columnar cells simulating in vivo morphology. This culture system should be valuable in delineating cellular processes relating to CFTR in gallbladder epithelium.     

A model of Salmonella colitis with features of diarrhea in SLC11A1 wild-type mice

BACKGROUND: Mice do not get diarrhea when orally infected with S. enterica, but pre-treatment with oral aminoglycosides makes them susceptible to Salmonella colitis. However, genetically susceptible ItyS mice (Nramp1(G169D) allele) die from systemic infection before they develop diarrhea, so a new model is needed to study the pathogenesis of diarrhea. We pretreated ItyR mice (Nramp1(G169)) with oral kanamycin prior to infecting them with virulent S. Typhimurium strain 14028s in order to study Salmonella-induced diarrhea. We used both a visual scoring system and the measurement of fecal water content to measure diarrhea. BALB/c.D2(Nramp1) congenic started losing weight 5 days post-infection and they began to die from colitis 10-14 days after infection. A SPI-1 (invA) mutant caused cecal, but not colonic inflammation and did not cause diarrhea. A phoP- mutant did not cause manifestations of diarrhea in either normal or NADPH-deficient (gp91(phox)) mice. However, strain 14028s caused severe colitis and diarrhea in gp91(phox)-deficient mice on an ItyR background. pmr A and F mutants, which are less virulent in orally infected BALB/c mice, were fully virulent in this model of colitis. CONCLUSIONS: S. enterica must be able to invade the colonic epithelium and to persist in the colon in order to cause colitis with manifestations of diarrhea. The NADPH oxidase is not required for diarrhea in Salmonella colitis. Furthermore, a Salmonella phoP mutant can be cleared from the colon by non-oxidative host defenses.     

Reproduction and fertility in wild-type and transgenic mice after orthotopic transplantation of cryopreserved ovaries from 10-d-old mice

Ovary cryopreservation and subsequent transplantation can enable researchers to preserve valuable transgenic animal strains. Some studies have indicated, however, that this process may impair ovary viability and recipient fertility. The authors investigated the effects of ovary vitrification followed by orthotopic transplantation in five strains of mice. They grafted fresh and frozen ovaries of 10-d-old mice into 4-week-old ovariectomized recipients. In addition to using wild-type strains (BALB/cAn and ICR/JCL), the authors used a transgene system that enabled them to identify whether offspring derived from the ovary of the recipient or that of the donor: they transplanted ovaries from one transgenic strain (LAP/rtTA) into wild-type C57BL/6J mice and into mice from a second transgenic strain (pTet/Cd226). The authors then determined the origin of the offspring born to these recipients using PCR. Ovary cryopreservation seemed to have no effect on the long-term fertility and reproductive characteristics of recipients and their offspring.     

Differential Akt activation in the photoreceptors of normal and rd1 mice

Retinitis pigmentosa is a blinding disease in which unknown mechanisms cause the degeneration of retinal photoreceptors. The retinal degeneration (rd1) mouse is a relevant model for this condition, since it carries a mutation also found in some forms of retinitis pigmentosa. To understand the degenerative process in the rd1 mouse, we must identify the survival and apoptosis-related signaling pathways in its photoreceptors and determine whether signaling differs from that in normal mice. The phosphatidylinositol 3-kinase/Akt kinase pathway promotes survival in several different cell types. The purpose of the present study has been to compare Akt activity in retinal cells of normal and rd1 mice. We have found that, in normal mice, Akt becomes activated in the retina in a developmentally regulated and cell-type-specific fashion, encompassing essentially all retinal cells. In most cell types, once Akt activation has begun, it remains in this state throughout life. An exception is seen in the rod photoreceptors, in which Akt is activated only transiently during their development. The rd1 retina behaves identically in all but one respect, namely that the activation of Akt in rod photoreceptors persists until these cells undergo apoptosis. Thus, Akt may participate in constitutive survival processes in retinal neurons, except in rod photoreceptors in which the role of this pathway may be restricted to the developmental period. However, Akt activation in the rods may be part of a defense mechanism initiated in response to insults, such as the retinal degeneration seen in the rd1 mouse.This work was supported by grants from the Foundation for Fighting Blindness, Stiftelsen för synskadade i fd Malmöhus län, 2nd ONCE International Award for New Technologies for the Blind, PRO-AGE-RET: QLK6-CT-2001-00385, PRO-RET: QLK6-CT-2000-00569, KMA, the Crafoord Foundation, and the Segerfalk Foundation.     

Retinol dehydrogenase (RDH12) protects photoreceptors from light-induced degeneration in mice

RDH12 has been suggested to be one of the retinol dehydrogenases (RDH) involved in the vitamin A recycling system (visual cycle) in the eye. Loss of function mutations in the RDH12 gene were recently reported to be associated with autosomal recessive childhood-onset severe retinal dystrophy. Here we show that RDH12 localizes to the photoreceptor inner segments and that deletion of this gene in mice slows the kinetics of all-trans-retinal reduction, delaying dark adaptation. However, accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor apoptosis were also observed in Rdh12(-/-) mice, suggesting that RDH12 plays a unique, nonredundant role in the photoreceptor inner segments to regulate the flow of retinoids in the eye. Thus, severe visual impairments of individuals with null mutations in RDH12 may likely be caused by light damage(1).     

Physiological and microfluorometric studies of reduction and clearance of retinal in bleached rod photoreceptors

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rods isolated from salamander retina by measuring its intrinsic fluorescence. Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction from the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after approximately 30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clearance of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching.     

Stereoselective disposition of S- and R-licarbazepine in mice

The stereoselective disposition of S-licarbazepine (S-Lic) and R-licarbazepine (R-Lic) was investigated in plasma, brain, liver, and kidney tissues after their individual administration (350 mg/kg) to mice by oral gavage. Plasma, brain, liver, and kidney concentrations of licarbazepine enantiomers and their metabolites were determined over the time by a validated chiral HPLC-UV method. The mean concentration data, attained at each time point, were analyzed using a non-compartmental model. S-Lic and R-Lic were rapidly absorbed from gastrointestinal tract of mouse and immediately distributed to tissues supplied with high blood flow rates. Both licarbazepine enantiomers were metabolized to a small extent, each parent compound being mainly responsible for the systemic and tissue drug exposure. The stereoselectivity in the metabolism and distribution of S- and R-Lic was easily identified. An additional metabolite was detected following R-Lic administration and S-Lic showed a particular predisposition for hepatic and renal accumulation. Stereoselective processes were also identified at the blood-brain barrier, with the brain exposure to S-Lic almost twice that of R-Lic. Another finding, reported here for the first time, was the ability of the mouse to perform the chiral inversion of S- and R-Lic, albeit to a small extent.     

Lobe-specific proteome changes in the dorsal-lateral and ventral prostate of TRAMP mice versus wild-type mice

The transgenic adenocarcinoma of mouse prostate (TRAMP) is the most widely used transgenic model for prostate cancer chemoprevention studies. Although two lobe‐specific lineages of carcinogenesis have been described, the molecular mechanisms are still poorly defined. Here, we concurrently profiled the proteome of dorsal‐lateral (DLP) and ventral (VP) prostate lobes of both TRAMP and littermate WT C57BL/6 mice of 18 wk by 2‐D LC‐MALDI‐TOF/TOF with iTRAQ labeling. A total of 483 and 748 proteins were identified at critical false discovery rates of 1 and 5%. In WT mice, 84 proteins were found to have different expression levels between DLP and VP. In TRAMP mice, 118 proteins significantly changed in DLP and/or VP during TRAMP carcinogenesis. Among them, 55 and 36 proteins were uniquely changed in DLP or VP lobe, respectively, and 27 proteins in both DLP and LP lobe. Ingenuity Pathway Analysis was able to segregate proteins changed in two lobes into different pathway networks. In addition to serving as reference for prostate proteomic profiles, our data suggest that different sets of proteins are involved in the carcinogenesis in DLP versus VP in the TRAMP model.     

Physiological and nutritional features of Corynebacterium pyogenes

Growth and acid metabolic products were similar when Corynebacterium pyogenes was grown aerobically or anaerobically in a serum-free medium (SFM). This indicated that C. pyogenes obtains energy for growth primarily by fermentative metabolism even under aerobic growth conditions. Growth yield was reduced by 90% in SFM minus glucose, 50% in SFM minus NaHCO3, 90% in SFM minus yeast extract, 100% in SFM minus Trypticase and yeast extract, and 30% in SFM minus haemin or Trypticase. Growth was not detectable when a known mixture of amino acids, vitamins, and nucleic acid bases were substituted for Trypticase and yeast extract in SFM; addition to the latter medium of a peptide source such as Trypticase or casitone supported good growth of the organism. When NaHCO3 was omitted from SFM and dissolved CO2 in the medium was rigorously excluded, growth was undetectable indicating that C. pyogenes has an obligate requirement for CO2 for growth. Succinate, formate and acetate were the major fermentation products in SFM, whereas in SFM minus HCO-3 or haemin, lactate was the major product and only small quantities of other acids accumulated.     

Quantification of the mechanical behavior of carotid arteries from wild-type, dystrophin-deficient, and sarcoglycan-delta knockout mice

As patients with muscular dystrophy live longer because of improved clinical care, they will become increasingly susceptible to many of the cardiovascular diseases that affect the general population. There is, therefore, a pressing need to better understand both the biology and the mechanics of the arterial wall in these patients. In this paper, we use nonlinear constitutive relations to model, for the first time, the biaxial mechanical behavior of carotid arteries from two common mouse models of muscular dystrophy (dystrophin-deficient and sarcoglycan-delta null) and wild-type controls. It is shown that a structurally motivated four-fiber family stress-strain relation describes the passive behavior of all three genotypes better than does a commonly used phenomenological exponential model, and that a Rachev-Hayashi model describes the mechanical contribution of smooth muscle contraction under basal tone. Because structurally motivated constitutive relations can be extended easily to model adaptations to altered hemodynamics, results from this study represent an important step toward the ultimate goal of understanding better the mechanobiology and pathophysiology of arteries in muscular dystrophy.     

Physiological characteristics of four methylotrophic bacteria and their potential use in single-cell protein production

Summary Four methylotrophic bacteria, isolated at the Kuwait Institute for Scientific Research, were able to grow on methanol as the only carbon source at a maximum temperature of 44°C. An optimized medium composition was obtained through intensive chemostat studies varying both macro- and micro-element concentrations. Various batch and chemostat experiments were carried out at different pH, temperature, dilution rate and methanol concentrations. The results showed optimum pH around 6.8, at temperatures of 37 to 40°C, dilution rate 0.2–0.3 h^–1 and methanol was found to be inhibitory at concentrations above 20 g l^–1. The performance of all four bacteria under chemostat conditions was similar. Chemostat fermentation experiments using the optimized medium at 40°C, pH 6.8, dilution 0.2 h^–1 and 10 g methanol/l in the feed gave a biomass yield coefficient of 0.42–0.44 g/g methanol, 78–79% crude protein content, 58–62% total amino acid content and 10–11.5% nucleic acid content. In conclusion all four methylotrophic strains has good potential for use in the production of single-cell protein.

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Caractéristiques physiologiques de quatre bactéries méthylotrophes et leur emploi potentiel dans la production de protéine uni-celluliare

Résumé Quatre bactéries méthylotrophes, isolées à l'Institut pour la Recherche Scientifique du Kuweit, se sont révélées capables de croître sur méthanol comme seule source de carbone à une température maximum de 44°C. Des études intensives en chémostat de variation des concentrations tant en macro-qu'en micro-éléments, ont permis d'obtenir une composition optimum du milieu. Diverses expériences en milieu non renouvelé et en chémostat ont été effectuées à différents pH, températures, taux de dilution et concentration en méthanol. Les résultats ont montré un optimum de pH vers 6.8, de température entre 37 et 40°C, de taux de dilution de 0.2 à 0.3 h^–1. Le méthanol s'est révélé inhibiteur aux concentrations supérieures à 20 g l^–1. La performance des quatres bactéries dans les conditions du chémostat se sont révélées très semblables. Les expériences de fermentátion en chémostat, utilisant le milieu optimum à 40°C, pH 6.8, taux de dilution de 0.2 h^–1 et à 10 g de méthanol par litre dans l'alimentation ont produit un coefficient de rendement en biomasse de 0.42 à 0.44 g de cellules par g de méthanol contenant de 78 à 79% de protéines, de 58 à 62% d'acides aminés totaux et de 10 à 11.5% d'acides nucléiques. En conclusion, les quatre bactéries méthylotrophes présentent une bonne potentialité pour être utilisées dans la production de protéine uni-cellulaire.

     

Extracellular electrical activity from the photoreceptors of midge

The ontogeny of photosensitivity has been studied in a holometabolous insect, the midgeChironomus ramosus. The life cycle of midges shifts from an aquatic environment to a non-aquatic environment. Extracellular electrical activity of photoreceptor organs was recorded at larval and adult stages. We found an increase in photosensitivity as the larva metamorphosed to the adult stage. This is the first report of changes in photosensitivity during the development of any insect described in an ecological context.     

Expression profiling of the developing testis in wild-type and Dazl knockout mice

Genetic understanding of male-factor infertility requires knowledge of gene expression patterns associated with normal germ cell differentiation. The mouse is one of the best models of mammalian fertility due to its well-characterized genetics and the existence of many infertile mutants both naturally occurring and experimentally induced. We used cDNA microarrays firstly to investigate normal gene expression in the wild-type (wt) testis and secondly to gain a better insight into the effect of the disruption of the Dazl gene on spermatogenesis. We constructed a cDNA microarray from a subtracted and normalized adult testis library and focused on six developmental time-points during the initial synchronous wave of spermatogenesis. The results suggest that in the wild-type testis, 89.5% of genes on our chip change expression dramatically during the time-course. To identify patterns in the gene-expression data, a k-means clustering algorithm and principal component analysis were used. In the Dazl knockout testes, the majority of genes remain at baseline levels of expression, because absence of Dazl has a severe effect on cell-types present in the testis. Although in the prepubescent Dazl-null mice the final point reached in germ cell development is the leptotene-zygotene stage, the microarray results suggest that lack of Dazl expression has a detectable effect on the mRNA complement of germ cells as early as day 5 when only type A spermatogonia are present. Mol. Reprod. Dev. 67: 26-54, 2004.