Effects of Histamine on Light Responses of Amacrine Cells in Tiger Salamander Retina

Using immunofluorescence, we showed that histamine receptor 1 is expressed by horizontal cell axons and a subset of amacrine cells in the tiger salamander retina. The effects of histamine on light responses of amacrine cells were studied in slice preparations. Histamine modulated the light responses of many salamander amacrine cells, depending upon the morphological type. The most pronounced effects of histamine were decreases in the light responses of broadly stratified amacrine cells, particularly those having medium-sized dendritic field diameters. To determine whether the effects of histamine were direct, Co⁺⁺ was substituted for Ca⁺⁺ in the extracellular medium to block synaptic transmission. Histamine still affected broadly stratified amacrine cells, but not narrowly stratified amacrine cells under these conditions. Taken together, these findings suggest that inhibitory interactions between strata of the IPL and within the classical receptive fields of the ganglion cells would be particularly sensitive to histamine released from retinopetal axons.     

Synaptic Transfer between Rod and Cone Pathways Mediated by AII Amacrine Cells in the Mouse Retina

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Functional Compartmentalization within Starburst Amacrine Cell Dendrites in the Retina

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Ganglion Cell and Displaced Amacrine Cell Density Distribution in the Retina of the Howler Monkey (Alouatta caraya)

Unlike all other New World (platyrrine) monkeys, both male and female howler monkeys (Alouatta sp.) are obligatory trichromats. In all other platyrrines, only females can be trichromats, while males are always dichromats, as determined by multiple behavioral, electrophysiological, and genetic studies. In addition to obligatory trichromacy, Alouatta has an unusual fovea, with substantially higher peak cone density in the foveal pit than every other diurnal anthropoid monkey (both platyrrhines and catarrhines) and great ape yet examined, including humans. In addition to documenting the general organization of the retinal ganglion cell layer in Alouatta, the distribution of cones is compared to retinal ganglion cells, to explore possible relationships between their atypical trichromacy and foveal specialization. The number and distribution of retinal ganglion cells and displaced amacrine cells were determined in six flat-mounted retinas from five Alouatta caraya. Ganglion cell density peaked at 0.5 mm between the fovea and optic nerve head, reaching 40,700–45,200 cells/mm². Displaced amacrine cell density distribution peaked between 0.5–1.75 mm from the fovea, reaching mean values between 2,050–3,100 cells/mm². The mean number of ganglion cells was 1,133,000±79,000 cells and the mean number of displaced amacrine cells was 537,000±61,800 cells, in retinas of mean area 641±62 mm². Ganglion cell and displaced amacrine cell density distribution in the Alouatta retina was consistent with that observed among several species of diurnal Anthropoidea, both platyrrhines and catarrhines. The principal alteration in the Alouatta retina appears not to be in the number of any retinal cell class, but rather a marked gradient in cone density within the fovea, which could potentially support high chromatic acuity in a restricted central region.     

Adenosine A2A Receptor Up-Regulates Retinal Wave Frequency via Starburst Amacrine Cells in the Developing Rat Retina

Background

Developing retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine''s regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A_2A receptor (A_2AR) regulates retinal waves and whether A_2AR regulation of retinal waves acts via presynaptic SACs.

Methodology/Principal Findings

We showed that A_2AR was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A_2AR decreased the frequency of spontaneous Ca²⁺ transients, suggesting that endogenous A_2AR may up-regulate wave frequency. To investigate whether A_2AR acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca²⁺ transient frequency was increased by expressing wild-type A_2AR (A_2AR-WT) in SACs, suggesting that A_2AR may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A_2AR-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC''s excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A_2AR may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A_2AR mutant (A_2AR-ΔC) in SACs, the wave frequency was reduced compared to the A_2AR-WT, but was similar to the control, suggesting that the full-length A_2AR in SACs is required for A_2AR up-regulation of retinal waves.

Conclusions/Significance

A_2AR up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full-length protein structure. Thus, by coupling with the downstream intracellular signaling, A_2AR may have a great capacity to modulate patterned spontaneous activity during neural circuit refinement.     

A Novel Zebrafish Gene Expressed Specifically in the Photoreceptor Cells of the Retina

We have identified and characterized a novel protein from adult zebrafish retina, which we named ES1. Database search revealed that the ES1 gene has significant similarity to two genes with unknown functions: theEscherichia colisigma cross-reacting protein 27a (scrp27a) and the human KNP-I/GT335.In situhybridization and immunohistochemistry experiments showed that both ES1 mRNA and protein are expressed specifically in adult photoreceptor cells. ES1 seems to be a cytoplasmic protein. An ES1-like antigen was also detected in photoreceptor cells of goldfish with anti-ES1 antibodies. The retina specific expression and the evolutionary conservation suggest that ES1 protein may be important for maintaining normal retina structure and function.     

Response Properties of a Newly Identified Tristratified Narrow Field Amacrine Cell in the Mouse Retina

Amacrine cells were targeted for whole cell recording using two-photon fluorescence microscopy in a transgenic mouse line in which the promoter for dopamine receptor 2 drove expression of green fluorescent protein in a narrow field tristratified amacrine cell (TNAC) that had not been studied previously. Light evoked a multiphasic response that was the sum of hyperpolarizing and depolarization synaptic inputs consistent with distinct dendritic ramifications in the off and on sublamina of the inner plexiform layer. The amplitude and waveform of the response, which consisted of an initial brief hyperpolarization at light onset followed by recovery to a plateau potential close to dark resting potential and a hyperpolarizing response at the light offset varied little over an intensity range from 0.4 to ~10^6 Rh*/rod/s. This suggests that the cell functions as a differentiator that generates an output signal (a transient reduction in inhibitory input to downstream retina neurons) that is proportional to the derivative of light input independent of its intensity. The underlying circuitry appears to consist of rod and cone driven on and off bipolar cells that provide direct excitatory input to the cell as well as to GABAergic amacrine cells that are synaptically coupled to TNAC. Canonical reagents that blocked excitatory (glutamatergic) and inhibitory (GABA and glycine) synaptic transmission had effects on responses to scotopic stimuli consistent with the rod driven component of the proposed circuit. However, responses evoked by photopic stimuli were paradoxical and could not be interpreted on the basis of conventional thinking about the neuropharmacology of synaptic interactions in the retina.     

Rax-CreERT2 Knock-In Mice: A Tool for Selective and Conditional Gene Deletion in Progenitor Cells and Radial Glia of the Retina and Hypothalamus

To study gene function in neural progenitors and radial glia of the retina and hypothalamus, we developed a Rax-CreER^T2 mouse line in which a tamoxifen-inducible Cre recombinase is inserted into the endogenous Rax locus. By crossing Rax-CreER^T2 with the Cre-dependent Ai9 reporter line, we demonstrate that tamoxifen-induced Cre activity recapitulates the endogenous Rax mRNA expression pattern. During embryonic development, Cre recombinase activity in Rax-CreER^T2 is confined to retinal and hypothalamic progenitor cells, as well as progenitor cells of the posterior pituitary. At postnatal time points, selective Cre recombinase activity is seen in radial glial-like cell types in these organs – specifically Müller glia and tanycytes – as well as pituicytes. We anticipate that this line will prove useful for cell lineage analysis and investigation of gene function in the developing and mature retina, hypothalamus and pituitary.     

Deletion of the Penicillin-Binding Protein 6 Gene of Escherichia coli

A strain of Escherichia coli with a deletion of the penicillin-binding protein 6 gene (dacC) has been constructed. The properties of this strain establish that the complete lack of penicillin-binding protein 6 has no marked effect on the growth of E. coli.     

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.     

内皮和造血系统特异性敲除Rictor基因对胎肝造血的影响

目的:研究Rictor基因对胚胎发育过程中胎肝造血的影响。方法:利用Cre-LoxP基因敲除系统,特异性在小鼠内皮(VEC-Cre)和造血(Vav1-Cre)系统敲除Rictor基因;通过流式细胞术分析特异敲除Rictor基因后小鼠胚胎第15 d胎肝中的各系细胞比例的变化,并进一步分析造血干细胞的变化。结果:利用VEC-Cre和Vav1-Cre小鼠敲除Rictor基因后,胎肝中各系细胞比例均有所减少,B细胞比例的减少较为明显,造血干细胞比例也明显减少。结论:Rictor基因敲除损害胎肝组织中造血干细胞的产生和各系细胞的分化。     

SPC-Cre-ERT2 Transgenic Mouse for Temporal Gene Deletion in Alveolar Epithelial Cells

Although several Cre-loxP-based gene knockout mouse models have been generated for the study of gene function in alveolar epithelia in the lung, their applications are still limited. In this study, we developed a SPC-Cre-ER^T2 mouse model, in which a tamoxifen-inducible Cre recombinase (Cre-ER^T2) is under the control of the human surfactant protein C (SPC) promoter. The specificity and efficiency of Cre-ER^T2 activity was first evaluated by crossing SPC-Cre-ER^T2 mouse with ROSA26R mouse, a β-galactosidase reporter strain. We found that Cre-ER^T2 was expressed in 30.7% type II alveolar epithelial cells of SPC-Cre-ER^T2/ROSA26R mouse lung tissues in the presence of tamoxifen. We then tested the tamoxifen-inducible recombinase activity of Cre-ER^T2 in a mouse strain bearing TSC1 conditional knockout alleles (TSC1^fx/fx). TSC1 deletion was detected in the lungs of tamoxifen treated SPC-Cre-ER^T2/TSC1^fx/fx mice. Therefore this SPC-Cre-ER^T2 mouse model may be a valuable tool to investigate functions of genes in lung development, physiology and disease.     

Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Müller Cells of the Retina

Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.     

Sez-6 may play an important role in neurite outgrowth through the PKCgamma signaling pathways

Seizure-related gene 6 (sez-6) was originally identified in a study of pentylenetetrazole-treated cortical neurons. Further studies on the structure and expression pattern suggested that Sez-6 may play an important role in neuronal development and function. In the present study, PC12 cells were used as a model to investigate the role of Sez-6 in neurite outgrowth. After a period of NGF treatment, the expression of Sez-6 in PC12 cells was increased. When Sez-6 expression was suppressed by the addition of an effective short hairpin RNA (shRNA) plasmid, the neurite outgrowth was significantly inhibited. In addition, we detected the expression level of protein kinase Cgamma (PKCgamma), and found that the PKCgamma protein level was reduced in the differentiated PC12 cells but increased in PC12 cells lacking Sez-6. Taken together, our results indicate that Sez-6 acts on the neurite outgrowth of PC12 cells likely through the PKCgamma signaling pathways.     

Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons

Development of appropriate dendritic arbors is crucial for neuronal information transfer. We show, using seizure-related gene 6 (sez-6) null mutant mice, that Sez-6 is required for normal dendritic arborization of cortical neurons. Deep-layer pyramidal neurons in the somatosensory cortex of sez-6 null mice exhibit an excess of short dendrites, and cultured cortical neurons lacking Sez-6 display excessive neurite branching. Overexpression of individual Sez-6 isoforms in knockout neurons reveals opposing actions of membrane-bound and secreted Sez-6 proteins, with membrane-bound Sez-6 exerting an antibranching effect under both basal and depolarizing conditions. Layer V pyramidal neurons in knockout brain slices show reduced excitatory postsynaptic responses and a reduced dendritic spine density, reflected by diminished punctate staining for postsynaptic density 95 (PSD-95). In behavioral tests, the sez-6 null mice display specific exploratory, motor, and cognitive deficits. In conclusion, cell-surface protein complexes involving Sez-6 help to sculpt the dendritic arbor, in turn enhancing synaptic connectivity.     

6-Phosphogluconate Dehydrogenase (6PGD) Gene Duplication in Kalimeris (Asteraceae)

Abstract The genus Kalimeris with a diagnostic character of short or inconspicuous pappus consists of two sections, Asteromoea and Cordifolium. As a result of 6PGD isozyme analysis, sect, Asteromoea, including 2 × and poly-ploid taxa from 5 × to 8 ×, show similar cytosolic isozyme multiplicity and share a monomorphic locus. The data suggest that gene duplication of polyploid members was derived from a common ancestor. K. miqueliana, belonging to sect. Cordifolium. also possessed a gene duplication in 6PGD, though significant differences were detected in electrophoretic mobility between the sections. The occurrence of gene duplication in East Asian diploid Astereae leaves intact the validity of the allopolyploid-origin hypothesis of n= 9, which was rejected by Gottlieb (1981a) in American Astereae.