Autoinhibition of serotonin cells: An intrinsic regulatory mechanism sensitive to the pattern of usage of the cells

After periods of high-frequency firing, the normal rhythmically active serotonin (5HT)-containing neurosecretory neurons of the lobster ventral nerve cord display a period of suppressed spike generation and reduced synaptic input that we refer to as “autoinhibition.” The duration of this autoinhibition is directly related to the magnitude and duration of the current injection triggering the high-frequency firing. More interesting, however, is that the autoinhibition is inversely related to the initial firing frequency of these cells within their normal range of firing (0.5–3 Hz). This allows more active 5HT neurons to resume firing after shorter durations of inhibition than cells that initially fired at slower rates. Although superfused 5HT inhibits the spontaneous firing of these cells, the persistence of autoinhibition in saline with no added calcium, in cadmium-containing saline, and in lobsters depleted of serotonin suggests that intrinsic membrane properties account for the autoinhibition. A similar autoinhibition is seen in spontaneously active octopamine neurons but is absent from spontaneously active γ-aminobutyric acid cells. Thus, this might be a characteristic feature of amine-containing neurosecretory neurons. The 5HT cells of vertebrate brain nuclei share similarities in firing frequencies, spike shapes, and inhibition by 5HT with the lobster cells that were the focus of this study. However, the mechanism suggested to underlie autoinhibition in vertebrate neurons is that 5HT released from activated or neighboring cells acts back on inhibitory autoreceptors that are found on the dendrites and cell bodies of these neurons.

Cloning, expression, and genetic mapping of Sema W, a member of the semaphorin family

The semaphorins comprise a large family of membrane-bound and secreted proteins, some of which have been shown to function in axon guidance. We have cloned a transmembrane semaphorin, Sema W, that belongs to the class IV subgroup of the semaphorin family. The mouse and rat forms of Sema W show 97% amino acid sequence identity with each other, and each shows about 91% identity with the human form. The gene for Sema W is divided into 15 exons, up to 4 of which are absent in the human cDNAs that we sequenced. Unlike many other semaphorins, Sema W is expressed at low levels in the developing embryo but was found to be expressed at high levels in the adult central nervous system and lung. Functional studies with purified membrane fractions from COS7 cells transfected with a Sema W expression plasmid showed that Sema W has growth-cone collapse activity against retinal ganglion-cell axons, indicating that vertebrate transmembrane semaphorins, like secreted semaphorins, can collapse growth cones. Genetic mapping of human SEMAW with human/hamster radiation hybrids localized the gene to chromosome 2p13. Genetic mapping of mouse Semaw with mouse/hamster radiation hybrids localized the gene to chromosome 6, and physical mapping placed the gene on bacteria artificial chromosomes carrying microsatellite markers D6Mit70 and D6Mit189. This localization places Semaw within the locus for motor neuron degeneration 2, making it an attractive candidate gene for this disease.

Enrichment for murine keratinocyte stem cells based on cell surface phenotype

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [3H]thymidine ([3H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [3H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of α6 integrin and low to undetectable expression of the transferrin receptor (CD71) termed α6briCD71dim cells, are enriched for epithelial stem cells because they represent a minor (≈8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing ≈70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed α6briCD71bri, representing the majority (≈60%) of basal keratinocytes that are actively cycling, and importantly contain ≈70% of [3H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71dim cells in the hair follicle bulge region, a well documented location for KSCs.

Suppression of trkB expression by antisense oligonucleotides alters a neuronal phenotype in the rod pathway of the developing rat retina.

trkB is the high-affinity receptor for brain-derived neurotrophic factor (BDNF), a trophic molecule with demonstrated effects on the survival and differentiation of a wide variety of neuronal populations. In the mammalian retina, trkB is localized to both ganglion cells and numerous cells in the inner nuclear layer. Much information on the role of BDNF in neuronal development has been derived from the study of trkB- and BDNF-deficient mutant mice. This includes an attenuation of the numbers of cortical neurons immunopositive for the calcium-binding proteins, parvalbumin, and calbindin. Unfortunately, these mutant animals typically fail to survive for > 24-48 hr after birth. Since most retinal neuronal differentiation occurs postnatally, we have devised an alternative scheme to suppress the expression of trkB in the retina to examine the role of BDNF on the postnatal development of neurons of the inner retina. Neonatal rats were treated with intraocular injection of an antisense oligonucleotide (1-2 microliters of 10-100 microM solution) targeted to the trkB mRNA. Immunohistochemistry with a polyclonal antibody to trkB showed that the expression of trkB in retinal neurons was suppressed 48-72 hr following a single injection. Northern blot analysis demonstrated that antisense treatment had no effect on the level of trkB mRNA, even after multiple injections. This suggests an effect of trkB antisense treatment on protein translation, but not on RNA transcription. No alterations were observed in the thickness of retinal cellular or plexiform layers, suggesting that BDNF is not the sole survival factor for these neurons. There were, however, alterations in the patterns of immunostaining for parvalbumin, a marker for the narrow-field, bistratified AII amacrine cell-a central element of the rod (scotopic) pathway. This was evidenced by a decrease in both the number of immunostained somata (> 50%) and in the intensity of immunolabeling. However, the immunostaining pattern of calbindin was not affected. These studies suggest that the ligands for trkB have specific effects on the neurochemical phenotypic expression of inner retinal neurons and in the development of a well-defined retinal circuit.

Retinal glial cell glutamate transporter is coupled to an anionic conductance.

Application of L-glutamate to retinal glial (Müller) cells results in an inwardly rectifying current due to the net influx of one positive charge per molecule of glutamate transported into the cell. However, at positive potentials an outward current can be elicited by glutamate. This outward current is eliminated by removal of external chloride ions. Substitution of external chloride with the anions thiocyanate, perchlorate, nitrate, and iodide, which are known to be more permeant at other chloride channels, results in a considerably larger glutamate-elicited outward current at positive potentials. The large outward current in external nitrate has the same ionic dependence, apparent affinity for L-glutamate, and pharmacology as the glutamate transporter previously reported to exist in these cells. Varying the concentration of external nitrate shifts the reversal potential in a manner consistent with a conductance permeable to nitrate. Together, these results suggest that the glutamate transporter in retinal glial cells is associated with an anionic conductance. This anionic conductance may be important for preventing a reduction in the rate of transport due the depolarization that would otherwise occur as a result of electrogenic glutamate uptake.

A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity.

We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI-1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 microM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine; hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se-carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent Km values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 microM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.

Radial and tangential dispersion patterns in the mouse retina are cell-class specific.

The retina is derived from a pseudostratified germinal zone in which the relative position of a progenitor cell is believed to determine the position of the progeny aligned in the radial axis. Such a developmental mechanism would ensure that radial arrays of cells which comprise functional units in the mature central nervous system are also clonally related. The present study has tested this hypothesis by using X chromosome-inactivation transgenic mosaic mice. We report that the retina shows a conspicuous distinction for clonally related neuroblasts of different laminar and functional fates: the rod photoreceptor, Müller, and bipolar cells are aligned in the radial axis, whereas the cone photoreceptor, horizontal, amacrine, and ganglion cells are tangentially displaced with respect to them. These results indicate that the dispersion of cell classes across the retinal surface is differentially constrained. Some classes of retinal neuroblast exhibit a significant tangential, as well as radial, component in their dispersion from the germinal zone, whereas others disperse only in the radial dimension. Consequently, the majority of radial columns within the mature retina must be derived from multiple progenitors. Because the cone photoreceptor, horizontal, amacrine, and ganglion cells establish nonrandom matrices in their cellular distributions within the respective retinal layers, tangential dispersion may be the means by which these matrices are constructed.

Whole-exome sequencing reveals ZNF408 as a new gene associated with autosomal recessive retinitis pigmentosa with vitreal alterations

Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies that cause visual impairment as a result of photoreceptor cell death. RP is heterogeneous, both clinically and genetically making difficult to establish precise genotype–phenotype correlations. In a Spanish family with autosomal recessive RP (arRP), homozygosity mapping and whole-exome sequencing led to the identification of a homozygous mutation (c.358_359delGT; p.Ala122Leufs*2) in the ZNF408 gene. A screening performed in 217 additional unrelated families revealed another homozygous mutation (c.1621C>T; p.Arg541Cys) in an isolated RP case. ZNF408 encodes a transcription factor that harbors 10 predicted C2H2-type fingers thought to be implicated in DNA binding. To elucidate the ZNF408 role in the retina and the pathogenesis of these mutations we have performed different functional studies. By immunohistochemical analysis in healthy human retina, we identified that ZNF408 is expressed in both cone and rod photoreceptors, in a specific type of amacrine and ganglion cells, and in retinal blood vessels. ZNF408 revealed a cytoplasmic localization and a nuclear distribution in areas corresponding with the euchromatin fraction. Immunolocalization studies showed a partial mislocalization of the p.Arg541Cys mutant protein retaining part of the WT protein in the cytoplasm. Our study demonstrates that ZNF408, previously associated with Familial Exudative Vitreoretinopathy (FEVR), is a new gene causing arRP with vitreous condensations supporting the evidence that this protein plays additional functions into the human retina.

Type 1 nitrergic (ND1) cells of the rabbit retina: Comparison with other axon-bearing amacrine cells

NADPH diaphorase (NADPHd) histochemistry labels two types of nitrergic amacrine cells in the rabbit retina. Both the large ND1 cells and the small ND2 cells stratify in the middle of the inner plexiform layer, and their overlapping processes produce a dense plexus, which makes it difficult to trace the morphology of single cells. The complete morphology of the ND1 amacrine cells has been revealed by injecting Neurobiotin into large round somata in the inner nuclear layer, which resulted in the labelling of amacrine cells whose proximal morphology and stratification matched those of the ND1 cells stained by NADPHd histochemistry. The Neurobiotin-injected ND1 cells showed strong homologous tracer coupling to surrounding ND1 cells, and double-labelling experiments confirmed that these coupled cells showed NADPHd reactivity. The ND1 amacrine cells branch in stratum 3 of the inner plexiform layer, where they produce a sparsely branched dendritic tree of 400-600 mum diameter in ventral peripheral retina. In addition, each cell gives rise to several fine beaded processes, which arise either from a side branch of the dendritic tree or from the tapering of a distal dendrite. These axon-like processes branch successively within the vicinity of the dendritic field before extending, with little or no further branching, for 3-5 mm from the soma in ventral peripheral retina. Consequently, these cells may span one-third of the visual field of each eye, and their spatial extent appears to be greater than that of most other types of axon-bearing amacrine cells injected with Neurobiotin in this study. The morphology and tracer-coupling pattern of the ND1 cells are compared with those of confirmed type 1 catecholaminergic cells, a presumptive type 2 catecholaminergic cell, the type 1 polyaxonal. cells, the long-range amacrine cells, a novel type of axon-bearing cell that also branches in stratum 3, and a type of displaced amacrine cell that may correspond to the type 2 polyaxonal cell. (C) 2004 Wiley-Liss, Inc.

The dendritic architecture of the cholinergic plexus in the rabbit retina: Selective labeling by glycine accumulation in the presence of sarcosine

The cholinergic amacrine cells in the rabbit retina slowly accumulate glycine to very high levels when the tissue is incubated with excess sarcosine (methylglycine), even though these cells do not normally contain elevated levels of glycine and do not express high-affinity glycine transporters. Because the sarcosine also depletes the endogenous glycine in the glycine-containing amacrine cells and bipolar cells, the cholinergic amacrine cells can be selectively labeled by glycine immunocytochemistry under these conditions. Incubation experiments indicated that the effect of sarcosine on the cholinergic amacrine cells is indirect: sarcosine raises the extracellular concentration of glycine by blocking its re-uptake by the glycinergic amacrine cells, and the excess glycine is probably taken-up by an unidentified low-affinity transporter on the cholinergic amacrine cells. Neurobiotin injection of the On-Off direction-selective (DS) ganglion cells in sarcosine-incubated rabbit retina was combined with glycine immunocytochemistry to examine the dendritic relationships between the DS ganglion cells and the cholinergic amacrine cells. These double-labeled preparations showed that the dendrites of the DS ganglion cells closely follow the fasciculated dendrites of the cholinergic amacrine cells. Each ganglion cell dendrite located within the cholinergic strata is associated with a cholinergic fascicle and, conversely, there are few cholinergic fascicles that do not contain at least one dendrite from an On-Off DS cell. It is not known how the dendritic co-fasciculation develops, but the cholinergic dendritic plexus may provide the initial scaffold, because the dendrites of the On-Off DS cells commonly run along the outside of the cholinergic fascicles. J. Comp. Neurol. 421:1-13, 2000. (C) 2000 Wiley-Liss, Inc.

Investigation into visual defects attributed to Vigabatrin

Vigabatrin (VGB) is a transaminase inhibitor that elicits its anitepileptic effect by increasing GABA concentrations in the brain and retina. - Assess whether certain factors predispose patients to develop severe visual field loss. - Develop a sensitive algorithm for investigating the progression of visual field loss. - Determine the most sensitive clinical regimen for diagnosing VGB-attributed visual field loss. - Investigate whether the reports of central retinal sparing are accurate. The investigations have resulted in a number of significant findings: - The anatomical evidence in combination with the pattern of visual field loss suggests that the damage induced by VGB therapy occurs at retinal level, and is most likely a toxic effect. - The quantitative algorithm, designed within the course of this investigation, provided increased sensitivity in determining the severity of visual field loss. - Maximum VGB dose predisposes patients to develop severe visual field loss. - The SITA Standard algorithm was found to be as sensitive and significantly faster, in diagnosing visual field defects attributed to VGB, when compared to the Full Threshold algorithm. The Full Threshold was found to be the most repeatable between visits. - The normal SWAP 10-2 database provided an effective method of differentiating SWAP defects. - SWAP, FDT and the mfERG have increased sensitivity in detecting visual field loss attributed to VGB. The pattern of visual field loss from these investigations suggests that VGB produces a diffuse effect across the retina including subtle central abnormalities and more severe peripheral defects. - Abnormalities detected using the mfERG have suggested that VGB adversely affects the photoreceptors Müller, amacrine and ganglion cells in the retina. An urgent review of the manufacturers recommended maximum dose for VGB is required.

An overview of eye-related photophobias

This chapter provides an overview of the various eye-related causes of photophobia and the likely mechanisms responsible. Photophobia is the experience of discomfort affecting the eyes as a result of exposure to light. It has a variety of causes, including the result of eye or brain disease, or it can be a side effect of various drugs or laser surgery. Photophobia can also be a symptom of a more serious disorder such as meningitis and therefore, requires appropriate investigation, diagnosis, and treatment. Trauma or disease affecting several structures of the eye are a common cause of photophobia and can be associated with: (1) the ocular adnexia, such as blepharitis and blepharospasm, (2) the cornea, including abrasion, ulcerative keratitis, and corneal dystrophy, (3) problems in eye development, such as aniridia, buphthalmos, coloboma, and aphakia, (4) various eye inflammations, including uveitis, and (5) retinal disorders, such as achromatopsia, retinal detachment, and retinal dystrophy. There may be two main explanations for photophobia associated with these conditions: (1) direct stimulation of the trigeminal nerve due to damage, disease, or excessive light entering the eye and (2) overstimulation of the retina including a specific population of light-sensitive ganglion cells.

Eye-related photophobia

This article provides an overview of the various eye-related causes of photophobia and the likely mechanisms responsible. Photophobia is an experience of discomfort affecting the eyes due to exposure to light. It has a variety of causes including the result of eye or brain disease, or it can be a side effect of various drugs or laser surgery. Photophobia can also be a symptom of a more serious disorder such as meningitis and therefore, requires appropriate investigation, diagnosis, and treatment. Trauma or disease affecting several structures of the eye are a common cause of photophobia and can be associated with: (1) the ocular adnexia, such as blepharitis and blepharospasm, (2) the cornea, including abrasion, ulcerative keratitis, and corneal dystrophy, (3) problems in eye development, such as aniridia, buphthalmos, coloboma, and aphakia, (4) various eye inflammations, including uveitis, and (5) retinal disorders, such as achromatopsia, retinal detachment, and retinal dystrophy. There may be two main explanations for eye-related photophobia: (1) direct stimulation of the trigeminal nerve due to damage, disease, or excessive light entering the eye and (2) overstimulation of the retina including a specific population of light-sensitive ganglion cells.

Dopamine and visually regulated eye growth in chick

Retinal image properties such as contrast and spatial frequency play important roles in the development of normal vision. For example, visual environments comprised solely of low contrast and/or low spatial frequencies induce myopia. The visual image is processed by the retina and it then locally controls eye growth. In terms of the retinal neurotransmitters that link visual stimuli to eye growth, there is strong evidence to suggest involvement of the retinal dopamine (DA) system. For example, effectively increasing retinal DA levels by using DA agonists can suppress the development of form-deprivation myopia (FDM). However, whether visual feedback controls eye growth by modulating retinal DA release, and/or some other factors, is still being elucidated. This thesis is chiefly concerned with the relationship between the dopaminergic system and retinal image properties in eye growth control. More specifically, whether the amount of retinal DA release reduces as the complexity of the image degrades was determined. For example, we investigated whether the level of retinal DA release decreased as image contrast decreased. In addition, the effects of spatial frequency, spatial energy distribution slope, and spatial phase on retinal DA release and eye growth were examined. When chicks were 8-days-old, a cone-lens imaging system was applied monocularly (+30 D, 3.3 cm cone). A short-term treatment period (6 hr) and a longer-term treatment period (4.5 days) were used. The short-term treatment tests for the acute reduction in DA release by the visual stimulus, as is seen with diffusers and lenses, whereas the 4.5 day point tests for reduction in DA release after more prolonged exposure to the visual stimulus. In the contrast study, 1.35 cyc/deg square wave grating targets of 95%, 67%, 45%, 12% or 4.2% contrast were used. Blank (0% contrast) targets were included for comparison. In the spatial frequency study, both sine and square wave grating targets with either 0.017 cyc/deg and 0.13 cyc/deg fundamental spatial frequencies and 95% contrast were used. In the spectral slope study, 30% root-mean-squared (RMS) contrast fractal noise targets with spectral fall-off of 1/f0.5, 1/f and 1/f2 were used. In the spatial alignment study, a structured Maltese cross (MX) target, a structured circular patterned (C) target and the scrambled versions of these two targets (SMX and SC) were used. Each treatment group comprised 6 chicks for ocular biometry (refraction and ocular dimension measurement) and 4 for analysis of retinal DA release. Vitreal dihydroxyphenylacetic acid (DOPAC) was analysed through ion-paired reversed phase high performance liquid chromatography with electrochemical detection (HPLC-ED), as a measure of retinal DA release. For the comparison between retinal DA release and eye growth, large reductions in retinal DA release possibly due to the decreased light level inside the cone-lens imaging system were observed across all treated eyes while only those exposed to low contrast, low spatial frequency sine wave grating, 1/f2, C and SC targets had myopic shifts in refraction. Amongst these treatment groups, no acute effect was observed and longer-term effects were only found in the low contrast and 1/f2 groups. These findings suggest that retinal DA release does not causally link visual stimuli properties to eye growth, and these target induced changes in refractive development are not dependent on the level of retinal DA release. Retinal dopaminergic cells might be affected indirectly via other retinal cells that immediately respond to changes in the image contrast of the retinal image.

Short-term retention of labeled chondrocyte subpopulations in stratified tissue-engineered cartilaginous constructs implanted in vivo in mini-pigs

It is likely that effective application of cell-laden implants for cartilage defects depends on retention of implanted cells and interaction between implanted and host cells. The objectives of this study were to characterize stratified cartilaginous constructs seeded sequentially with superficial (S) and middle (M) chondrocyte subpopulations labelled with fluorescent cell tracking dye PKH26 (*) and determine the degree to which these stratified cartilaginous constructs maintain their architecture in vivo after implantation in mini-pigs for 1 week. Alginate-recovered cells were seeded sequentially to form stratified S*/M (only S cells labelled) and S*/M* (both S and M cells labelled) constructs. Full-thickness defects (4 mm diameter) were created in the patellofemoral groove of adult Yucatan mini-pigs and filled with portions of constructs or left empty. Constructs were characterized biochemically, histologically, and biomechanically, and stratification visualized and quantified, before and after implant. After 1 week, animals were sacrificed and implants retrieved. After 1 week in vivo, glycosaminoglycan and collagen content of constructs remained similar to that at implant, whereas DNA content increased. Histological analyses revealed features of an early repair response, with defects filled with tissues containing little matrix and abundant cells. Some implanted (PKH26-labeled) cells persisted in the defects, although constructs did not maintain a stratified organization. Of the labelled cells, 126 +/- 38% and 32 +/- 8% in S*/M and S*/M* constructs, respectively, were recovered. Distribution of labelled cells indicated interactions between implanted and host cells. Longer-term in vivo studies will be useful in determining whether implanted cells are sufficient to have a positive effect in repair.