Effect of a 30 kD protein from tectal extract of rat on cultured retinal neuron.

For the first time we have shown here that the constituent from tectal extract (Te) which can support and promote the survival and growth of the retinal ganglion cell is a 30 kD protein. (i) Using MTT colorimetric microassay to measure the optical density for the survival and growth of the cultured retinal neuron, it was found that the optical densities for the experimental cultures with either Te, or its 10-30 kD fraction, or its greater than or equal to 30 kD fraction were 2-4 times that of the control culture without Te (P less than 0.01). This indicated that experimental cultures were more active in growth. (ii) The retinal neurons cultured on Te Phast gels showed that large retinal neurons (greater than 18 microns) grew only on the gel region containing the 30 kD protein. The retrograde prelabelled with horseradish peroxidase (HRP) for retinal ganglion cells indicated that these surviving neurons grown on Te Phast gel were HRP positive, i.e. retinal ganglion cells. (iii) The retinal explants cultured with 30 kD gels at a close distance of 1 mm apart revealed that many neurite outgrowths, up to 400 microns, extended from the retinal explants towards 30 kD gels, and that many individual cells and tissue masses migrated out from the explants and grew onto 30 kD gels. They were stained anti-neuron specific enolase and anti-Thy 1.1 positive, indicating they are retinal ganglion cells. Therefore, we concluded that the 30 kD protein is the neuronotrophic factor in the tectal extract specific for retinal ganglion cells.     

High Resolution MALDI Imaging Mass Spectrometry of Retinal Tissue Lipids

Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has the ability to provide an enormous amount of information on the abundances and spatial distributions of molecules within biological tissues. The rapid progress in the development of this technology significantly improves our ability to analyze smaller and smaller areas and features within tissues. The mammalian eye has evolved over millions of years to become an essential asset for survival, providing important sensory input of an organism’s surroundings. The highly complex sensory retina of the eye is comprised of numerous cell types organized into specific layers with varying dimensions, the thinnest of which is the 10 μm retinal pigment epithelium (RPE). This single cell layer and the photoreceptor layer contain the complex biochemical machinery required to convert photons of light into electrical signals that are transported to the brain by axons of retinal ganglion cells. Diseases of the retina, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, occur when the functions of these cells are interrupted by molecular processes that are not fully understood. In this report, we demonstrate the use of high spatial resolution MALDI IMS and FT-ICR tandem mass spectrometry in the Abca4 ^–/– knockout mouse model of Stargardt disease, a juvenile onset form of macular degeneration. The spatial distributions and identity of lipid and retinoid metabolites are shown to be unique to specific retinal cell layers.

Microfluidic devices for culturing primary mammalian neurons at low densities

Microfluidic devices have been used to study high-density cultures of many cell types. Because cell-to-cell signaling is local, however, there exists a need to develop culture systems that sustain small numbers of neurons and enable analyses of the microenvironments. Such cultures are hard to maintain in stable form, and it is difficult to prevent cell death when using primary mammalian neurons. We demonstrate that postnatal primary hippocampal neurons from rat can be cultured at low densities within nanoliter-volume microdevices fabricated using polydimethylsiloxane (PDMS). Doing so requires an additional fabrication step, serial extractions/washes of PDMS with several solvents, which removes uncrosslinked oligomers, solvent and residues of the platinum catalyst used to cure the polymer. We found this step improves the biocompatibility of the PDMS devices significantly. Whereas neurons survive for > or = 7 days in open channel microdevices, the ability to culture neurons in closed-channel devices made of untreated, native PDMS is limited to < or = 2 days. When the closed-channel PDMS devices are extracted, biocompatibility improves allowing for reliable neuron cultures at low densities for > or = 7 days. Comparisons made to autoclaved PDMS and native, untreated PDMS reveal that the solvent-treated polymer is superior in sustaining low densities of primary neurons in culture. When neuronal affinity for local substrates is observed directly, we find that axons localize to channel corners and prefer PDMS surfaces to glass in hybrid devices. When perfusing the channels with media by gravity flow, cultured hippocampal neurons survive for > or = 11 days. Extracting PDMS improves biocompatibility of microfluidic devices and thus enables the study of differentiation of identifiable neurons and the characterization of local extracellular signals.     

Effects of Lead on Temporal Response Properties of Retinal Ganglion Cells in Developing Rats

Neonatal rats have taken in lead, during the period from their parturition to their weaning, from the milk of dams fed with water containing 0.2% lead acetate solutions. The alterations in the temporal response properties of retinal ganglion cells in adult rats (90 days) following the lead exposure at their developing stage have been studied. The results of this investigation demonstrate that the lead exposure in neonatal rats causes decreases in the optimal temporal frequency, bandwidth at half amplitude, temporal resolution and response phase of the retinal ganglion cells in adult rats. Compared with the sustained cells, the transient cells have a much greater alteration in temporal response properties.     

The photochemical reaction cycle of retinal reconstituted bacteriorhodopsin

The function of three types of bacteriorhodopsins was compared: the wild-type, the bleached and retinal reconstituted and retinal deficient bacteriorhodopsin after retinal addition. The apparent pK(a) of the proton acceptor group for the bleached BR and retinal deficient BR shifted toward higher pH values compared to the wild-type BR. Fitting the photocycle model to the absorption kinetic signals for all three proteins showed the existence of the same intermediates, but the time-dependent concentration of the intermediates was different. Although measurements were made at pH 7, the absorption kinetics and photoelectric signals in both retinal reconstituted samples acted as wild-type bacteriorhodopsin at significantly higher pH. Below pH 3 the retinal deficient and reconstituted sample bleached. These results suggested that the added retinal was not able to rebind in the same position in the protein as in native bacteriorhodopsin. This points out that care should be taken, when bleached bacteriorhodopsin is reconstituted with different retinal analogs.     

镧对仔鼠嗅感觉神经元分化中β—Ⅲ微管蛋白的作用

为观察发育期氯化镧暴露对仔鼠嗅觉功能及β-Ⅲ微管蛋白表达的影响,将16只Wistar孕鼠随机分为两组（对照组、氯化镧组）,对照组母鼠饮用蒸馏水,氯化镧组饮用0．25％氯化镧溶液;仔鼠出生后25d采用嗅觉迷宫试验检测仔鼠嗅觉功能的差异;于出生后28d采用免疫荧光和免疫印迹观察仔鼠β-Ⅲ微管蛋白在嗅上皮的表达变化。结果表明,氯化镧组仔鼠在嗅觉迷宫试验寻找食物的时间较对照组长,差异具有统计学意义（P〈0．05）;氯化镧组下调仔鼠嗅上皮β-Ⅲ微管蛋白在嗅感觉神经元的表达。提示镧暴露损害子代嗅觉功能,可能与镧损害嗅上皮β-Ⅲ微管蛋白的表达有关。     

ORCADIAN OSCILLATORS AND PHOTORECEPTORS IN THE GASTROPOD,APLYSIA*

Abstract— The sea slug, Aplysia, is a useful model system for research on the neurophysiology of circadian integration. The animal contains several circadian oscillators and several photoreceptors. Each eye contains a circadian oscillator as well as photoreceptors. The ocular oscillators can be entrained by extraocular photoreceptors as well as their own ocular photoreceptors. The abdominal ganglion probably contains another oscillator but it has been much more difficult to manipulate in the laboratory than have the oscillators in the eyes. There is also a circadian rhythm in overt behavioral activity. This rhythm is controlled in part by extraocular oscillators and extraocular photoreceptors and in part by the eyes. In exerting their influence on the behavioral rhythm, the eyes appear to act in the capacity of oscillators and not merely as photoreceptors. Although neurons in the retina have neurosecretory morphology, the entire influence of the eyes on the behavioral rhythm appears to be mediated by nerve signals which travel in the optic nerve. As yet there is no evidence to suggest that any two oscillators in Aplysia are internally coupled. There is also no evidence yet for hormonal coupling between photoreceptors and oscillators or between oscillators and rhythmic outputs.     

A microfluidic chip capable of switching W/O droplets to vertical laminar flow for electrochemical detection of droplet contents

Analysis of droplet contents is a key function involved in droplet-based microfluidic systems. Direct electrochemical detection of droplet contents suffers problems such as relatively poor repeatability, interference of capacitive current and relatively poor detectability. This paper presents a novel hybrid polydimethylsiloxane-glass chip for highly sensitive and reproducible amperometric detection of droplet contents. By wettability-patterning of the channel surface of the hybrid chip, water in oil droplets generated in the upstream part of the central channel can be switched to a two-phase vertical laminar flow (i.e., a continuous oil stream flowing atop a continuous aqueous stream) in the downstream part of the channel. The vertical laminar flow keeps the analyte in the underneath-flowing aqueous stream in direct contact with the sensing electrodes located on the bottom surface of the channel. Therefore, steady-state current signals with high sensitivity (1.2 A M⁻¹ cm⁻² for H₂O₂), low limit of detection (0.12 μM, S/N = 2), and good reproducibility (RSD 1.1% at 0.3 mM H₂O₂) were obtained. The methods for patterning of the inner channel surface are presented, and the behaviors of the microchip in flow profile switching and amperometric detection are discussed. The application of the developed microchip to enzyme kinetics study is also demonstrated.     

Fabrication of a Retinal‐protein Photoreceptor Array

A pixel‐architecture film of retinal proteins was prepared by an approach combining chemical, physical and biological technologies. Oriented multilayers of purple membrane composed of bacteriorhodopsin (BR) and lipids were patterned on an array of gold electrode pixels. In order to improve stability and resolution, the gene engineering technique was employed to make a mutant of the protein BR by replacing the 36^th amino acid residue from aspartic acid to cysteine with a thiol end group ready to react with gold; electric sedimentation was used to guarantee the high probability of formation of the Au‐S bond and meanwhile to orient BR; further chemical crosslinking was introduced among layers of purple membranes to significantly enhance photoelectrical signals while keeping high stability. The non‐bound BR region was eventually washed out by detergent, and the remaining BR pixels were thus detergent resistant due to chemical crosslinking among BR layers and covalent binding between the multilayer and the substrate. The protein array was confirmed to keep photoelectrical activity.     

不同产地金线莲根茎和叶中多糖含量对比

考察了不同产地金线莲根茎和叶中多糖含量。应用超声辅助法和微波-超声协同萃取法提取金线莲根茎和叶中多糖,用苯酚-硫酸法测定其含量。微波-超声协同萃取法提取优于超声辅助法提取,金线莲根茎部的多糖含量高于叶中多糖含量,福建华安产金线莲多糖含量最高。金线莲中多糖含量为0.8509%～6.400%,具有开发药用的价值。     

A three-dimensional flexible microprobe array for neural recording assembled through electrostatic actuation

We designed, fabricated and tested a novel three-dimensional flexible microprobe to record neural signals of a lateral giant nerve fiber of the escape circuit of an American crayfish. An electrostatic actuation folded planar probes into three-dimensional neural probes with arbitrary orientations for neuroscientific applications. A batch assembly based on electrostatic forces simplified the fabrication and was non-toxic. A novel fabrication for these three-dimensional flexible probes used SU-8 and Parylene technology. The mechanical strength of the neural probe was great enough to penetrate into a bio-gel. A flexible probe both decreased the micromotion and alleviated tissue encapsulation of the implant caused by chronic inflammation of tissue when an animal breathes or moves. The cortex consisted of six horizontal layers, and the neurons of the cortex were arranged in vertical structures; the three-dimensional microelectrode arrays were suitable to investigate the cooperative activity for neurons in horizontal separate layers and in vertical cortical columns. With this flexible probe we recorded neural signals of a lateral giant cell from an American crayfish. The response amplitude of action potentials was about 343 μV during 1 ms period; the average recorded data had a ratio of signal to noise as great as 30.22 ± 3.58 dB. The improved performance of this electrode made feasible the separation of neural signals according to their distinct shapes. The cytotoxicity indicated a satisfactory biocompatibility and non-toxicity of the flexible device fabricated in this work.     

Comparisons of the structural and chemical properties of melanosomes isolated from retinal pigment epithelium, iris and choroid of newborn and mature bovine eyes

Melanosomes were isolated from the retinal pigment epithelium (RPE), iris and choroid of mature (age >2 years) and newborn (age <1 week) bovine eyes. Scanning electron microscopy was utilized to analyze the morphology of the melanosomes, which were found to vary among different tissues and different ages. While the total content of amino acids differs slightly (ranging from 9% to 15% by mass), the distributions of the amino acids are similar. The pheomelanin content is low in the choroid and the RPE (0.1-0.5%), and moderate in the iris (<2%); therefore, the major melanin component of bovine eye melanosomes is eumelanin, independent of the shape of the melanosomes. The yields of pyrrole-2,3,5-tricarboxylic acid from melanosomes decrease in the following order: choroid > iris > RPE, and exhibit decreasing yields with age. 13C solid-state nuclear magnetic resonance (NMR) spectroscopic analysis of iris and choroid melanosomes indicates the same trends. These observations suggest that the 5,6-dihydroxyindole-2-carboxylic acid contents decrease in the following order: choroid > iris > RPE, and decrease with age. Moreover, the 13C solid-state NMR spectra show (1) for the same age samples, the CH:Cq ratio for choroid is larger than that for iris melanosomes; and (2) an increase in the concentration of carbonyl groups with age within each type of melanosome.     

DISTRIBUTION OF 5-AMINOLEVULINIC ACID-INDUCED PORPHYRINS IN NODULOULCERATIVE BASAL CELL CARCINOMA

Abstract— Microscopic fluorescence photometry incorporating a light-sensitive thermo-electrically cooled charge-coupled device (CCD) camera was employed to investigate the fluorescence distribution of 5-aminolevulinic acid (ALA)-induced porphyrins in 22 patients with a total number of 52 noduloul-cerative basal cell carcinomas (BCC) after topical ALA application with or without dimethylsulfoxide (DMSO)/ethylenediaminetetraacetic acid (EDTA) or after intravenous administration of ALA. Both localization patterns and amounts of ALA-induced porphyrins in the BCC were studied. The ALA-induced porphyrins were localized only in the superficial layers of the noduloulcerative BCC lesions after topical application of 20% ALA alone for 3 h. However, both the penetration of ALA into deep lesions and the production of the ALA-induced porphyrin fluorescence were increased after topical administration of 20% ALA and 20% DMSO/4% EDTA for 3 h. Prior treatment with 99% DMSO for 15 min further enhanced the ALA penetration into the BCC lesions after topical application of the ALA/DMSO/EDTA mixture and produced more ALA-induced porphyrins by a factor of about three compared with those treated with ALA alone. The penetration of ALA into the deep BCC lesions could also be increased by prolonging the time of topical application of 20% ALA/4% EDTA to 29–48 h (without DMSO). Intravenous injection of ALA led to a more homogeneous distribution of the ALA-derived porphyrins in the whole noduloulcerative BCC lesions.     

Microfluidic hydrogel layers with multiple gradients to stimulate and perfuse three-dimensional neuronal cell cultures

We present a simple and easy to handle PDMS microfluidic device for neuronal cell culture studies in three-dimensional hydrogel scaffolds. The hydrogel is structured in parallel layers to reconstruct cell layers close to the natural environment. Dissociated cortical neurons of embryonic rats have been cultured in 0.5% w/v agarose including 0.2% w/v alginate. The cells formed neurite networks through neighboring cell free hydrogel layers. The cell culture showed neurite outgrowth in the microfluidic channel over more than seven days in vitro without perfusion. Culturing neurons in hydrogel layers surrounded by a liquid phase containing culture medium resulted in denser neuronal networks.     

Device for co-culture of sympathetic neurons and cardiomyocytes using microfabrication

Rat superior cervical ganglion (SCG) neurons and ventricular myocytes (VMs) were co-cultured separately in a minichamber placed on a microelectrode-array (MEA) substrate. The minichamber, fabricated photolithographically using polydimethylsiloxane (PDMS), had 2 compartments, 16 microcompartments and 8 microconduits. The SCG neurons were seeded into one of the compartments and all of the microcompartments using a glass pipette controlled by a micromanipulator and a microinjector. The VMs were seeded into the other compartment. Three days after seeding of the VMs, the SCG neurons were still confined to one compartment and all of the microcompartments, and the neurites of the SCG neurons had connected with the VMs via the microconduits. Constant-voltage stimulation, using a train of biphasic square pulses (1 ms at +1 V, followed by -1 ms at 1 V), was applied to the SCG neurons in the microcompartments using 16 electrodes. Evoked responses were observed in several electrodes while electrical stimulation was applied to the SCG neurons. Two-way analysis of variance (ANOVA) revealed that the frequency of the stimulation pulses had significant effects in increasing the beat rate of the VMs, and that the interaction between the frequency and the number of the pulses also had a significant effect on the ratio. No significant increases in the beat rate were observed when propranolol, a β-adrenergic receptor antagonist, was added to the culture medium. These results suggest that synaptic pathways were formed between the SCG neurons and the VMs, and that this co-culture device can be utilized for studies of network-level interactions between sympathetic neurons and cardiomyocytes.     

Visual functional effects of constant blue light in a retinal degenerate rat model

Retinal degenerative conditions increase susceptibility to light damage, but rapid retinal degeneration (RD) models show less susceptibility to cyclic dim light. We investigated whether constant blue light (BL) exposure can eliminate the residual visual responses in a comparatively rapid RD rat model. Pigmented rhodopsin mutant S334ter line-3 rat pups (21 days old) were exposed for 5-6 consecutive days to constant BL. Visual behavior was evaluated with an optokinetic head tracking apparatus. Electrophysiological recordings were made from the superior colliculus (SC). S-antigen, red-green opsin and rhodopsin immunoreactive residual photoreceptors were counted. Following BL exposure, head tracking was significantly reduced at 0.25 cycles degree(-1) in 38-day-old line 3 rats. With a 0.125 cycles degree(-1) stimulus, the head tracking performance of 80-day-old BL rats were similar to that of 220-day-old no-BL-treated line-3 rats. SC recordings also revealed a significant decrease in the residual photoreceptor activity. Histological evaluation showed reduction of the rod population in the central area of the light-damaged retina. Exposure to constant BL considerably reduces the residual visual responses in a rapid degenerating RD rat model.     

Functional expression, targeting and Ca2+ signaling of a mouse melanopsin-eYFP fusion protein in a retinal pigment epithelium cell line

Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11-cis retinal or all-trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin.     

Shank 2 expression coincides with neuronal differentiation in the developing retina

The retinal activity for vision requires a precise synaptic connectivity. Shank proteins at postsynaptic sites of excitatory synapses play roles in signal transmission into the postsynaptic neuron. However, the correlation of Shank 2 expression with neuronal differentiation in the developing retina remains to be elucidated regardless of previous evidences of Shank 2 expression in retina. Herein, we demonstrated that with progression of development, Shank 2 is initially detected in the inner plexiform layer at P2, and then intensively detected in inner plexiform layer, outer plexiform layer, and ganglion cell layer at P14, which was closely colocalized to the neurofilament expression. Shank 2 was, however, not colocalized with glial fibrillary acidic protein. Shank 2 expression was increased in the differentiated retinoblastoma cells, which was mediated by ERK 1/2 activation. Moreover, Shank 2 expression was colocalized with neurofilament at the dendritic region of cells. In conclusion, our data suggests that Shank 2 is expressed in the neurons of the developing retina and could play a critical role in the neuronal differentiation of the developing retina.     

Melanopsin triggers the release of internal calcium stores in response to light

Melanopsin is the photopigment that confers photosensitivity upon intrinsically photosensitive retinal ganglion cells (ipRGCs). This subset of retinal ganglion cells comprises less than 2% of all RGCs in the mammalian retina. The paucity of melanopsin-positive cells has made studies on melanopsin signaling difficult to pursue in ipRGCs. To address this issue, we have established several cell lines consisting of a transformed human embryonic kidney cell line (HEK293) stably expressing human melanopsin. With these cell lines, we have investigated the intracellular rise in calcium triggered upon light activation of melanopsin. Our human melanopsin-expressing cells exhibit an irradiance-dependent increase in intracellular calcium. Control cells expressing human melanopsin, where the Schiff-base lysine has been mutated to alanine, show no responses to light. Chelating extracellular calcium has no effect on the light-induced increase in intracellular calcium suggesting that calcium is mobilized from intracellular stores. This involvement of intracellular stores has been confirmed through their depletion by thapsigargin, which inhibits a subsequent light-induced increase in intracellular calcium. Addition of the nonselective cation channel blocker lanthanum does not alter light-induced rises in intracellular calcium, further supporting that melanopsin triggers a release of internal calcium from internal stores. HEK293 cells stably expressing melanopsin have proven to be a useful tool to study melanopsin-initiated signaling.     

CO2 Induces Symmetry Breaking in Layered Dipeptide Crystals

Control of symmetry breaking of materials provides large opportunities to regulate their properties and functions. Herein, we report breaking the symmetry of layered dipeptide crystals by utilizing CO₂ to induce the adjacent monomolecular layers to stack from the opposite to the same direction. The role of CO₂ is to cover the interlayer interaction sites and force the dipeptides to adsorb at asymmetric positions. Further, the dipeptide crystals exhibit far superior piezoelectricity after symmetry breaking and the piezoelectric voltage generated from the dipeptide-based generators becomes more than 500 % higher than before. This work reveals a potential route to engineer structures and properties of layered materials and provides a deep insight into the control of non-covalent interactions.