Specification of neuropeptide Y phenotype in visual cortical neurons by leukemia inhibitory factor.

Building the complex mammalian neocortex requires appropriate numbers of neurochemically specified neurons. It is not clear how the highly diverse cortical interneurons acquire their distinctive phenotypes. The lack of genetic determination implicates environmental factors in this selection and specification process. We analysed, in organotypic visual cortex cultures, the specification of neurons expressing neuropeptide Y (NPY), a potent anticonvulsant. Endogenous brain-derived neurotrophic factor and neurotrophin 4/5 play no role in early NPY phenotype specification. Rather, the decision to express NPY is made during a period of molecular plasticity during which differentiating neurons with the potential to express NPY compete for the cytokine leukemia inhibitory factor which is produced in the cortex, but is negatively regulated by thalamic afferences. The neurons that fail in this competition are parvalbuminergic basket and chandelier neurons, which express NPY transiently, but will not acquire a permanent NPY expression. They switch into a facultative NPY expression mode, and remain responsive to the neurotrophins which modulate NPY expression later in development.

Epigenetic factors regulate the NPY expression in rat cortical neurons.

The NPY phenotype expressed in a subset of rat neocortical neurons is influenced by a variety of epigenetic factors. In the present study, we analyzed the role of synaptically driven spontaneous bioelectric (action potential) activity (SBA) and neurotrophic factors. Our model systems are organotypic monocultures of visual cortex which either grow as spontaneously active cultures or as activity-blocked cultures to which neurotrophic factors can be applied via the medium. NPY mRNA expressing neurons are detected by in situ hybridization and are quantified as a percentage of all neurons. In spontaneously active cultures, about 7% of all neurons express NPY mRNA. This expression is regulated by SBA, because expression is reduced to about 2% by different activity blockade paradigms. When putative NPY neurons differentiate under activity blockade, they are unable to restitute the NPY expression during a subsequent period of SBA. A restitution of the NPY phenotype in 6-7% of the neurons after a transient blockade of activity is only possible when neurons were initially allowed to differentiate in the presence of SBA. We then analyzed whether neurotrophic factors known to promote NPY expression can do so in the absence of SBA. Neurotrophin-4/5 and leukemia inhibitory factor, but not brain-derived neurotrophic factor and neurotrophin-3, stimulate the NPY phenotype in the absence of SBA. In situ hybridization in combination with immuno-fluorescence reveals that NPY-ir neurons express the receptors trkB or LIFRbeta, but not trkC. This coexpression pattern explains why neurotrophin-4/5 and leukemia inhibitory factor are efficient regulators of the NPY-expression. Our results suggest that the NPY expression in neocortical neurons depends on epigenetic factors: spontaneous activity and neurotrophic factors modulate the expression and are thus involved in shaping the neurochemical architecture of the cerebral cortex.

NT-4/5 and LIF, but not NT-3 and BDNF, promote NPY mRNA expression in cortical neurons in the absence of spontaneous bioelectrical activity.

Epigenetic factors are known to influence the differentiation of neocortical neurons. The present study analyses the role of spontaneous bioelectrical activity (SBA) and neurotrophic factors on the expression of neuropeptide Y (NPY) in rat visual cortical neurons using organotypic monocultures prepared from newborn animals and in situ hybridization to detect the NPY messenger ribonucleic acid (mRNA). Spontaneously active cortex cultures display NPY mRNA expression in about 7% of all cortical neurons from 10 days in vitro (DIV) on. Blocking the SBA by chronic application of 10 mM Mg2+ for 3-30 DIV reduces the percentage of NPY neurons to about 2%. Allowing an initial phase of SBA (1-20 DIV) followed by an SBA blockade (for 21-50 DIV) results in 2% labelled neurons, indicating a dramatic reduction of NPY mRNA expression in the absence of SBA. Surprisingly, the reverse experiment (a period of SBA blockade for 1-20 DIV followed by a period of SBA recovery for 21-40 DIV) does not cause an upregulation of NPY mRNA expression. However, allowing cultures to differentiate as spontaneously active cultures, then applying a transient period of SBA blockade which is followed by a second period of SBA, does rescue the NPY mRNA expression in 7% of the cortical neurons. We conclude that SBA is a main trigger for NPY mRNA expression and it is particularly important during an early postnatal period of differentiation. We then analysed whether neurotrophic factors known to modulate cortical neuropeptide expression are able to do so in the absence of SBA. Supplementing chronically blocked cultures with the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and the cytokine, leukaemia inhibitory factor (LIF), reveals that BDNF and NT-3 are unable to increase the percentage of NPY neurons. In contrast, LIF and NT-4/5 increase the percentage of NPY neurons to 4 and 6-7%, respectively. Moreover, neurons treated with NT-4/5 display a very high level of NPY mRNA expression in somata and in the dendritic trees. The data suggest a complex interplay and a hierarchy of epigenetic factors in regulating the neurochemical architecture of the developing neocortex.

Surface-confined living radical polymerization for coatings in capillary electrophoresis.

Surface-confined living radical polymerization is shown to be a controlled means of covalently bonding both linear and cross-linked polymer films on silica. CuCl/bipyridine initiates radical formation through atom transfer with a self-assembled monolayer of benzyl chloride, onto which polymer then grows. The polymerization is intrinsically confined to the surface, avoiding problems associated with polymer formed in the solution. The surface-confined polymerization scheme is generally applicable to radical polymerization of vinyl monomers and was studied here for the case of acrylamide. Infrared spectroscopy shows that the film growth is controllable, and atomic force microscopy reveals that smooth films are prepared. The surface-confinement polymerization scheme was tested for both linear and cross-linked polyacrylamide. Capillary electrophoresis of strongly basic proteins confirms that the coated capillaries provide the high efficiency expected for polyacrylamide. The cross-linked coating exhibits higher reproducibility with respect to migration time than does the linear coating. Surface-confined living radical polymerization prepares linear and cross-linked polymer films without danger of clogging narrow capillaries and will ultimately facilitate cross-laboratory comparisons by enabling control of film thickness.

Mixed self-assembled monolayers in chemical separations.

Chemical separations of many biomolecules and pharmaceuticals are limited by their electrostatic interaction with the surfaces of the separation medium. Mixed self-assembled monolayers of octadecyl and methyl chains organize into a dense, two-dimensionally cross-linked network over the chromatographic silica surface to reduce acid dissociation of the surface silanols. Molecular models predict that two-dimensional cross-linking is sterically possible for pure methylsiloxane monolayers, silicon-29 nuclear magnetic resonance measurements show that cross-linking predominates for mixed monolayers of primarily methylsiloxane, and chromatographic measurements confirm that electrostatic interactions are reduced when the monolayer is primarily methylsiloxane. Chromatographic separation of genetic variants of a highly charged protein, cytochrome c, demonstrates the promise of self-assembled monolayers in separations of biomolecules.

Electrostatic Interactions between Ru(bpy)(3)(2+) and Chromatographic Surfaces.

In HPLC, the zones of organic bases tail when silica-based stationary phases are used because the analytes and the surface are oppositely charged. In developing new stationary phases to achieve lower surface charge, a measure of surface charge is needed. The choice of a suitable analyte to quantitate electrostatic interactions is complicated by the acid-base equilibrium of the analyte itself and by nonelectrostatic interactions between the analyte and the surface, which alter the charge-induced tailing. This paper describes the study of the pH dependence of adsorption to isolate electrostatic interaction and the use of a cationic probe, tris(2,2'-bipyridine)ruthenium chloride (Ru(bpy)(3)(2+)), to sense surface charge without the complication of the probe's acid-base equilibria. The paper further describes the application of Gouy-Chapman theory to reveal the surface charge density. The results confirm that type A silica is considerably more acidic than type B silica and that horizontal polymerization makes type A silica perform as well as type B silica.

Spectroscopic Probing of Mixed-Mode Adsorption of Ru(bpy)(3)(2+) to Silica.

Evanescent wave excitation of fluorescence was used to study the adsorption of Ru(bpy)(3)(2+) from aqueous solution to three types of surfaces: bare silica, a dimethylethylsiloxane (C(2)) monolayer on silica, and a dimethyloctadecylsiloxane (C(18)) monolayer on silica. The solution pH was varied to investigate the nonpolar and electrostatic contributions to the free energy of adsorption for each surface. The pH dependence of the adsorption showed that the pK(a) is the same for each of the three surfaces, consistent with earlier conclusions that the acidity of derivatized silica surfaces is due to areas of exposed silica. The free energies of adsorption for the bare silica surface, -26.2(±0.2) kJ/mol at pH 8, was attributed to electrostatic interactions alone. The free energy of adsorption for the C(2) and C(18) surfaces, -28.5(±0.1) and -31.5(±0.1) kJ/mol, respectively, were found to have both electrostatic and nonpolar contributions, with the latter being larger by 50% for the C(2) surface and 100% for the C(18) surface. Using Gouy-Chapman theory, the surface charge densities on each of the three surfaces, calculated from the electrostatic interaction energy of Ru(bpy)(3)(2+), were found to be within the range of literature values: 8.8(±0.1) × 10(-)(7) mol/m(2) for bare silica and 1.7(±0.1) × 10(-)(7) mol/m(2) for both the C(18) and C(2) surfaces. The results demonstrate that a cationic dye can be used to probe the silanol activity of chemically modified silica surfaces. The results support the picture that these chemically modified silica surfaces are acidic due to molecular-scale areas of contact between the bare silica substrate and the aqueous phase.

Detector nonlinearity in frequency-domain fluorometry.

Frequency-domain fluorometry relies on the measurement of the phase and amplitudes of the Fourier components of the time-dependent fluorescence signal. Experimental results that show that a conventional photomultiplier is subject to intensity-dependent phase shifts are presented. The measurements indicate that this is a problem well below the maximum linear current of the photomultiplier response. These results have important implications in frequency-domain fluorescence anisotropy experiments, in which the parallel and the perpendicular components of the emission intensity are inherently different from one another: a phase shift can be introduced by the photomultiplier.

Time-Resolved Fluorescence Intensity and Anisotropy Decays of 2,5-Diphenyloxazole by Two-Photon Excitation and Frequency-Domain Fluorometry.

We report the first time-resolved fluorescence measurements of the intensity and anisotropy decays resulting from two-photon excitation. A 10-GHz frequency-domain fluorometer (Rev. Sci. Instrum 1990, 61, 2331), equipped with two focal lenses and an emission monochromator, was used for steady-state and time-resolved measurements of PPO fluorescence. The emission spectra and the intensity decays observed with single- and two-photon excitation were essentially identical. The steady-state limiting anisotropy r 0 of PPO in glycerol at -5 °C measured for two-photon excitation is significantly higher than that observed for one-photon excitation. The r 0 value of 0.54 for two-photon excitation is well in excess of the theoretical maximum of 0.4 for single-photon excitation. A similar value of r 0 ≃ 0.50 was obtained from the frequency-domain anisotropy data with two-photon excitation of PPO in methanol, butanol, and propylene glycol at 20 °C. These higher values of r 0 indicate that two-photon excitation results in a more highly oriented photoselected population, which can increase the resolution of rotational correlation times and/or complex anisotropy decays. The anisotropy resolution can still be increased by using global analysis of anisotropy decays measured with single- and two-photon excitation.