bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunogold localization of plant surface arabinogalactan‐proteins using glycerol liquid substitution and scanning electron microscopy
Authors: ŠAMAJ  , ENSIKAT  , BALUŠKA  , KNOX  , BARTHLOTT  , VOLKMANN
Published: March 2003

We have studied the spatial distributions of arabinogalactan‐protein (AGP) epitopes on the surface of maize embryogenic calli and roots using monoclonal antibodies JIM4 and MAC207. For this purpose, a new immunogold‐scanning electron microscopy (SEM) method was employed which is based on liquid substitution of samples with glycerol. Using this method, we were able to show that the AGP epitopes are distributed along callus and root surfaces and they decorate filamentous structures. In callus cells, the JIM4 epitope was specifically enriched in the outer extracellular layers covering compact clusters of embryogenic meristematic callus cells. In roots, the MAC207 epitope was abundant on the root epidermal surface corresponding to the outer root pellicle, but was only occasionally found on the mucilage layer covering the root cap cells. Silver‐enhanced gold particles, indicating AGP epitopes, were often linearly arranged suggesting that AGPs associate with filamentous structures both on the surface of embryogenic calli and root epidermal cells. These results indicate that AGPs are components of the outer extracellular layers and networks that cover the surface of roots and cells undergoing somatic embryogenesis.
Electronic structure analysis of (In, Ga, Al) N heterostructures on the nanometre scale using EELS
Authors: LAKNER H. , RAFFERTY B. , BROCKT G.
Published: March 2003

In this work the local electronic structure of MOVPE‐grown (In, Ga, Al) N heterostructures has been investigated by electron energy loss spectroscopy (EELS). The cold field‐emission scanning transmission electron microscope (VG HB501) used was equipped with a dedicated parallel EELS system which provides high dispersions at an energy resolution of 0.35 eV with the use of subnanometre electron probes the spatial resolution of the measurements depends on the physical localization of the scattering process itself and thus is in the order of nanometres.

The low‐loss region of the energy spectra gives information on plasmon excitations and transitions across the bandgap. The main problem on looking at the bandgap region of EELS spectra is to separate the bandgap signal from the fading tail of the zero‐loss peak. High energy resolution and application of suitable deconvolution routines for removal of the zero‐loss peak extract improved information from this energy region.

Thus the EEL spectra of different group III nitrides reveal the onset of the bandgap itself and the characteristic shape of the joint density of states. From these results the local optical properties can be deduced via a Kramers–Kronig transformation.

The data obtained show detailed structure on the energy scale and are in excellent agreement with optical ellipsometric results. In comparison with these techniques EELS methods yield a superior spatial resolution of better than 10 nm which enables detailed investigation of the effect of local defects and boundaries on the optical properties.
Resolution beyond the Rayleigh limit using beam displacement
Authors: Somekh  , Liu  , See
Published: March 2003

Two fluorescence microscope systems which claim to achieve resolution beyond the Rayleigh limit have recently been described. These systems operate using two displaced beams focused on the sample and produce the image from the region of overlap. We describe an analogous system, which performs in a similar manner but does not rely on fluorescence. The imaging performance of these systems is analysed and we show that they all give improved resolution although, crucially, the optical bandwidth is not increased. These systems merely attenuate the lower spatial frequencies and, although such systems can be useful and operate in a manner analogous to pupil plane filters, it is important to appreciate that they do not offer true superresolution, contrary to the impression given in previous papers.
Nano‐slit probes for near‐field optical microscopy fabricated by focused ion beams
Authors: Danzebrink H. U. , Dziomba TH. , Sulzbach T. , Ohlsson O. , Lehrer C. , Frey L.
Published: March 2003

The near‐field probes described in this paper are based on metallized non‐contact atomic force microscope cantilevers made of silicon. For application in high‐resolution near‐field optical/infrared microscopy, we use aperture probes with the aperture being fabricated by focused ion beams. This technique allows us to create apertures of sub‐wavelength dimensions with different geometries. In this paper we present the use of slit‐shaped apertures which show a polarization‐dependent transmission efficiency and a lateral resolution of < 100 nm at a wavelength of 1064 nm. As a test sample to characterize the near‐field probes we investigated gold/palladium structures, deposited on an ultrathin chromium sublayer on a silicon wafer, in constant‐height mode.
Ultrafast nonlinear sub‐wavelength solid immersion spectroscopy at T = 8 K: an alternative to nonlinear scanning near‐field optical microscopy
Authors: Vollmer M. , Giessen H. , Stolz W. , Rühle W. W. , Knorr A. , Koch S. W. , Ghislain L. , Elings V.
Published: March 2003

Pump–probe measurements were performed with a sub‐wavelength spatial resolution of 355 nm and a temporal resolution of 130 fs in a multiple quantum well sample at T = 8 K. A solid immersion lens was used to increase the spatial resolution to 0.41 λ, demonstrating that the limit of conventional microscopy was surpassed.
Fluorescence imaging with a laser trapping scanning near‐field optical microscope
Authors: Sugiura T. , Kawata S. , Okada T.
Published: March 2003

We investigated fluorescence imaging using a near‐field scanning optical microscope which uses a laser‐stabilized gold nanoparticle as a near‐field probe. This microscope is suitable for observations of biological specimens in aqueous solutions because the probe particle is held by a noncontact force exerted by a laser beam. Theoretical calculations based on Mie scattering theory are presented to evaluate the near‐field enhancement by a gold particle of 40 nm diameter. We also present fluorescence images of a single fluorescent bead and discuss the near‐field contribution to the fluorescence image in this type of microscope.
Imaging of optical disc using reflection‐mode scattering‐type scanning near‐field optical microscopy
Authors: Yamaguchi M. , Sasaki Y. , Sasaki H. , Konada T. , Horikawa Y. , Ebina A. , Umezawa T. , Horiguchi T.
Published: March 2003

A phase‐change optical disc was observed using a reflection‐mode scattering‐type scanning near‐field optical microscope (RS‐SNOM). In an a.c.‐mode SNOM image, the 1.2 μm × 0.6 μm recording marks were successfully observed although the data were recorded on the groove. In contrast, no recording marks could be resolved in a d.c.‐mode SNOM image. These results are in good agreement with those from a numerical simulation using the finite difference time domain method. The resolution was better than 100 nm with a.c.‐mode SNOM operation and the results indicate that recording marks in phase‐change optical media can be directly observed with the RS‐SNOM.
Towards better scanning near‐field optical microscopy probes — progress and new developments
Authors: Heinzelmann H. , Freyland J. M. , Eckert R. , Huser TH. , Schürmann G. , Noell W. , Staufer U. , De Rooij N. F.
Published: March 2003

Several approaches are described with the aim of producing near‐field optical probes with improved properties. Focused ion beam milling allows the fabrication of small apertures in a controlled fashion, resulting in probes with excellent polarization properties and increased transmission. Microfabrication processes are described that allow the production of apertures of 30–50 nm, facilitating the mass‐fabrication of apertured tip structures that can be used in a combined force/near‐field optical microscope. Finally, possible future developments are outlined.
Fluorescence photobleaching‐based image standardization for fluorescence microscopy
Authors: Ghauharali  , Brakenhoff
Published: March 2003

A method is presented for the standardization of images acquired with fluorescence microscopy, based on the knowledge of spatial distributions proportional to the microscope's absolute excitation intensity and fluorescence detection efficiency distributions over the image field. These distributions are determined using a thin fluorescent test layer, employed under practically mono‐exponential photobleaching conditions. It is demonstrated that these distributions can be used for (i) the quantitative evaluation of differences between both the excitation intensity and the fluorescence detection efficiency of different fluorescence microscopes and (ii) the standardization of images acquired with different microscopes, permitting the deduction of quantitative relationships between images obtained under different imaging conditions.
Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules
Authors: Greulich K. O. , Pilarczyk G. , Hoffmann A. , Meyer Zu Hörste G. , Schäfer B. , Uhl V. , Monajembashi S.
Published: March 2003

Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.
Thin sections for hard tissue histology: a new procedure
Authors: Caropreso S. , Bondioli L. , Capannolo D. , Cerroni L. , Macchiarelli R. , Condò S. G.
Published: March 2003

We describe a simple method by which thin sections (∼100 µm) from modern and archaeological teeth and bones can be obtained. A detailed embedding–cutting–mounting procedure is proposed, suggesting the use of a dental adhesive system, composite resins and conventional embedding resins, with the aims of improving the quality of the sections and substantially reducing the steps and time needed to prepare specimens for histological analysis. The introduction of this dental materials‐based system allows an accurate positioning of the sample embedded inside the resin, prevents cracks and distortions of the section during the cutting phase and generally improves mounting sections on slides.
Digital imaging in transmission electron microscopy
Authors: Fan G. Y. , Ellisman M. H.
Published: March 2003

The digital revolution currently under way, as evidenced by the rapid development of the Internet and the world‐wide‐web technologies, is undoubtedly impacting the field of transmission electron microscopy (TEM). Digital imaging systems based on charge‐coupled device (CCD) technologies, with pixel array size up to 2 k × 2 k at the present and increasing, are available for TEM applications and offer many attractions. Is it time to phase out film cameras on TEMs and close the darkrooms for good? This paper reviews digital imaging technologies for TEM at different voltages, and contrasts the performance of digital imaging systems with that of TEM film. The performance characteristics of CCD‐based digital imaging systems, as well as methods for assessing them, are discussed. Other approaches to digital imaging are also briefly reviewed.
Adaptive aberration correction in a two‐photon microscope
Authors: Neil M. A. A. , Juškaitis R. , Booth M. J. , Wilson T. , Tanaka T. , Kawata S.
Published: March 2003

We demonstrate aberration correction in two‐photon microscopy. Specimen‐induced aberrations were measured with a modal wavefront sensor, implemented using a ferro‐electric liquid crystal spatial light modulator (FLCSLM). Wavefront correction was performed using the same FLCSLM. Axial scanned (xz) images of fluorescently labelled polystyrene beads using an oil immersion lens show restored sectioning ability at a depth of 28 µm in an aqueous specimen.
The correlation between polarization modulated near‐field optical images and the anisotropy of the probe
Authors: Wei P. K. , Fann W. S.
Published: March 2003

The true anisotropic images taken with the polarization modulation near‐field optical microscope are often influenced by the linear dichroism of the tapered fibre probe. In this paper, we develop a new method to separate the anisotropic image form probe's dichroism. Our calculations show that the near‐field optical image is simply a vector sum of the sample's dichroism and the probe's dichroism, when the probe's anisotropy is small. With this result, we demonstrate the true anisotropic images of poly(phenylene vinylene) (PPV) thin films. The PPV films show non‐uniform mesoscale dichroic domains with average domain size, ∼ 0.3 mm, and the coefficient for linear dichroism is 1.25 × 104 cm−1.
Three‐dimensional analysis of light propagation through uncoated near‐field fibre probes
Authors: Müller R. , Lienau C.
Published: March 2003

The near‐field emission from uncoated tapered fibre probes is investigated for different probe geometries. The three‐dimensional model calculations are based on Maxwell's curl equations and describe the propagation of a 10 fs optical pulse (λ = 805 nm) through tapers of different lengths and different diameters of the taper exit. The numerical evaluation is done with a finite difference time domain code. Two tapers with cone angles of 50°, with taper lengths of 1.5 µm and 1.0 µm and exit diameters of 100 nm and 520 nm, respectively, are considered. We find that without sample the short taper with large exit diameter optimizes both light transmission and spatial resolution. In the presence of a sample with a high dielectric constant, however, the spatial near‐field distribution changes drastically for both taper geometries. We find a pronounced increase in spatial resolution, down to about 250 nm inside the medium. This collimation of the near‐field distribution arises from interferences between emitted and reflected light from the sample surface and from a collimation effect that the field experiences in the high‐index semiconductor material. The combination of high spatial resolution and transmission and collection efficiencies makes such probes interesting for spectroscopic investigations, as demonstrated by recent experiments.
SNOM/STM using a tetrahedral tip and a sensitive current‐to‐voltage converter
Authors: Heimel J. , Fischer U. C. , Fuchs H.
Published: March 2003

Scanning near‐field optical microscopes (SNOM) using the tetrahedral‐tip (T‐tip) with scanning tunnelling microscopy (STM) distance control have been realized in transmission and reflection mode. Both set‐ups used ordinary STM current‐to‐voltage converters allowing measurement of metallic samples. In the transmission mode, a resolution of 10 nm to 1 nm with regard to material contrast can be achieved on binary metal samples. Because of the great near‐field optical potential of the T‐tip with respect to the optical resolution, it is a challenging task to find out whether these results can be transferred to non‐metallic sample systems as well. This paper reports on a newly designed SNOM/STM transmission mode set‐up using the tetrahedral‐tip. It implements a sensitive current‐to‐voltage converter to widen the field of measurable sample systems. Beyond this, mechanical and optical measuring conditions are substantially improved compared to previous set‐ups. The new set‐up provides a basis for the routine investigation of metal nanostructures and adsorbed organic monolayers at resolutions in the 10 nm range.
Mapping and manipulating whispering gallery modes of a microsphere resonator with a near‐field probe
Authors: Götzinger S. , Demmerer S. , Benson O. , Sandoghdar V.
Published: March 2003

We report high spatial resolution mapping of high‐Q whispering gallery modes in microsphere resonators with a near‐field probe. We present experimental results on the effect of Q‐factor degradation when the probe interacts with the evanescent field and discuss future applications of our experimental set‐up for realization of novel nanolasers and nano light‐emitting‐diodes.
Local field enhancement effects for nanostructuring of surfaces
Authors: Münzer H.‐J. , Mosbacher M. , Bertsch M. , Zimmermann J. , Leiderer P. , Boneberg J.
Published: March 2003

We report on a method that allows the nanostructuring of surfaces with intense laser pulses. For this purpose isolated polystyrene spheres with diameters in the order of the laser wavelength were deposited on a silicon or glass surface. Illumination with short and ultrashort laser pulses produced holes underneath these particles. Calculations of the field near the particles make clear that geometrical optics, that is, focusing by a spherical lens, as well as near‐field effects, contribute to the size and shape of these holes. This technique can be utilized for the parallel structuring of large surface areas with a single laser shot.
Micromechanical and structural properties of a pennate diatom investigated by atomic force microscopy
Authors: Almqvist N. , Delamo Y. , Smith B. L. , Thomson N. H. , Bartholdson Å. , Lal R. , Brzezinski M. , Hansma P. K.
Published: March 2003

The mechanisms behind natural nanofabrication of highly structured silicas are increasingly being investigated. We have explored the use of a standard Nanoscope III Multimode atomic force microscope (AFM) to study the silica shell of diatoms. The delicate structures of the shell surface of the diatom Navicula pelliculosa (Bréb.) Hilse were imaged and the shell's micromechanical properties were measured semi‐quantitatively with a resolution down to approximately 10 nm. The technique to measure elasticity and hardness with the AFM was demonstrated to be useable even on these hard glass‐like surfaces. Different experimental configurations and evaluation methods were tested. They gave a consistent result of the shell micromechanical properties. The first results showed that the diatom shell's overall hardness and elasticity was similar to that of known silicas. However, regions with different mechanical properties were distinguished. The elastic modulus varied from 7 to 20 GPa, from 20 to 100 GPa and from 30 to hundreds of GPa depending on the location. In general, the hardness measurements showed similar spatial differences. The hardness values ranged from 1 to 12 GPa but one specific part of the shell was even harder. Hence, certain localized regions of the shell were significantly harder or more elastic. These regions coincide with known characteristic features and mechanisms appearing at the different stages of the shell's growth. These results show that this method serves as a complementary tool in the study of silica biomineralization, and can detect eventual crystalline phases.
Improved precision of quantitating calcium in biological electron probe analysis
Authors: Ho R. , Jia Z. , Somlyo A. P. , Shao Z.
Published: March 2003

We describe the use of direct multiple least‐squares fitting of (unfiltered) biological X‐ray spectra. Test of this method on computer‐generated spectra and experimental specimens shows that, for the average biological specimen, direct fitting improves the precision of Ca measurements by an approximate factor of 1.6 for a given electron dose. The influence of non‐statistical, instrumental sources of measurement errors is considered, and we show a simple method for reducing the effect of these sources of error in the directly fitted spectra.
A simple method for SEM examination of sectioned diatom frustules
Authors: Massé G. , Poulin M. , Belt S. T. , Robert J. ‐M. , Barreau A. , Rincé Y. , Rowland S. J.
Published: March 2003

We describe an innovative yet straightforward method to obtain high quality thin sections of diatom exoskeletons for observation by scanning electron microscopy (SEM). The use of this new technique allows for clear observations of some ultrastructural valve features, including the raphe, which are generally difficult to observe and describe accurately using transmission electron microscopy analysis of thin sections or SEM of randomly fractured diatom valves. In addition, because this method involves the complete removal of the organic content of the diatom cells, resulting in clean and mostly undisturbed skeletal thin cross‐sections, even the intact valvar structures of weak girdle bands can be studied.
Comparison of the axial resolution of practical Nipkow‐disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment
Authors: Egner A. , Andresen V. , Hell S. W.
Published: March 2003

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross‐talk between adjacent imaging channels. We demonstrate that a time‐multiplexed non‐linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single‐photon confocal system. The background becomes irrelevant for thin (< 15 µm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.
Chemical reactions and morphological stability at the Cu/Al2O3 interface
Authors: Scheu C. , Klein S. , Tomsia A. P. , Rühle M.
Published: March 2003

The microstructures of diffusion‐bonded Cu/(0001)Al2O3 bicrystals annealed at 1000 °C at oxygen partial pressures of 0.02 or 32 Pa have been studied with various microscopy techniques ranging from optical microscopy to high‐resolution transmission electron microscopy. The studies revealed that for both oxygen partial pressures a 20–35 nm thick interfacial CuAlO2 layer formed, which crystallises in the rhombohedral structure. However, the CuAlO2 layer is not continuous, but interrupted by many pores. In the samples annealed in the higher oxygen partial pressure an additional reaction phase with a needle‐like structure was observed. The needles are several millimetres long, ∼10 µm wide and ∼1 µm thick. They consist of CuAlO2 with alternating rhombohedral and hexagonal structures. Solid‐state contact angle measurements were performed to derive values for the work of adhesion. The results show that the adhesion is twice as good for the annealed specimen compared to the as‐bonded sample.
In vivo three‐dimensional imaging of plants with optical coherence microscopy
Authors: Reeves A. , Parsons R. L. , Hettinger J. W. , Medford J. I.
Published: March 2003

Achieving the ability to non‐destructively, non‐invasively examine subsurface features of living multicellular organisms at a microscopic level is currently a challenge for biologists. Optical coherence microscopy (OCM) is a new photonics‐based technology that can be used to address this challenge. OCM takes advantage of refractive properties of biological molecules to generate three‐dimensional images that can be viewed with a computer. We describe new data processing techniques and a different visualization algorithm that substantially improve OCM images. We have applied OCM imaging, in conjunction with these improvements, to a variety of structures of plants, including leaves, flowers, ovules and germinating seeds, and describe the visualization of cellular and subcellular structures within intact plants. We present evidence, based on detailed examination of our OCM images, comparisons to classical plant anatomy studies, and current knowledge of light scattering by cells and their components, that we can distinguish nuclei, organelles and vacuoles. Detailed examination of vascular tissue, which contains cells with elaborate wall structure, shows that cell walls produce no significant OCM signal. These improvements to the visualization process, together with the powerful non‐invasive, non‐destructive aspects of the technology, will broaden the application of OCM to questions in studies of plants as well as animals.
A prism combination for near isotropic fluorescence excitation by total internal reflection
Authors: Wakelin S. , Bagshaw C. R.
Published: March 2003

Total internal reflection fluorescence (TIRF) microscopy is finding increasing application for selectively detecting molecules at or near a glass–water surface. As with all fluorescence methods, the efficiency of excitation of a fluorophore is potentially sensitive to the polarization state of the source. In TIRF, s‐polarized excitation produces an evanescent field that is perpendicular to the incident plane (ydirection), whereas p‐polarized light generates a more complex pattern but one dominated by a field that is vertical to the surface (zdirection). Thus, fluorophores whose absorption dipoles are fixed in thexdirection are not favourably aligned for excitation. Here we describe a beam‐splitting prism arrangement that allows excitation by two orthogonal beams, thus giving isotropic excitation in thex–yplane with s‐polarized light. With linearly polarized light at the magic angle, near isotropic excitation in three dimensions should be achieved. This prism design should find application in polarized fluorescence microscopy to investigate the rotational motions of macromolecules or to minimize flickering of fluorescence emission arising from molecular rotations in single molecule studies.
Ethanol Reduces the Duration of Single Evoked Spikes by a Selective Inhibition of Voltage‐Gated Calcium Currents in Acutely Dissociated Supraoptic Neurons of the Rat
Authors: Widmer  , Lemos  , Treistman
Published: March 2003

The effects of ethanol were studied on single evoked spikes recorded at 20 °C with the perforated‐patch method in acutely dissociated rat supraoptic neurons. In seven out of eight neurons, ethanol (50 mM) significantly reduced the spike duration by selectively decreasing the decay time (82±2% of the control), leaving the amplitude and rise time unaffected. Resting potential and threshold did not change. Similarly, CdCl2 at a concentration of 100 μM, which blocks all voltage‐activated calcium current in the supraoptic neurons, reduced the decay time of single evoked spikes (76±3% of the control, n=10) without modifying the other above‐mentioned parameters. In addition, exposure to 100 μM CdCl2 prevented any subsequent effect of 50 mM ethanol (n=5). Exposure to apamin (10 nM) and iberiotoxin (10 nM) did not have any effect on single evoked spikes. Because these concentrations are effective in blocking, respectively, small (SK) and large (BK) conductance calcium‐dependent potassium channels in these neurons, this result shows that these currents are not involved in either the shaping of single evoked spikes or the actions of ethanol on spike shape. The sustained component of whole‐cell recorded calcium current measured at −10 mV (hp −60 mV) was inhibited by ethanol in a dose‐dependent manner, with a significant effect detectable at 25 mM. Exposure to 50 mM ethanol significantly reduced the sustained current to 70±5% of the control (n=12), without any apparent shift of the current‐voltage relationship. Control exposure of the neurons to either 50 mM urea or 50 mM sucrose did not affect the voltage‐gated calcium currents. We conclude that ethanol reduces the duration of single evoked spikes by a specific inhibition of voltage‐activated calcium currents. The results suggest that, in addition to its direct effects on release of vasopressin and oxytocin from neurohypophysial terminals, ethanol could also affect hormonal release via changes in firing patterns arising in the cell bodies.
Anorectic Effect of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) in Rats: Lack of Evidence for Involvement of Hypothalamic Neuropeptide Gene Expression
Authors: Mizuno Yumiko , Kondo Kunikazu , Terashima Yasuhiro , Arima Hiroshi , Murase Takashi , Oiso Yutaka
Published: March 2003

We investigated the effect of centrally administered pituitary adenylate cyclase activating polypeptide (PACAP) on feeding in rats, and the involvement of hypothalamic neuropeptide gene expression using in situ hybridization. lntracerebroventricular injection of PACAP (1000 pmol/rat) significantly decreased food intake in a dose‐dependent manner. In PACAP‐treated rats, neuropeptide Y (NPY) mRNA levels in the arcuate nucleus and galanin mRNA levels in the paraventricular nucleus increased, and corticotropin‐releasing hormone (CRH) mRNA levels in the paraventricular nucleus decreased. In rats fasted for 72 h, NPY mRNA levels increased, and CRH mRNA levels decreased, but galanin mRNA levels were unchanged. These results indicate that the anorectic function of PACAP is not mediated by NPY or CRH, and that PACAP increases galanin synthesis.
Stationary Organotypic Cultures of Oxytocin and Vasopressin Magnocellular Neurones from Rat and Mouse Hypothalamus
Authors: House  , Thomas  , Kusano  , Gainer
Published: March 2003

Rat and mouse hypothalami from postnatal animals containing highly differentiated neurones survive very well in long‐term (>15 days in vitro, DIV) stationary organotypic cultures. Magnocellular oxytocin (OT) and vasopressin (VP) neurones are present in identifiable paraventricular (PVN), supraoptic (SON) and accessory (ACC) nuclei in these cultures. After 15 DIV in standard medium immunocytochemistry revealed 427±63 OT cells and 217±27 VP cells per cultured rat hypothalamus, and 380±72 OT cells and 622±91 VP cells per cultured mouse hypothalamus. Following a 7‐day adaptation period in standard culture medium containing serum, the rat slice‐explants survived very well after subsequent transfer to defined, serum‐ free media (SFM) for an additional 8 days. The number of OT cells surviving in SFM was 612±147 OT cells per cultured rat hypothalamus. Only 0.5% of the magnocellular OT and VP neurones in the cultures appeared to express both peptides. Experiments on c‐fosgene expression in these cultures showed that while only 12% of the magnocellular OT and VP neurones contained barely detectable Fos protein in their nuclei under control conditions, potassium depolarization of these cultures for 3 h produced intense c‐fos expression in 87–91% of these cells. Thus, magnocellular neurones in these cultures are sufficiently stable and responsive to permit long‐term physiological and gene expression studies to be done under defined media conditions.
Role of Non‐NMDA Receptors in Osmotic and Glutamate Stimulation of Vasopressin Release: Effect of Rapid Receptor Desensitization
Authors: Sladek  , Badre  , Morsette  , Sidorowicz
Published: March 2003

Previous studies demonstrated that the increase in vasopressin (VP) release and induction of VPmRNA content by osmotic stimulation was blocked by kynurenic acid, a non‐specific antagonist of excitatory amino acid (EAA) receptors. In order to identify the type of EAA receptor involved, perifused explants of the hypothalamo‐neurohypophyseal system (HNS) were exposed to a ramp increase in osmolality (40 mOsm over 6 h achieved by increasing NaCl) in the presence and absence of 10 μm 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX), an antagonist of non‐n‐methyl‐d‐aspartate (NMDA) excitatory amino acid receptors. Vasopressin release and VP mRNA content were significantly increased by exposure to the osmotic stimulus. 6,7‐dinitroquinoxaline‐2,3‐dione inhibited osmotically stimulated VP release (F=16.65, P=0.0008) without significantly reducing basal release. It also prevented the osmotically stimulated increase in VP mRNA content (P<0.05). Although these results implicated glutamate, the primary endogenous ligand for EAA receptors, in the regulation of VP, exogenous glutamate was ineffective in stimulating VP release from HNS explants in either low‐Mg2+ or Mg2+‐replete medium. However, blockade of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptor desensitization with cyclothiazide (100 μm) caused a marked increase in VP release in response to 100 μm glutamate, and blockade of kainate receptor desensitization with concanavalin A resulted in a small, but significant increase in VP release in response to 1 m
m glutamate. These results support a role for non‐NMDA receptor activation in osmotic regulation of VP release.
Glutamatergic Induction of CREB Phosphorylation and Fos Expression in Primary Cultures of the Suprachiasmatic Hypothalamus In Vitro is Mediated by Co‐Ordinate Activity of NMDA and Non‐NMDA Receptors
Authors: Schurov  , McNulty*  , Best  , Sloper  , Hastings
Published: March 2003

Exposure of Syrian hamsters to light 1 h after lights‐off rapidly (10 min) induced nuclear immunoreactivity (–ir) to the phospho‐Ser133 form of the Ca2+/cAMP response element (CRE) binding protein (pCREB) in the retinorecipient zone of the suprachiasmatic nuclei (SCN). Light also induced nuclear Fos‐ir in the same region of the SCN after 1 h. The glutamatergic N‐methyl‐
d‐aspartate (NMDA) receptor blocker MK801 attenuated the photic induction of both factors. To investigate glutamatergic regulation of pCREB and Fos further, tissue blocks and primary cultures of neonatal hamster SCN were examined by Western blotting and immunocytochemistry in vitro. On Western blots of SCN tissue, the pCREB‐ir signal at 45 kDa was enhanced by glutamate or a mixture of glutamatergic agonists (NMDA, amino‐methyl proprionic acid (AMPA), and Kainate (KA)), whereas total CREB did not change. Glutamate or the mixture of agonists also induced a 56 kDa band identified as Fos protein in SCN tissue. In dissociated cultures of SCN, glutamate caused a rapid (15 min) induction of nuclear pCREB‐ir and Fos‐ir (after 60 min) exclusively in neurones, both GABA‐ir and others. Treatment with NMDA alone had no effect on pCREB‐ir. AMPA alone caused a slight increase in pCREB‐ir. However, kainate alone or in combination with NMDA and AMPA induced nuclear pCREB‐ir equal to that induced by glutamate. The effects of glutamate on pCREB‐ir and Fos‐ir were blocked by antagonists of both NMDA (MK801) and AMPA/KA (NBQX) receptors. In the absence of extracellular Mg2+, MK801 blocked glutamatergic induction of Fos‐ir. However, the AMPA/KA receptor antagonist was no longer effective at blocking glutamatergic induction of either Fos‐ir or pCREB‐ir, consistent with the model that glutamate regulates gene expression in the SCN by a co‐ordinate action through both NMDA and AMPA/KA receptors. Glutamatergic induction of nuclear pCREB‐ir in GABA‐ir neurones was blocked by KN‐62 an inhibitor of Ca2+/Calmodulin (CaM)‐dependent kinases, implicating Ca2+‐dependent signalling pathways in the glutamatergic regulation of gene expression in the SCN.
Characterization of 17β‐Hydroxysteroid Dehydrogenase Activity in Brain Tissue: Testosterone Formation in the Human Temporal Lobe
Authors: Steckelbroeck  , Stoffel‐Wagner  , Reichelt  , Schramm  , Bidlingmaier  , Siekmann  , Klingmüller
Published: March 2003

Sex steroids exert important effects on the central nervous system (CNS). Although the formation of 17β‐hydroxysteroid dehydrogenase (17β‐HSD) metabolites in the CNS was discovered almost 30 years ago, conclusive studies concerning 17β‐HSD activity in the human brain are still lacking. Therefore, we investigated 17β‐HSD in vitro activity in human temporal lobe biopsies of 13 women and 13 men using radioactively labelled androstenedione, testosterone, oestrone and 17β‐oestradiol and compared it to that in human placenta, liver, testis and prostate. We could demonstrate androgenic and oestrogenic 17β‐HSD activities in all tissues under investigation. The reduction of androstenedione and oestrone in brain was NADPH dependent with a broad pH optimum between 6.5 and 9.0, whereas the oxidation of testosterone and 17β‐oestradiol was NAD dependent with a pH optimum of ≥9.0. Using optimum cofactors sex differences of brain 17β‐HSD activities were not observed. Conversion of androstenedione, testosterone, oestrone and 17β‐oestradiol was significantly higher in the subcortical white matter than in the cerebral cortex. We could demonstrate a significant formation of testosterone in the brain tissue of all patients under investigation. Substrate specificity and cofactor requirement patterns as well as pH optima and kinetic properties suggest the occurrence of 17β‐HSD type 3 and type 4 in the human temporal lobe.
Stress‐Induced Suppression of Pulsatile Luteinising Hormone Release in the Female Rat: Role of Vasopressin
Authors: Cates  , Forsling  , O’byrne
Published: March 2003

Insulin‐induced hypoglycaemic (IIH) stress evokes the release of arginine vasopressin (AVP) and suppresses luteinising hormone (LH) pulses in a number of species, a phenomenon augmented by the presence of oestradiol (E2). The aim of this study was to test the hypothesis that AVP not only disrupts pulsatile LH secretion in the female rat, but specifically mediates the effect of IIH stress on suppressing LH release. The role of E2 in augmenting the disruptive effect of AVP on LH secretion was also addressed. Rats were ovariectomized (OVX) and fitted with intracerebroventricular (i.c.v.) and intravenous (i.v.) cannulae. For experiments requiring comparisons of neuroendocrine responses in the presence and absence of E2, animals were implanted subcutaneously with E2 or oil‐filled capsules respectively. AVP (5 μg) administered via the i.c.v. cannula suppressed LH secretion by decreasing LH pulse amplitude without affecting LH pulse frequency, an effect that was blocked by central administration of an AVP antagonist (25 μg). This inhibitory response was evident only in E2‐replaced OVX rats, thus suggesting a sensitizing influence of the gonadal steroid. In the AVP‐deficient Brattleboro rats, IIH stress did not interrupt pulsatile LH secretion as demonstrated in Long Evans and Wistar controls. While these data might suggest a pivotal role for AVP in stress‐induced suppression of LH release, central administration of an AVP antagonist did not prevent the interruption of LH pulses in response to IIH stress. Furthermore, it would appear that AVP is not primarily involved in hypoglycaemic stress‐induced suppression of pulsatile LH secretion since central administration of very high doses of AVP resulted in a suppression of LH pulse amplitude and not frequency, while hypoglycaemic stress caused an interruption of LH pulses.
Melanin‐Concentrating Hormone, Melanocortin Receptors and Regulation of Luteinizing Hormone Release
Authors: Murray  , Adan  , Walker  , Baker  , Thody  , Nijenhuis  , Yukitake  , Wilson
Published: March 2003

Melanin‐concentrating hormone (MCH) is a neuropeptide, identified by its ability to either mimic or antagonize the melanin‐dispersing action ofα‐melanocyte stimulating hormone (αMSH) on skin melanophores. MCH andαMSH also have antagonistic actions in the brain affecting feeding behaviour, aggression, anxiety, arousal and reproductive function through the release of luteinizing hormone (LH). It is not clear, however, how they exert their opposite effects in the central nervous system (CNS). One possibility is that they act via a common receptor. In this study we have examined the effect of a number of MC receptor antagonists, with relative selectivity for the MC3, 4 and 5 subtypes, on the actions of MCH on LH release. We confirmed that bilateral administration of MCH (100 and 200 ng/side) into the medial preoptic area of oestrogen‐primed (oestradiol benzoate 5 μg) ovariectomized anaesthetized rats, stimulated the release of LH. This effect was blocked by the concomitant administration into the medial preoptic area of the MC4/5 antagonist ([d‐Arg8]ACTH(4–10) and the MC3/5 antagonist ([Ala6]ACTH(4–10)—both at 500 ng/side—but not by the MC3/4 antagonist, SHU9119 (200 ng/side). Furthermore, the MC3 agonist [Nle3]‐γ2 MSH failed to affect LH release. These results indicate that the MC3 and MC4 receptors are not involved in mediating the action of MCH but are consistent with an action via the MC5 subtype. Preputial glands, which express MC5 receptors, were also stimulated by MCH which is in keeping with this idea. In HEK293 cells transfected with the MC5 receptor MCH increased the production of IP3. However, it was much less potent thanαMSH and unlikeαMSH, had no effect on the production of cAMP. MCH (10−10to 10−5 M) also failed to displace I125NDP‐MSH from cells transfected with MC5 receptors indicating that it was not acting as a competitive antagonist and its binding site was distinct from that ofαMSH. Thus while MCH may function as an agonist at the MC5 receptor, its stimulation of LH release is more likely to be mediated via a specific MCH receptor that has common properties with the MC5 receptor.
Hypervascularization in the Magnocellular Nuclei of the Rat Hypothalamus: Relationship with the Distribution of Aquaporin‐4 and Markers of Energy Metabolism
Authors: Badaut J. , Nehlig A. , Verbavatz J.‐M. , Stoeckel M.‐E. , Freund‐Mercier M.‐J. , Lasbennes F.
Published: March 2003

In the magnocellular nuclei of the hypothalamus, there is a rich vascular network for which the function remains to be established. In the supraoptic nucleus, the high vascular density may be one element, which together with the water channel aquaporin‐4 expressed in the astrocytes, is related to a role in osmoreception. We tested the osmoreception hypothesis by studying the correlation between vascular and cellular densities in the paraventricular nucleus and the supraoptic nucleus. Whether aquaporin‐4 is likely to contribute to osmoreception was tested by studying the distribution in the magnocellular nuclei of the hypothalamus. The high vascular density may also reflect a high metabolic activity due to the synthesis of vasopressin and oxytocin. This metabolic hypothesis was tested by studying the regional cytochrome oxidase histochemistry, the local cerebral blood flow, and the density of glucose transporter type‐1 in the supraoptic and paraventricular nuclei. All the magnocellular nuclei were characterized by an extended and intense aquaporin‐4 labelling and a weak cytochrome oxidase histochemistry. The highest vascular density was found in the supraoptic nucleus and the magnocellular regions of the paraventricular nucleus. The local cerebral blood flow rates were surprisingly low in the paraventricular nucleus and the supraoptic nucleus in comparison to the cerebral cortex. Furthermore in these nuclei, the antibody for glucose transporter type‐1 revealed two populations of vessels differing by their labelling intensity. The similarities observed between the different nuclei suggest that, in the hypothalamus, all magnocellular regions sense the plasma osmolarity. The low local cerebral blood flow, and the patterns of glucose transporter type‐1 labelling and cytochrome oxidase histochemistry suggest that the high vascularization of these hypothalamic nuclei is not related to a high metabolic capacity in basal conditions.
Agouti Related Protein in the Rat Adrenal Cortex: Implications for Novel Autocrine Mechanisms Modulating the Actions of Pro‐Opiomelanocortin Peptides
Authors: Bicknell A. B. , Lomthaisong K. , Gladwell R. T. , Lowry P. J.
Published: March 2003

Agouti related protein (AgRP) is a recently discovered melanocortin receptors (MCR) antagonist implicated in the control of feeding behaviour. Expression of AgRP has been shown to be localized by in situ hybridization to the arcuate nucleus and median eminence of the brain, where it acts as an antagonist to the MC3 and MC4 receptors, while in the periphery the only significant expression was located in the adrenal medulla. As AgRP is only a weak antagonist of the MC2 and MC5 receptors, which are expressed principally by adipocytes and in the adrenal cortex, the question arizes as to the function of peripheral AgRP. In this study, we investigated the expression of AgRP in the rat adrenal and suggest that it is expressed in the adrenal cortex and not as previously described in the medulla. We also show that AgRP mRNA expression is upregulated in the adrenal during fasting and in the contralateral gland following unilateral adrenalectomy but not during chronic stress. These results indicate an as yet undefined role for AgRP in the periphery and are supportive of the suggestion that a further melanocortin receptor exists.
Progestin Regulation of Galanin and Prolactin Gene Expression in Oestrogen‐Induced Pituitary Tumours
Authors: Piroli G. G. , Cassataro J. , Pietranera L. , Grillo C. A. , Ferrini M. , Lux‐Lantos V. , De Nicola A. F.
Published: March 2003

Galanin is a peptide widely distributed in the hypothalamic‐pituitary axis. In the female rat pituitary, galanin is mainly present in lactotrophs, where it regulates their secretion and proliferation. Galanin expression is increased in oestrogen‐induced prolactinomas, and it has been proposed that oestrogen effects on lactotroph function and proliferation could be mediated by galanin. Previous studies from our laboratory demonstrated that the synthetic progestin levonorgestrel antagonizes pituitary tumorigenesis of rats given oestrogen, reducing the number of proliferating cells and increasing cell death by nonapoptotic mechanism(s). To elucidate the role of galanin in levonorgestrel effects on the tumours, we examined galanin and prolactin mRNA and peptide expression in prolactinomas of rats receiving the progestin. Levonorgestrel reduced the pituitary weight and serum prolactin concentrations in oestrogen‐treated rats. Galanin mRNA expression (determined by in situ hybridization), and the number of galanin expressing cells (determined by immunocytochemistry) were also reduced by the progestin in tumour‐bearing rats. However, neither prolactin mRNA content, nor the number of prolactin‐expressing cells, were modified by levonorgestrel treatment of oestrogen‐receiving rats. The present study suggests that levonorgestrel controls pituitary growth by diminishing galanin expression. In contrast, changes in serum prolactin concentration seem to be more related to the reduction in tumour size, since the reduction in galanin expression was not large enough to regulate prolactin mRNA expression or the percentage of lactotrophs.