bluuebunny/crossref_metadata_2025 huggingface dataset:
Apertureless near‐field optical microscopy via local second‐harmonic generation
Authors: Zayats A. V. , Sandoghdar V.
Published: March 2003

We describe an apertureless scanning near‐field optical microscope (SNOM) based on the local second‐harmonic generation enhancement resulting from an electromagnetic interaction between a probe tip and a surface. The imaging mechanisms of such apertureless second‐harmonic SNOM are numerically studied. The technique allows one to achieve strongly confined sources of second‐harmonic light at the probe tip apex and/or surface area under the tip. First experimental realization of this technique has been carried out using a silver‐coated fibre tip as a probe. The experiments reveal a strong influence of the tip–surface interaction as well as polarization of the excitation light on images obtained with apertureless second‐harmonic SNOM. The technique can be useful for studying the localized electromagnetic excitations on surfaces as well as for visualization of lateral variations of linear and nonlinear optical properties of surfaces.
Study of energy transfer from excited TPD to Alq in organic electroluminescent devices by time‐resolved fluorescence spectroscopy using a scanning near‐field optical atomic force microscope
Authors: Yamamoto N. , Muramatsu H. , Do L.‐M. , Han E.‐M. , Kato T. , Fujihira M.
Published: March 2003

We demonstrate the direct measurement of molecular diffusion at organic/organic interfaces of organic electroluminescence devices by use of a scanning near‐field optical atomic force microscope. Our preliminary study shows that the degradation of an electroluminescence device is partly caused by crystallization of the organic layers. Because the initial stage of degradation cannot be observed by microscopic methods, nanoscale optical properties of the interface in multilayer systems are currently receiving a great deal of attention. Defects of organic electroluminescence devices were investigated using a scanning near‐field optical atomic force microscope. This instrument is capable of measuring both a topographic and a fluorescence image at the same time. The defect area and other areas are clearly observed and time‐resolved near‐field fluorescence spectra demonstrate emission of the different species. These results suggest that defects occur at the organic solid interface, and that energy transfer occurs from excited TPD, as donor, to Alq, as acceptor.
Near‐field spectroscopy of single quantum dots at room temperature
Authors: Ikeda K. , Matsuda K. , Saito H. , Nishi K. , Saiki T.
Published: March 2003

The observation of photoluminescence spectra of self‐assembled single InGaAs quantum dots at room temperature was performed under weak excitation conditions using a near‐field scanning optical microscope. Operation in illumination‐collection mode with a highly sensitive double‐tapered optical fibre probe enabled detection of weak photoluminescence signals at room temperature with high efficiency and high spatial resolution. Each single quantum dot was imaged with a spatial resolution of about 250 nm, which corresponded to a quarter of the wavelength of the photoluminescence from quantum dots. The photoluminescence yields of individual quantum dots were widely distributed and were found to decrease with photoluminescence energy. This result serves as a clue to be pursued for better understanding of the thermal excitation of the carrier from confined states in quantum dots.
Photoluminescence properties of multiple stacked planes of GaN/AlN quantum dots studied by near‐field optical microscopy
Authors: Gucciardi P. G. , Vinattieri A. , Colocci M. , Damilano B. , Grandjean N. , Semond F. , Massies J.
Published: March 2003

We have studied the photoluminescence properties of GaN quantum dots with submicrometre lateral resolution by means of near‐field scanning optical microscopy. The instrument operated at room temperature and was implemented for near‐ultra‐violet spectroscopy in the illumination‐mode configuration. The analysed sample consisted of several stacked planes of GaN/AlN quantum dots grown by molecular beam epitaxy on Si(111) substrate. The photoluminescence maps showed islands in the micrometre range emitting at different wavelengths, confirming the atomic force microscopy studies on the morphology of similar uncapped samples.
Dynamic experimentation on the confocal laser scanning microscope: application to soft‐solid, composite food materials
Authors: Plucknett K. P. , Pomfret S. J. , Normand V. , Ferdinando D. , Veerman C. , Frith W. J. , Norton I. T.
Published: March 2003

Confocal laser scanning microscopy (CLSM) is used to follow the dynamic structural evolution of several phase‐separated mixed biopolymer gel composites. Two protein/polysaccharide mixed gel systems were examined: gelatin/maltodextrin and gelatin/agarose. These materials exhibit ‘emulsion‐like’ structures, with included spherical particles of one phase (i.e. polymer A) within a continuous matrix of the second (i.e. polymer B). Compositional control of these materials allows the phase order to be inverted (i.e. polymer B included and polymer A continuous), giving four basic variants for the present composites. Tension and compression mechanical tests were conducted dynamically on the CLSM, with crack/microstructure interactions investigated using a notched compact tension geometry. Gelatin/maltodextrin composites exhibit a ‘pseudo‐yielding’ stress/strain response in both tension and compression, when the gelatin‐rich phase is continuous, which was attributed to debonding of the particle/matrix interface. This behaviour is significantly less apparent for both the gelatin/agarose composites, and the maltodextrin continuous gelatin/maltodextrin composites, with these materials responding in a nominally linear elastic manner.

Values of the interfacial fracture energy for selected compositions of the two biopolymer systems were determined by 90° peel testing, where a gelatin layer was peeled from either a maltodextrin or agarose substrate. For biopolymer layers ‘cast’ together, a value of 0.2 ± 0.2 J m−2 was obtained for the fracture energy of a gelatin/maltodextrin interface, while a significantly higher value of 6.5 ± 0.2 J m−2 was determined for a gelatin/agarose interface.

The interfacial fracture energy of the two mixed systems was also determined following an indirect elastomer composite debonding model. An interfacial fracture energy of ∼0.25 J m−2 was determined using this approach for the gelatin continuous gelatin/maltodextrin composite, which compares favourably with the value calculated directly by peel testing (i.e. ∼0.2 J m−2). A somewhat higher value was estimated for the gelatin continuous gelatin/agarose system (1.0–2.0 J m−2), using this model, although there are severe limitations to this approach for this mixed gel system. In the present case, it is believed that the differing mechanical response of the two mixed biopolymer systems, when the gelatin phase is continuous, arises from the order of magnitude difference in interfacial fracture energy. It is postulated that polymer interdiffusion may occur across the interface for the gelatin/agarose system, to a significantly greater extent than for interfaces between gelatin and maltodextrin, resulting in a higher interfacial fracture energy.
High‐resolution constant‐height imaging with apertured silicon cantilever probes
Authors: Dziomba T. , Danzebrink H. U. , Lehrer C. , Frey L. , Sulzbach T. , Ohlsson O.
Published: March 2003

We present high‐resolution aperture probes based on non‐contact silicon atomic force microscopy (AFM) cantilevers for simultaneous AFM and near‐infrared scanning near‐field optical microscopy (SNOM). For use in near‐field optical microscopy, conventional AFM cantilevers are modified by covering their tip side with an opaque aluminium layer. To fabricate an aperture, this metal layer is opened at the end of the polyhedral probe using focused ion beams (FIB). Here we show that apertures of less than 50 nm can be obtained using this technique, which actually yield a resolution of about 50 nm, corresponding to λ/20 at the wavelength used. To exclude artefacts induced by distance control, we work in constant‐height mode. Our attention is particularly focused on the distance dependence of resolution and to the influence of slight cantilever bending on the optical images when scanning at such low scan heights, where first small attractive forces exerted on the cantilever become detectable.
Shaping the reflection near‐field optical probe: finite domain time difference modelling and fabrication using a focused ion beam
Authors: Spajer M. , Parent G. , Bainier C. , Charraut D.
Published: March 2003

An optical fibre ending in a trihedral tip is proposed as a convenient probe for reflection near‐field optical microscopy in emission/collection mode. Its shape is obtained by ion milling. A first example of manufacturing and numerical models using the bi‐dimensional FDTD method is presented. It confirms the strong influence of the facet angle on the intensity reflected by the probe, which is predicted by a rough analysis. This method can help us to optimize the ‘reflection probe’ by reducing this offset signal.
Non‐optical tip–sample distance control method for scanning near‐field optical microscopy using a piezoresistive micro cantilever
Authors: Muramatsu H. , Egawa A. , Homma K. , Kim J.‐M. , Takahashi H. , Shirakawabe Y. , Shimizu N.
Published: March 2003

A piezoresistive micro cantilever is applied to monitor the displacement of an optical fibre probe and to control tip–sample distance. The piezoresistive cantilever was originally made for a self‐sensitive atomic force microscopy (AFM) probe and has dimensions of 400 µm length, 50 µm width and 5 µm thickness with a resistive strain sensor at the bottom of the cantilever. We attach the piezoresistive cantilever tip to the upper side of a vibrating bent optical fibre probe and monitor the resistance change amplitude of the strain sensor caused by the optical fibre displacement. By using this resistance change to control the tip–sample distance, the two‐cantilever system successfully provides topographic and near‐field optical images of standard samples in a scanning near‐field optical microscopy (SNOM)/AFM system. A resonant characteristic of the two‐cantilever system is also simulated using a mechanical model, and the results of simulation correspond to the experimental results of resonance characteristics.
Rapid contrasting of ultrathin sections using microwave irradiation with heat dissipation
Authors: Hernández‐Chavarría F. , Vargas‐Montero M.
Published: March 2003

The use of microwave irradiation (MWI) to accelerate fixation, dehydration and contrasting (staining) for electron microscopy has been applied to the development of rapid methods to process biological samples in electron microscopy. A simple explanation is that the reduced time in those procedures is due to heating. In this paper we propose a contrasting method for thin sections that avoids the thermal effects of MWI. Grids with thin sections of mouse kidney, the dinoflagellate Alexandrium monilatum, spermatophores of the fly Archicepsis diversiformis, the bacteria Acinetobacter calcoaceticum and Enterobacter cloacae were placed into Beem capsules and stained with uranyl acetate and lead citrate, while immersed in an ice‐water bath, and irradiated for periods ranging from 30 s to 2 min. After each contrasting procedure, the Beem capsule was filled with distilled water to wash the grids under MWI with the same irradiation time as used to contrast. Good results were obtained on irradiating for 1 min and the temperature of the Beem capsule was maintained around 5 °C.
In situ transmission electron microscopy studies and real‐time digital imaging
Authors: Alani R. , Pan M.
Published: March 2003

A newly designed CCD camera has been utilized for real‐time and static image acquisitions. The performance of the camera is demonstrated for heating/cooling in‐situ TEM experiments performed on a commercial high strength aluminium alloy using a double tilt heating holder. The real‐time digital imaging capability of the new camera should facilitate the in‐situ TEM that is now re‐establishing itself as a strategic tool for materials characterization.
High‐voltage electron microscope high‐temperature in situ straining experiments to study dislocation dynamics in intermetallics and quasicrystals
Authors: Messerschmidt U.
Published: March 2003

The dynamic behaviour of dislocations in several intermetallic alloys, studied by in situ straining experiments in a high‐voltage electron microscope, is compared at room temperature and at high temperatures. In contrast to room temperature, the dislocations move viscously at high temperatures, which is explained by diffusion processes in the dislocation cores. In quasicrystals, the viscous dislocation motion can be interpreted by models on the cluster scale.
A simple technique to measure the movements of the microscope stage along the x and y axes for stereological methods
Authors: Kaplan S. , Canan S. , Aslan H. , Ünal B. , Sahi˙n B.
Published: March 2003

Measurements of microscope stage movements in the x and y directions are of importance for some stereological methods such as the optical disector and optical fractionator. The length of stage movements can be measured with great precision and accuracy using a suitable motorized stage, which is generally a computer‐assisted instrument. This type of equipment is generally too expensive for and not readily available in many laboratories. This paper describes a simple method to measure the movements of the microscope stage along the x and y directions, which can be used for purposes such as systematic uniform random sampling. It needs a microscope attachment consisting of two dial indicators; one of them is used to measure the amount of stage movement along the x‐axis and the other measures the amount of movement along the y‐axis. Movements of the stage on the micrometre‐scale can be measured easily using this device.
Scanning force images through the ‘Milliscope’– a probe microscope with very wide scan range
Authors: Mariani T. , Ascoli C. , Baschieri P. , Frediani C. , Musio A.
Published: March 2003

The effectiveness and adequacy of a home‐built scanning force microscope (SFM) able to cover a volume of ∼1.2 × 1.2 × 0.13 mm3 (X × Y × Z) were tested on calibrating objects, as well as on cytological and histological samples. The instrument was designed for matching the magnification range of an optical microscope (∼ 20–1200×) but its dynamics were one or two orders of magnitude higher, thanks to a lateral resolution of about 10 nm. Images ranging in size from 1.2 × 1.2 mm2 to 1 × 1 µm2 showed a quality comparable to that given by other SFMs on similar materials. The ‘Milliscope’ is a curious but effective imaging tool whose operating range overlaps at one extreme with a goldsmith's eyepiece, and at the other with an electron microscope. The intrinsic limits of scanning probe techniques and of the available SFM cantilevers prevented us taking complete advantage of the wide height range of our scanner. However, our results show that an instrument having a very wide scan area, obtained through simple, inexpensive and intrinsically linear techniques, can give a good performance even at small scan sizes. This encourages us to develop wide scan instruments, which could further increase the already extensive use of scanning force microscopy in biology.
Higher‐order Laue zone line contrast in large‐angle convergent‐beam electron diffraction around a dislocation
Authors: Toda A. , Ikarashi N. , Ono H.
Published: March 2003

The physical picture of higher‐order Laue zone (HOLZ) line contrast in a large‐angle convergent‐beam electron diffraction pattern around a dislocation, which is used for determining the Burgers vector, was examined. To evaluate the analytical expression of diffracted wave amplitude, we introduced an approximate form of the atomic displacement field of a dislocation. We showed that the four features of the HOLZ line contrast, that is, splitting, fading, bending and periodical contrast can be explained by analysis of the atomic displacement field. The localized lattice plane bending around a dislocation core made a HOLZ line split, fade and bend. However, we found that the periodical contrast of a HOLZ line was produced by the change of phase difference of the atomic displacement field between the crystals above and below the slip plane across the dislocation line.
A simple method for orienting silk and other flexible fibres in transmission electron microscopy specimens
Authors: Trancik J. E. , Czernuszka J. T. , Merriman C. , Viney C.
Published: March 2003

When microstructures are characterized by transmission electron microscopy (TEM), the interpretation of results is facilitated if the material can be sectioned in defined orientations. In the case of fibres, it is especially useful if transverse and longitudinal sections can be obtained reliably. Here we describe a procedure for orienting spider silk and other flexible fibres for TEM investigation. Prior to embedding in epoxy resin, the silk is wound around a notched support made from polyester film. No glue is required. After the silk and its supporting film have been embedded and the resin has been cured the film can be peeled away to reveal nearly perfectly orientated silk threads. Both transverse and longitudinal sections can then be cut with a microtome. The method can be extended to obtain sections at any intermediate orientation.
Tracing DiO‐labelled tumour cells in liver sections by confocal laser scanning microscopy
Authors: Timmers M. , Vermijlen D. , Vekemans K. , Zanger R. DE , Wisse E. , Braet F.
Published: March 2003

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO‐labelled colon carcinoma cells (CC531s) in 100 µm thick liver sections. In vitro and in vivo experiments revealed that DiO‐labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO‐labelled CC531s cells could be traced all over the tissue depth and anti‐metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine‐phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO‐labelled cells in tissue sections.
Automatic fluorescent tag detection in 3D with super‐resolution: application to the analysis of chromosome movement
Authors: Thomann D. , Rines D. R. , Sorger P. K. , Danuser G.
Published: March 2003

In this paper, we describe an algorithmic framework for the automatic detection of diffraction‐limited fluorescent spots in 3D optical images at a separation below the Rayleigh limit, i.e. with super‐resolution. We demonstrate the potential of super‐resolution detection by tracking fluorescently tagged chromosomes during mitosis in budding yeast. Our biological objective is to identify and analyse the proteins responsible for the generation of tensile force during chromosome segregation. Dynamic measurements in living cells are made possible by green fluroescent protein (GFP)‐tagging chromosomes and spindle pole bodies to generate cells carrying four fluorescent spots, and observe the motion of the spots over time using 3D‐fluorescence microscopy. The central problem in spot detection arises with the partial or complete overlap of spots when tagged objects are separated by distances below the resolution of the optics. To detect multiple spots under these conditions, a set of candidate mixture models is built, and the best candidate is selected from the set based on χ2‐statistics of the residuals in least‐square fits of the models to the image data.

Even with images having a signal‐to‐noise ratio (SNR) as low as 5–10, we are able to increase the resolution two‐fold below the Rayleigh limit. In images with a SNR of 5–10, the accuracy with which isolated tags can be localized is less than 5 nm. For two tags separated by less than the Rayleigh limit, the localization accuracy is found to be between 10 and 20 nm, depending on the effective point‐to‐point distance. This indicates the intimate relationship between resolution and localization accuracy.
Brain Angiotensin II Modulates Sympathoadrenal and Hypothalamic Pituitary Adrenocortical Activation during Stress
Authors: Jezova Daniela , Ochedalski Tomasz , Kiss Alexander , Aguilera Greti
Published: March 2003

Angiotensin II (Ang II) type‐1 (AT1) receptors are present in areas of the brain controlling autonomic nervous activity and the hypothalamic‐pituitary‐adrenal (HPA) axis, including CRH cells in the hypothalamic paraventricular nucleus (PVN). To determine whether brain AT1 receptors are involved in the activation of the HPA axis and sympathetic system during stress, we studied the effects of acute immobilization stress on plasma catecholamines, ACTH and corticosterone, and mRNA levels of CRH and CRH receptors (CRH‐R) in the PVN in rats under central AT1 receptor blockade by the selective antagonist, Losartan. While basal levels of epinephrine, norepinephrine and dopamine in plasma were unaffected 30 min after icv injection of Losartan (10 μg), the increases after 5 and 20 min stress were blunted in Losartan treated rats (P<0.05 for norepinephrine, and P<0.01 for epinephrine and dopamine, vs controls). Basal or stress‐stimulated plasma ACTH and corticosterone levels were unaffected by icv Losartan treatment. Using in situ hybridization studies, basal levels of CRH mRNA and CRH‐R mRNA in the PVN were unchanged after icv Losartan. While Losartan had no effect on the increases in CRH‐R mRNA levels 2 or 3 h after 1 h immobilization, it prevented the increases in CRH mRNA. The blunted plasma catecholamine responses after central AT1 receptor blockade indicate that endogenous Ang II in the brain is required for sympathoadrenal activation during immobilization stress. While Ang II appears not to be involved in the acute secretory response of the HPA axis, it may play a role in regulating CRH expression in the PVN.
Effect of Photostimulation on Concentrations of Plasma Prolactin in Castrated Bantams (Gallus domesticus)
Authors: Sreekumar Kannoth , Sharp Peter
Published: March 2003

The annual breeding cycle of ‘unimproved’ breeds of domestic chicken, including the bantam, at temperate latitudes, is terminated by decreasing daylength in autumn and is initiated in late winter, while daylengths are still short. Observations on photoperiodic birds that terminate seasonal breeding by the development of long day photorefractoriness suggest that the photoinduced pattern of prolactin secretion is associated with the pattern of gonadal growth and regression. It was predicted that, if there is a causal relationship between photoinduced changes in prolactin secretion and gonadal function in birds then, in the bantam, the pattern of prolactin secretion observed after photostimulation would not be the same as in birds terminating breeding by the development of long day photorefractoriness. Experiments were carried out on surgically castrated bantams to avoid confounding the effects of photostimulation and the stimulatory actions of testicular hormones on prolactin secretion. Transfer of photosensitive castrated bantams from 8 to 14, 16, 18 or 20 h light/day initially stimulated prolactin release and, subsequently, after 20–30 days, concentrations of plasma prolactin progressively decreased. After 148 days of photostimulation, concentrations of plasma prolactin approached but were still higher than short day controls. Transfer of photosensitive castrated bantam cockerels from 8 to 12 h light/day stimulated a slower increase in plasma prolactin that subsequently remained higher than in other photostimulated groups. A further 4 h increase in photoperiod in the birds exposed for 148 days to 12 or 16 h light/day resulted, respectively, in a transitory increase and no increase in prolactin secretion. Recovery of photosensitivity for prolactin release was observed in the birds transferred to 18 or 20 h light/day for 148 days after treatment with 8 h light/day for 35 days. Attempts to obtain an independent hormonal correlate of the prolactin responses to photostimulation by measurement of plasma luteinizing hormone (LH) were unsuccessful. The concentration of plasma LH in castrated bantams did not change in response to a change in photoperiod. These observations show that the photoinduced pattern of prolactin release in the bantam, a species which terminates seasonal breeding in response to decreasing daylength, is the same as that in birds which terminate seasonal breeding by the development of long day photorefractoriness. It is concluded that the photoinduced pattern of prolactin secretion in birds can be dissociated from the neuroendocrine mechanisms controlling the termination of seasonal breeding.
Developmental Expression and Regulation of Aromatase– and 5α‐Reductase Type 1 mRNA in the Male and Female Mouse Hypothalamus
Authors: Karolczak Magdalena , Küppers Eva , Beyer Cordian
Published: March 2003

Androgen metabolites synthesized by neural aromatase and 5α–reductase are implicated in many aspects of mammalian brain development and, in particular, in the masculinization of distinct central nervous system structures and brain functions. The present study was designed to determine (1) the developmental profile of aromatase‐ and 5α–reductase type I mRNA expression in the mouse hypothalamus and (2) to relate ontogenetic sex differences in aromatase activity which have been described in the past to sex‐specific aromatase gene expression. In addition, we analysed the effect of androgens on the perinatal regulation of hypothalamic aromatase and 5α–reductase type I mRNA expression. By applying semiquantitative reverse transcription‐polymerase chain reaction analysis, we found hypothalamic aromatase mRNA expression to be developmentally regulated and to display sex differences at birth and on postnatal day 15 with higher mRNA levels in males. Newborn males and females, which were treated in utero with the androgen receptor antagonist cyproterone actetate, exhibited significantly reduced aromatase mRNA levels compared with untreated controls. In contrast to aromatase, expression levels of hypothalamic 5α–reductase mRNA did not reveal a clear‐cut developmental profile or sex differences, and no regulatory role for androgens in controlling 5α–reductase mRNA expression was found. In conclusion, these results demonstrate perinatal sex differences in hypothalamic aromatase‐ but not 5α–reductase gene expression and suggest that sex differences in perinatal aromatase activity are reflected by corresponding differences in mRNA levels. Androgens are found to control brain estrogen formation pretranslationally at the level of aromatase gene expression. Our findings imply that sex differences in androgen availability and responsiveness are important regulatory factors for aromatase expression in the developing male hypothalamus.
Glutamate Pathways Mediate Somatostain Responses to Glucose in Normal and Diabetic Rat Hypothalamus
Authors: Issa  , Lewis  , Ham  , Scanlon
Published: March 2003

We investigated the role of hypothalamic glutamate receptors in mediating the stimulatory effect of low glucose (<5 mM) on somatostatin release. We also studied whether alteration in glutamate release might contribute to the reduced hypothalamic somatostatin response to low glucose observed in diabetic (Goto‐Kakizaki) rat hypothalami. Hypothalamic somatostatin release in response to incubation with 1 mM d‐glucose was inhibited by the ionotropic glutamate receptor antagonists MK801, D‐AP5 and DNQX but not by the metabotropic antagonists L‐AP3 or MCPG. The release of somatostatin was increased by the ionotropic agonists NMDA, AMPA and kainate but not by metabotropic agonists t‐ACPD or L‐AP4. Basal and peak glutamate release in response to incubation with 1 mM glucose, were significantly lower from GK hypothalami There were no significant differences in the basal or stimulated release of serine and GABA. These data indicate that ionotropic NMDA/AMPA/kainate receptors and not metabotropic receptors mediate the effects of glucose on rat hypothalamic somatostatin release. Reduced hypothalamic somatostatin release in response to low glucose in diabetic (Goto‐Kakizaki) rats may well be secondary, at least in part, to reduced glutamate release.
Activation by Systemic Angiotensin II of Neurochemically Identified Neurons in Rat Hypothalamic Paraventricular Nucleus
Authors: Dawson Corrine A. , Jhamandas Jack H. , Krukoff Teresa L.
Published: March 2003

Using the immunohistochemical localization of the protein product of the immediate early gene, c‐fos, to localize activated neurons in the paraventricular nucleus of the hypothalamus (PVN), we studied the chemical phenotypes of neurons activated by circulating angiotensin II (AII). We determined the proportions of activated PVN neurons that expressed AII type I receptor‐like immunoreactivity (AT1‐L) or the neurohormones vasopressin (VP) and oxytocin (OXY). In addition, we identified activated PVN neurons that putatively produce nitric oxide (NO) on the basis of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH‐d). Conscious rats received intravenous AII infusions at a rate sufficient to elevate mean arterial pressure by 40–60 mmHg for 90 min; control rats received infusions of vehicle. Brains were prepared for double immunohistochemistry [Fos‐like immunoreactivity (FLI)/AT1‐L, FLI/VP or FLI/OXY] or FLI/NADPH‐d histochemistry. Systemic AII infusions led to activation of 149±14 PVN neurons per section. In contrast, control animals showed activation of 21±6 PVN neurons per section. AII infusions elicited the activation of the following numbers of chemically identified PVN neurons per section: AT1‐L, 24±5; VP, 26±5; OXY, 11±2; NADPH‐d, 22±4. Control animals had few activated PVN neurons per section. For each of the chemically identified populations of PVN neurons, the following proportions were activated: AT1‐L, 12.5%; VP, 15.2%; OXY, 7.2%; NADPH‐d, 17.3%. The results suggest that PVN neurons producing the AT1 receptor, VP, OXY, and NO, participate in the mediation of the central responses to circulating AII.
Daily and Circadian Variations in 2‐[125I]‐Iodomelatonin Binding Sites in the Pike Brain (Esox Iucius)
Authors: Gaildrat Pascaline , Ron Benny , Falcón Jack
Published: March 2003

The fish pineal organ, through its 24 h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g. reproduction, growth). We have investigated the binding of 2‐[125I]iodomelatonin to whole brain membrane preparations from pikes (Esox lucius L., teleost) maintained for 24–48 h under different photoperiodic conditions. Specific binding was stable, reversible, saturable and sensitive to the presence of a GTP analogue. Scatchard analysis revealed one class of binding sites. Displacement experiments suggested the presence of two components with affinities in the femtomolar and nanomolar range of concentrations, respectively. The Bmax exhibited monophasic nycthemeral variations, with higher values at the light‐to‐dark transition (34.0±4.5 fmol/mg protein) and low values during the second half of night (10.0±1.0 fmol/mg protein). Under the same conditions, the KD exhibited biphasic variations: values were low during daytime and at the middle of the dark phase (approximately 100 pM); they were high at the beginning (approximately 225 pM) and at the end (approximately 330 pM) of the night. These variations were maintained under constant light (LL) and constant darkness (DD). Thus, the variations in the number and affinity of the melatonin binding sites were controlled by circadian oscillators, synchronized by the photoperiod. The nature of these oscillators is not known. Therefore, in fish, we suggest that the photodependent effects of melatonin result from the circadian variations of both its production by the pineal and its binding sites in the brain.
Influence of Carbon Monoxide, and its Interaction with Nitric Oxide, on the Adrenocorticotropin Hormone Response of the Normal Rat to a Physico‐Emotional Stress
Authors: Turnbull Andrew V. , Kim C. Kwon , Lee Soon , Rivier Catherine L.
Published: March 2003

We determined whether the gas carbon monoxide (CO) altered the adrenocorticotropin hormone (ACTH) response to mild inescapable electrofootshocks, and whether it interacted with nitric oxide (NO). Peripheral injection of the NO synthase (NOS) inhibitor Nwnitro‐l‐arginine‐methylester (l‐NAME), a compound which readily crosses the blood–brain barrier, produced the expected blunting of the ACTH response to the shocks. This effect was mimicked by other arginine analogues such as l‐nitroarginine (l‐NNA) and NG‐methyl‐l‐arginine (NMMA). The subcutaneous (s.c.) administration of the heme oxygenase (HO) blockers tin mesoporphyrin (SnMP) or tin protoporphyrin (SnPP) significantly decreased brain HO levels, indicating that both compounds had penetrated the brain. Blood pressure showed a modest increase in response to SnMP, and no change after SnPP. SnMP and SnPP both decreased shock‐induced ACTH release, though the magnitude of this effect was slightly less than that of l‐NAME. The influence of SnPP was further augmented in rats with concomitant blockade of NO formation, which suggests that both NO and CO are necessary for the full response of this axis to electrofootshocks. Finally, the ability of SnPP to significantly blunt the expression of the mRNA for the immediate early gene NGFI‐B in the paraventricular nucleus (PVN) of rats exposed to shocks, indicates that the influence of CO was exerted on hypothalamic neuronal activity. Collectively, our results show that NO and CO exert a stimulatory effect on the HPA axis response to mild electrofootshocks, and that at least part of this influence takes place on hypothalamic neurons and/or their afferents.
Aromatase in Developing Sensory Sytems of the Rat Brain
Authors: Horvath  , Wikler
Published: March 2003

Sex differences in the rat brain are dependent, in part, on oestrogen exposure during specific developmental perinatal periods. The availability of oestrogen requires precursor androgen and the presence of intraneuronal aromatase. To examine sites of oestrogen formation and action in the brain, immunocytochemical and biochemical localization of aromatase in the rat brain were determined between embryonic day 14 and postnatal day 20. Aromatase‐immunolabelled neuronal profiles were present in hypothalamic, cortical and limbic regions. Surprisingly, aromatase immunoreactivity was also observed in non‐limbic regions of the immature brain where it was previously unsuspected. Among these regions, aromatase staining was robust in developing sensory systems, including primary afferents of the olfactory, trigeminal, vestibulocochlear, and visual systems. To determine whether this aromatase is functional in these systems, i.e. converts testosterone to estradiol, the trigeminal nerve was dissected from the hindbrain of perinatal animals and studied for enzyme activity by the tritium release method. The dpm/mg protein/h tritium release in these tissues equalled that of hypothalamic or limbic controls, indicating that these sensory areas are sites of in‐situ estradiol synthesis. Our data suggests that aromatase (estradiol)‐dependent mechanisms may play a role in the differentiation and maturation of sensory pathways, which, in turn, may contribute to sex differences in the activity of these systems.
Effects of Osmotic Pressure and Brain‐Derived Neurotrophic Factor on the Survival of Postnatal Hypothalamic Oxytocinergic and Vasopressinergic Neurons in Dissociated Cell Culture
Authors: Kusano  , House  , Gainer
Published: March 2003

Neurons from hypothalamic paraventricular nuclei (PVN) and supraoptic nuclei (SON) from postnatal day 6–8 rats were enzymatically dissociated and separately maintained in monolayer cultures for 14 days. The osmotic pressure of the culture medium, based on Neurobasal medium (Life Technologies), was varied (255, 300 and 330 mOsm/l) by adjustment using mannitol. The survival of oxytocin (OT), vasopressin (VP) and oxytocin‐vasopressin (OT/VP) coexpressing neurons were studied under these varied conditions, and the identification of the cell phenotypes in the cultures was carried out by using double‐label immunofluorescence. Under control osmolar conditions (300 mOsm/l) equivalent numbers of OT and VP neurons were found in the SON (P=0.8398) and PVN (P=0.4721) cultures. The OT neurons’ survival did not change in 255 or 330 mOsm media in the SON cultures, but the VP neurons in the SON cultures were significantly increased in 255 mOsm/l medium as compared to control (300 mOsm/l) medium (P=0.0088). No significant changes were found in VP neuron survival in SON cultures between the 300–330 mOsm/l media (P=0.2372). Similar data were obtained for the VP neurons in PVN‐derived cultures, but the OT neurons in these cultures survived significantly better at 300 mOs/l than at 255 mOsm/l (P<0.0001), but were not significantly different at 330 mOsm/l (P=0.1208). In general, the VP neurons were more vulnerable than OT neurons to increases of culture medium osmolarity with respect to their survival. The number of OT/VP coexpressing neurons was greater in SON‐derived cell cultures as compared to PVN‐derived cell cultures, and their numbers were higher in the lower osmolarity media. The effects of adding brain‐derived neurotrophic factor (BDNF) to the culture medium on survival were determined. BDNF significantly increased the numbers of all three types of neurons in both PVN and SON cell cultures (P=0.0001–0.0060). The phenotypically identified cells, cultured in the 300 mOsm/l medium, responded by depolarization or hyperpolarization when transferred to hypertonic or hypotonic perfusion salines, respectively.
Strain Differences in Corticosteroid Receptor Efficiencies and Regulation in Brown Norway and Fischer 344 Rats
Authors: Marissal‐Arvy  , Morméde  , Sarrieau
Published: March 2003

During the dark phase of the diurnal cycle, and during recovery from restraint stress, Brown Norway (BN) rats secrete less corticosterone than Fischer 344 (F344) rats. These strains also display different levels of corticosteroid receptors in the hippocampus, and of plasma transcortin. Because corticosteroid receptors, plasma transcortin and corticosterone secretion are mutually regulated, we examined brain and pituitary mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) expression and some of the parameters modulated by these receptors (i.e. body and thymus weight, fluid intake, plasma transcortin) in BN and F344 rat strains, by comparing the effects of either hormone deprivation by long‐term (21 days) adrenalectomy (ADX), or chronic elevation of corticosterone given in drinking fluid to ADX rats. In BN rats, body weight gain and fluid intake were insensitive to corticosterone deprivation, suggesting that MR‐related mechanisms are constitutively active in this strain. Body weight (b.w.) gain, plasma transcortin and thymus weight were reduced to a greater extent by chronic corticosterone in BN rats than in F344 rats, possibly as a consequence of higher free, active fraction of plasma corticosterone due to lower plasma transcortin concentrations and/or a greater efficiency of GR‐related mechanisms in BN rats. F344 rats displayed twofold higher brain and pituitary MR levels than BN rats, whereas tissue‐and strain‐specific regulations were observed for GR levels. The differences in MR levels observed between BN and F344 strains cannot completely explain the differences in corticosterone actions, suggesting that strain differences in response to ADX or corticosterone treatment result from variable receptor efficiencies.
Leptin Effects on the Expression of Type‐2 CRH Receptor mRNA in the Ventromedial Hypothalamus in the Rat
Authors: Nishiyama  , Makion  , Asaba  , Hasimoto
Published: March 2003

The product of the ob gene, leptin, is thought to act in the hypothalamus to reduce food intake and body weight (b.w.) in rats and mice; however, the mechanisms of leptin action in the brain have not been fully elucidated. Corticotropin‐releasing hormone (CRH) is a potent anorectic neuropeptide, and its type‐2 receptor (CRHR‐2) in the ventromedial hypothalamus (VMH) appears to play an important role in the expression of this anorectic effect. We explored here the impact of systemic leptin administration on CRH mRNA expression in the hypothalamic paraventricular nucleus (PVN) and CRHR‐2 mRNA expression in the VMH in male rats, using in‐situ hybridization histochemistry. The expression of CRH mRNA in the PVN and CRHR‐2 mRNA in the VMH were increased at 2 h and 6 h, respectively, after a single intraperitoneal injection of leptin (1.0 mg/kg). Continuous subcutaneous infusion of leptin (1.2 mg/kg/day) via an osmotic minipump for 5 days increased the expression of CRHR‐2 mRNA in the VMH, but not the expression of CRH mRNA in the PVN, compared with vehicle treatment. The rats that received the single or continuous administration of leptin showed reductions of food intake and b.w. compared with vehicle‐treated rats. These results are consistent with our previous findings that the expression of CRHR‐2 mRNA in the VMH is positively correlated with plasma leptin concentrations under various conditions, and highlight the importance of circulating leptin for the regulation of VMH CRHR‐2 mRNA. The present results also raise the possibility that leptin reduces food intake and b.w. at least partially due to the enhancement of the anorectic effect of CRH via increased PVN CRH expression and/or VMH CRHR‐2 expression.
Differential Expression of c‐fos mRNA Within Neurocircuits of Male Hamsters Exposed to Acute or Chronic Defeat
Authors: Kollack‐Walker  , Don  , Watson  , Akil
Published: March 2003

Chronic exposure to stress has been implicated in physical and mental illness, and such experiences can produce alterations in the connectivity and number of neurones within the brain to variations in the expression of specific genes. The purpose of this study was to determine how repeated exposure to social defeat affects neuronal activation patterns within the male Syrian hamster brain. Toward this end, the levels c‐fos mRNA were compared among three groups: (1) handled controls (HC); (2) acutely defeated males (AD); and (3) chronically defeated males (15 min aggression daily, 7 days) exposed to an acute challenge (CD). Plasma glucocorticoids were also measured and compared among groups as an index of neuroendocrine activity. The results show a selective pattern of habituation of immediate early gene expression within the brains of chronically defeated males. In particular, c‐fos mRNA levels were significantly decreased within the paraventricular nucleus of the hypothalamus (PVN), supraoptic nucleus of the hypothalamus, septohypothalamic nucleus, intermediate subdivision of the lateral septum, central amygdaloid nucleus, and the amygdalohippocampal area in the CD group exposed to an acute challenge when compared to males defeated only once. In contrast, c‐fos expression within the anterior and ventromedial nuclei of the hypothalamus, dorsal periaqueductal grey, dorsal raphe, cuneiform nucleus, and locus coeruleus did not differ between AD and CD groups. Similarly, plasma levels of cortisol and corticosterone in CD group were equivalent to those observed after a single defeat experience. We discuss the possibility that decreased expression of c‐fos mRNA within the PVN and other brain regions of defeated animals–in the presence of elevated adrenal steroids–may reflect a state of molecular plasticity that could alter neurotransmission within the limbic‐hypothalamo‐pituitary‐adrenocortical axis. In contrast, brain areas that maintain relatively high levels of c‐fos mRNA following repeated defeat may reflect processes less likely to adapt such as defensive behaviour.
Prior Exposure To Oxytocin Mimics the Effects Of Social Contact and Facilitates Sexual Behaviour In Females
Authors: Cushing  , Carter
Published: March 2003

The purpose of this study was to determine whether pretreatment with oxytocin could mimic the effects of social contact and enhance sexual receptivity in female prairie voles. Female prairie voles require prolonged exposure to males to become sexually active and oxytocin has been shown to play a major role in the establishment of social bonds between males and females. Therefore, we hypothesized that prior exposure to exogenous oxytocin, in the absence of males, would enhance sexual activity in females. Two experiments were conducted to test this hypothesis. Experiment 1 examined the capacity of oxytocin to enhance sexual behaviour in females undergoing natural oestrus. Sexually naive female prairie voles received a daily subcutaneous injection of 20 μg oxytocin or isotonic saline for 5 days before being placed with a sexually experienced male for 48 h. Females treated with oxytocin were significantly more likely to mate during this period than saline‐treated females. In experiment 2 the ability of oxytocin to increase subsequent sensitivity of sexually naive females to oestradiol was tested. Females that received oxytocin pretreatment, as in experiment 1, followed by oestradiol displayed a significant increase in sexual receptivity when compared to females treated with saline and oestradiol or oestradiol only. The results supported the hypothesis that prior exposure to oxytocin can mimic the effects of social contact, and can facilitate sexual receptivity by increasing the sensitivity of females to very low doses of oestradiol.
Potentiation Effect of Vasopressin on Melatonin Secretion as Determined by Trans‐Pineal Microdialysis in the Rat
Authors: Barassin  , Kalsbeek  , Saboureau  , Bothorel  , Vivien‐Roels  , Malan  , Buijs  , Pevet
Published: March 2003

The mammalian pineal gland is known to receive a noradrenergic innervation originating from the superior cervical ganglion which corresponds to the primary regulatory input for melatonin synthesis. However, many peptidergic fibers containing peptides such as vasopressin and oxytocin have also been found in the rat pineal gland. The present study was performed to investigate the possible role of vasopressin and oxytocin on melatonin secretion in vivo. Therefore, both neuropeptides were delivered for 2 h through a trans‐pineal microdialysis probe directly into the gland at different times during the nocturnal phase of the light:dark cycle. At the same time pineal dialysates were collected continuously. Melatonin concentrations were measured by radioimmunoassay. Melatonin synthesis potentiation was achieved when vasopressin was infused locally in the pineal, during the onset of nocturnal melatonin secretion. In order to assess the possible role of a physiological increase of endogenous circulating vasopressin on pineal metabolism, melatonin synthesis was recorded in the same animals before and after a prolonged dehydration period. Night time melatonin concentration was increased after the water deprivation vs control conditions. Contrary to that, oxytocin seems not to affect pineal metabolism in the rat since no significant change was observed on melatonin secretion in response to a local oxytocin infusion. These results show that vasopressin can modulate melatonin synthesis in the rat pineal whereas no effect was obtained with oxytocin, at least under the present experimental conditions.