Owe Orwar

Enriched environment increases neurogenesis in the adult rat dentate gyrus and improves spatial memory.

The fetal and even the young brain possesses a considerable degree of plasticity. The plasticity and rate of neurogenesis in the adult brain is much less pronounced. The present study was conducted to investigate whether housing conditions affect neurogenesis, learning, and memory in adult rats. Three-month-old rats housed either in isolation or in an enriched environment were injected intraperitoneally with bromodeoxyuridine (BrdU) to detect proliferation among progenitor cells and to follow their fate in the dentate gyrus. The rats were sacrificed either 1 day or 4 weeks after BrdU injections. This experimental paradigm allows for discrimination between proliferative effects and survival effects on the newborn progenitors elicited by different housing conditions. The number of newborn cells in the dentate gyrus was not altered 1 day after BrdU injections. In contrast, the number of surviving progenitors 1 month after BrdU injections was markedly increased in animals housed in an enriched environment. The relative ratio of neurogenesis and gliogenesis was not affected by environmental conditions, as estimated by double-labeling immunofluorescence staining with antibodies against BrdU and either the neuronal marker calbindin D28k or the glial marker GFAp, resulting in a net increase in neurogenesis in animals housed in an enriched environment. Furthermore, we show that adult rats housed in an enriched environment show improved performance in a spatial learning test. The results suggest that environmental cues can enhance neurogenesis in the adult hippocampal region, which is associated with improved spatial memory.

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy.

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.

Selective introduction of antisense oligonucleotides into single adult CNS progenitor cells using electroporation demonstrates the requirement of STAT3 activation for CNTF-induced gliogenesis.

We have developed a novel method in which antisense DNA is selectively electroporated into individual adult neural progenitor cells. By electroporation of antisense oligonucleotides against signal transducer and activator of transcription 3 (STAT3) we demonstrate that ciliary neurotrophic factor (CNTF) is an instructive signal for astroglial type 2 cell fate specifically mediated via activation of STAT3. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway induced only a transient increase in glial fibrillary acidic protein (GFAP) expression, and inhibition of this signaling pathway did not block the induction by CNTF of glial differentiation in progenitor cells. In addition we show that microelectroporation is a new powerful method for introducing antisense agents into single cells in complex cellular networks.

Manipulating the biochemical nanoenvironment around single molecules contained within vesicles

A method to study single-molecule reactions confined in a biomimetic container is described. The technique combines rapid vesicle preparation, optical trapping and fluorescence confocal microscopy for performing simultaneous single-vesicle trapping and single-molecule detection experiments. The collisional environment between a single enzyme and substrate inside a vesicle is characterized by a Brownian dynamics Monte Carlo simulation. q 1999 Elsevier Science B.V. All rights reserved.

Patch-Clamp Detection of Neurotransmitters in Capillary Electrophoresis

Gamma-aminobutyrate acid, L-glutamate, and N-methyl-D-aspartate were separated by capillary electrophoresis and detected by the use of whole-cell and outside-out patch-clamp techniques on freshly dissociated rat olfactory interneurons. These neuroactive compounds could be identified from their electrophoretic migration times, unitary channel conductances, and power spectra that yielded corner frequencies and mean single-channel conductances characteristic for each of the different agonist-receptor interactions. This technique has the sensitivity to observe the opening of a single ion channel for agonists separated by capillary electrophoresis.

Determination of Photodestruction Quantum Yields Using Capillary Electro-Phoresis: Application to o-Phthalalde-Hyde/β-Mercaptoethanol-Labeled Amino Acids

Photodestruction quantum yields, Φ D , were determined for o-phthalaldehyde/β-mercaptoethanol-labeled protein amino acids in aqueous alkaline solution using trans-azobenzene actinometry. The Φ D values ranged from 0.020 to 0.062 ; the highest were obtained for amide-containing amino acid derivatives (Gln and Asn ; Φ D = 0.062 and 0.054, respectively). The average maximal number of fluorescent photons obtained from the chromophores ranged from 6.4 to 19.0 per molecule. We show that Φ D values can be determined with the help of an internal standard using capillary electrophoresis with laser-induced fluorescence detection. Generally, good agreement between the Φ D values obtained in this way and Φ D values obtained using trans-azobenzene actinometry was found. Furthermore, we show that knowledge of the analytes photochemical properties assists identification of components separated by capillary electrophoresis.

Confining and Probing Single Molecules in Synthetic Liposomes

As organisms, we are amazingly complex living laboratories. As we move, breathe, think, and eat, seemingly endless chemical reactions and interactions occur inside us. The test tubes, beakers, and flasks used to separate and selectively mix the myriad of reactants involved are cells, vesicles, and organelles. Taking the analogy further, whereas chemists typically mix chemicals milliliters or more in volume, biological systems carry out their biochemistry in containers that are femtoliters or less in volume. As researchers we assume, with good reason, that the material surfaces of our laboratory test tubes do not substantially affect the kinetics we measure. This assumption might not hold were we to shrink our containers to the femtoliter scale. At such a small scale, collision rates between reactants and their container walls become significant [1], and the inner surface, particularly in biological containers, is chemically complex. The bilayers of cells and organelles are composed of a variety of lipids. These varieties assemble into domains [2] in a process partly controlled by the transmembrane proteins in them [3]. Cellular and organellar control of chemical reactions may thus come, in part, from alterations in the composition and arrangement of the molecular species making up the bilayer membrane [4]. How do systematic alterations to the bilayer composition of a liposome alter the kinetics of reactions within the liposome interior? We may find that the potential physiological significance of lipid domains within bilayers to the kinetics of in-plane reactions [5] (i.e., for proteins and other molecules moving within the bilayer) has applications to molecules within liposomes that interact with the inner bilayer surface.

Electrical and optical methods for the manipulation and analyses of single cells

This paper describes the use of focused electric fields and focused optical fields for the high-resolution manipulation of single cells. A focused electric field, obtained with the use of ultramicroelectrodes (tip diameter approximately 5 μm), is used to electroporate and electrofuse individual cells selectively and with high spatial resolution. A focused optical field, in the form of an optical tweezer, is used to isolate single organelles from a cell as well as to position liposomes incorporated with receptors and transporters along the cell for the high-resolution sampling and probing of the cellular microenvironment.

Patch Clamp Detection of Neuroreceptor Modulators in Capillary Electrophoresis

Many areas of research in the life sciences have been hampered by the inability to analyse microenvironments for important biologically active species. For example, the identities of many fast acting neurotransmitter substances in the mammalian brain are unknown, even after at least thirty years of active research, and endogenous ligands to numerous orphan receptors (e.g., plasma membrane opiate receptors) already cloned, remain to be discovered. During the past twenty years, development in instrumentation for microanalysis has resulted in techniques capable of sample handling in the low femtoliter range (10–15 L). Yet the development of detection devices for bioactive compounds that are compatible with these instrumentations has been extremely slow.